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OULU 2009

A 524

François Pujol

EXPERIMENTS ON FATTY ACIDS CHAIN ELONGATION AND GLYCAN FLIPPINGIN THE ER MEMBRANE

FACULTY OF SCIENCE,DEPARTMENT OF BIOCHEMISTRY,UNIVERSITY OF OULU;BIOCENTER OULU,UNIVERSITY OF OULU

A 524

ACTA

François Pujol

A C T A U N I V E R S I T A T I S O U L U E N S I SA S c i e n t i a e R e r u m N a t u r a l i u m 5 2 4

FRANÇOIS PUJOL

EXPERIMENTS ON FATTY ACIDS CHAIN ELONGATION AND GLYCAN FLIPPING IN THE ER MEMBRANE

Academic dissertation to be presented with the assent ofthe Faculty of Science of the University of Oulu for publicdefence in Raahensali (Auditorium L10), Linnanmaa,on 27 March 2009, at 12 noon

OULUN YLIOPISTO, OULU 2009

Copyright © 2009Acta Univ. Oul. A 524, 2009

Supervised byDoctor Alexander KastaniotisProfessor Kalervo Hiltunen

Reviewed byDoctor Maurizio MolinariDoctor Peter Richard

ISBN 978-951-42-9068-8 (Paperback)ISBN 978-951-42-9069-5 (PDF)http://herkules.oulu.fi/isbn9789514290695/ISSN 0355-3191 (Printed)ISSN 1796-220X (Online)http://herkules.oulu.fi/issn03553191/

Cover designRaimo Ahonen

OULU UNIVERSITY PRESSOULU 2009

Pujol, François, Experiments on fatty acids chain elongation and glycan flipping inthe ER membraneFaculty of Science, Department of Biochemistry, University of Oulu, P.O.Box 3000, FI-90014University of Oulu, Finland; Biocenter Oulu, University of Oulu, P.O. Box 5000, FI-90014University of Oulu, Finland Acta Univ. Oul. A 524, 2009Oulu, Finland

Abstract

Very long chain fatty acids (VLCFA) are essential molecules that take part in many differentcellular processes such as membrane pore stabilization, membrane trafficking and signalingpathways.

The fatty acid elongation pathway in yeast has been studied for about a decade. As part of ourwork on cellular VLCFA elongation, we identified and characterized the condensing enzyme aswell as ketoacyl reductases of the elongation pathway in cotton.

In order to identify the yeast 3-hydroxyacyl-CoA dehydratase, we introduced a redundancy inthis function by engineering a chimera consisting of the two first predicted transmembranedomains of Elo3p and the hydratase2 domain of Candida tropicalis Mfe2p. Yeast harboring thechimeric construct were subjected to random mutagenesis, and screened for mutants whosesurvival was dependent on the chimera. The mutants isolated contained RFT1 mutations andexhibited a defect in protein glycosylation, but no VLCFA deficiencies.

The N-linked glycosylation pathway is well conserved in eukaryotes. Glycan synthesis occurson the ER membrane; first on the cytoplasmic side up to Dol-PP-GlcNAc2Man5, which is thentranslocated to the ER luminal side in an Rft1p-dependent flipping process. The core glycan isfurther extended to Dol-PP-GlcNAc2Glc3Man9, and then transferred to an asparagine side chainof the nascent polypeptide to be glycosylated.

It was found that the Elo3'-hydratase2 chimera acts as a multicopy suppressor of the Rft1pdeficiency. The subsequent studies elucidated new aspects of Rft1p function, as well as a hithertounder-appreciated role of the ER associated protein degradation process in the maintenance of ERintegral membrane complexes and the physical integrity of the membrane.

The functionality of the human Rft1p homologue was demonstrated using a yeastcomplementation assay. A mutant variant from a patient was analyzed, aiding in the identificationand characterization of the first reported case of a glycosylation deficiency in humans caused bya defective RFT1 allele.

Keywords: condensation, ER, ERAD, flipping, N-linked glycosylation, RFT1,Very long chain fatty acid

« La plus perdue de toutes les journées est celle où l’on a pas ri »

Nicolas de Chamfort, Maximes et pensées, caractères et anecdotes

« The most wasted of days are those during which we do not laugh »

6

7

Acknowledgements

This work has been carried out at Department of biochemistry and Biocenter Oulu,

University of Oulu during the years 2002–2009, and was supported by the Sigrid

Juselius Foundation and the Academy of Finland.

I would like to express my deepest gratitude to my supervisors, Ph.D.

Alexander Kastaniotis for his careful guidance and wide expertise, and Professor

Kalervo Hiltunen for its everlasting enthusiasm and bottomless well of knowledge.

They both contributed greatly to the completion of the projects, and more

importantly helped me in making my first steps in the world of science with their

great support.

I wish to thank all the professors, group leaders, researchers and staff

members for the friendly and positive working atmosphere. They all have been

very helpful, and contributed to the scientific results obtained.

I wish to address a special thank to Professor Yu-Xian Zhu for the successful

collaboration in the isolation of new genes in cotton, Professor Yong-Mei Qin for

her great enthusiasm and help when visiting the group, and Chun-Yang Hu for the

great moment in and outside the lab and for trying to teach me some Chinese.

I am indebted to Professor Markus Aebi for the fruitful collaboration. I would

like also to express my warmest thanks to Christine Neupert for her

communicative passion and eagerness to solve the glycan flipping mistery.

I am grateful to Maurizio Molinari and Peter Richard for their careful

revision of this thesis. Their encouraging comments helped me to improve my

work. Dr. Deborah Kaska is acknowledged for her help is the language revision.

I am very thankful to my colleagues for the nice discussions and great

atmosphere in the lab: Eija, Fumi, Kaija, Katja, Katri, Laura, Maija, Miia, Silke,

Tonja, Aare, Antti, Ilkka, Samuli, Tatu, Tuomo, Vasily and Zhi-Jun and all former

members of the KH group, an especially those of you who shared the same office

and faced yeast fun and frustration. I also would like to thank the staff members

for overall maintenance of equipments and facilities, and excellent help and

advises with official matters and technical assistance.

I would like to thank Teemu for the fun we had, and for his generous help

regarding computers and their mysteries.

I wish to address a special thank to Aino-Maija, Florence, Morgane, Antti,

Pekka, Sharat and Ville for all the shared activities which greatly helped me

relaxing and especially when playing tennis and floorball. All these moments

8

contributed to keep me balanced and healthy (despite all the very nice and joyful

dinner we had).

I would like to thank Aino-Maija, Tiina and Antti for the challenging

questions regarding my mother tongue. Answering all the questions was not

always easy, but I always took a great pleasure in finding the best answer possible.

Je voudrais dire un grand merci à Florence, Morgane et Sharat pour les très

bons moments passés en votre compagnie lors de repas ou autres activités. Votre

proximité et présence dans les moments les plus difficiles comme dans les

meilleurs moments, tout comme vos encouragements ont été pour moi très

importants tout au long de ces années.

J’adresse mes plus chaleureux remerciements à Benjamin et Tatiana ainsi

qu’à Sébastien, Ségolène, Anaïs et Lisa pour tous les merveilleux moments passés

ensemble lors de nos diverses retrouvailles. Ces instants, toujours trop courts, font

partis des meilleurs événements de ma vie.

Je souhaite tout particulièrement remercier mes parents, ma petite sœur

Céline et Emeric pour m’avoir toujours encouragé, mais surtout pour m’avoir

apporté leur soutien à chaque étape importante et dans les moments les plus

difficiles tout au long de ma vie.

Thank you all, this work would have not been accomplished without your

contribution and support.

9

Abbreviations

26:0 Standard numerical fatty acid nomenclature, in which the number

preceding the colon represents the number of carbons in the

hydrocarbon chain, and the number following the colon indicates

the number of double bonds in the hydrocarbon chain.

CDG congenital disorders of glycosylation

cDNA Complementary deoxyribonucleic acid

CM Chloroform/methanol

CMW Chloroform/methanol/water

CoA Coenzyme A

CPY Carboxypeptidase Y

DNA Deoxyribonucleic acid

Dpa Day post anthesis

DTT Dithiothreitol

E. coli Escherichia coli

EDEM ER degradation enhancing α-mannosidase-like lectin

ELO3 Elongase3

ELO3’ Truncated version of ELO3 consisting of the two first

transmembrane domains

EMS Ethane methyl sulfonate

ER Endoplasmic reticulum

ERAD Endoplasmic reticulum associated degradation

ERAD-C Endoplasmic reticulum associated degradation cytosolic

ERAD-L Endoplasmic reticulum associated degradation lumenal

FA Fatty acid

FAS Fatty acid synthase

FPP Farnesyl diphosphate

GFP Green fluorescent protein

GSL Glycosphingolipid

GPL Glycerophospholipids

H2 Hydratase2

HMGR 3-hydroxy-3-methylglutaryl-coenzyme A reductase

HPLC High performance liquid chromatography

IL Interleukine

kb Kilobase

kDa Kilodalton

10

LiAc Lithium acetate

LCB Long chain base

LLO Lipid linked oligosaccharides

MFE2 Peroxisomal multifunctional enzyme type 2

mYFP Monomeric Yellow fluorescent protein

NADP+ Nicotinamide adenine dinucleotide phosphate, oxidized form

NADPH Nicotinamide adenine dinucleotide phosphate, reduced form

NLG Asparagine-linked glycosylation

NV Nucleus-vacuole

ORF Open reading frame

OST Oligosaccharyltransferase

PCR Polymerase chain reaction

PDB Protein data bank

PMN Piecemeal microautophagy of the nucleus

RNA Ribonucleic acid

S. cerevisiae Saccharomyces cerevisiae

SDS-page Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SL Sphingolipid

TE Tris EDTA

VLCFA Very long chain fatty acid

YFP Yellow fluorescent protein

11

List of original articles

This thesis is based on the following articles:

I Qin YM, Pujol FM, Shi YH, Feng JX, Liu YM, Kastaniotis AJ, Hiltunen JK

& Zhu YX (2005) Cloning and functional characterization of two cDNAs

encoding NADPH-dependent 3-ketoacyl-CoA reductases from developing cotton

fibers. Cell Res 15(6): 465–473.

II Qin YM, Pujol FM, Hu CY, Feng JX, Kastaniotis AJ, Hiltunen JK & Zhu YX

(2007) Genetic and biochemical studies in yeast reveal that the cotton fibre-

specific GhCER6 gene functions in fatty acid elongation. J Exp Bot 58(3):

473–481.

III Pujol FM*, Neupert C*, Kastaniotis AJ, Savilahti H, Hiltunen JK & Aebi M

(2009) Artificially generated Rft1p-independent flipping sufficient for

N-linked glycan synthesis. Manuscript.

IV Haeuptle MA*, Pujol FM*, Neupert C, Winchester B, Kastaniotis AJ, Aebi M

& Hennet T (2008) Human RFT1 deficiency leads to a disorder of N-linked

glycosylation. Am J Hum Genet 82(3): 600–606.

* Equal contribution

12

13

Contents

Abstract Acknowledgements 7 Abbreviations 9 List of original articles 11 Contents 13 1 Introduction 16 2 Review of the literature 18

2.1 Sphingolipids .......................................................................................... 18 2.1.1 Simple sphingolipids .................................................................... 19 2.1.2 Glycosphingolipids....................................................................... 19

2.2 Very long chain fatty acids...................................................................... 20 2.3 Very long chain fatty acid synthesis........................................................ 21

2.3.1 The ketoacyl-synthase step........................................................... 23 2.3.2 The 3-Ketoreductase step ............................................................. 25 2.3.3 The dehydratase step .................................................................... 25 2.3.4 The enoylreductase step ............................................................... 26

2.4 Protein glycosylation............................................................................... 27 2.4.1 Overview of the asparagine-linked glycosylation pathway.......... 27

Dolichol synthesis 27 The asparagine-linked oligosaccharide synthesis pathway 30 Flipping of lipid-linked oligosaccharides 33 Oligosaccharyl transferase 36 2.4.2 Physiological role of N-linked glycosylation ............................... 38

Role of protein N-glycosylation 40 Lectin chaperones and glucose on the A branch 42 Protein degradation and Man8-GlcNac2 specific lectin 43 2.4.3 Glycosylation defect and congenital disorders ............................. 48

2.5 Yeast as a model organism to study essential genes................................ 48 3 Aim of the study 51 4 Material and Methods 53

4.1 Yeast strains, culture media and plasmid construction............................ 53 4.2 Hydratase2 enzymatic assay ................................................................... 53 4.3 Random mutagenesis .............................................................................. 53

4.3.1 Chemical mutagenesis .................................................................. 53 4.3.2 Transposon mutagenesis............................................................... 54

14

Transpososome assembly 54 Yeast electroporation 54

4.4 Yeast genomic DNA library transformation............................................ 55 4.5 Western blot ............................................................................................ 55 4.6 Pulse chase and LLO preparation............................................................ 55 4.7 Semi-quantitative reverse transcription (RT)-PCR analysis.................... 56 4.8 Real time quantitative (QRT)-PCR analysis of GhCER6

expression in different cotton tissues ...................................................... 56 4.9 RNA in situ hybridization ....................................................................... 57 4.10 Preparation of ER extracts from yeast cells ............................................ 57 4.11 Elongase assays of cotton 3-ketoacyl-CoA reductases

overexpressed in yeast cells .................................................................... 57 4.12 Lentiviral mediated hRFT1 expression ................................................... 58 4.13 Human patient mutation analysis ............................................................ 58

5 Results 59 5.1 Identification of cotton orthologs to yeast enzymes involved in

the VLCFA elongation pathway in cotton ............................................... 59 5.1.1 Identification of cotton 3-ketoacyl-CoA reductases ..................... 59 5.1.2 Identification of cotton tissue specific 3-ketoacyl-CoA

synthase ........................................................................................ 59 5.2 A genetic screen designed to identify the 3-hydroxyacyl-CoA

dehydratase located in the yeast ER........................................................ 60 5.2.1 Random mutagenesis and colony-color-sectoring screen

for identification of mutants dependent on the chimera ............... 61 5.2.2 Transposon random mutagenesis in yeast..................................... 62 5.2.3 Investigation of the chimera function ........................................... 63 5.2.4 Lipid-linked oligosaccharide profiles ........................................... 66 5.2.5 Analyzing human patient mutation and yeast

complementation .......................................................................... 67 6 Discussion 69

6.1 Identification and characterization of genes encoding NADPH-

dependent 3-ketoacyl-CoA reductase from developing cotton

fibers........................................................................................................ 69 6.2 Identification of a cotton fiber-specific fatty acid elongation

condensing enzyme ................................................................................. 69 6.3 Investigation of the flipping mechanism of the lipid linked

glycan in the yeast N-linked glycosylation pathway. .............................. 70

15

6.4 Implication to the general membrane flipping process ........................... 74 6.5 Identification and analysis of a human patient mutation in the N-

linked glycosylation pathway.................................................................. 75 7 Conclusions 77 References 79 Original articles 99

16

1 Introduction

Fatty acids (FAs) are essential molecules composed of oxygen, carbon and

hydrogen, and are used in many different ways in cells. They can function in

energy storage, and as structural elements or building blocks for the synthesis of

more complex molecules. They also have regulatory roles in biological processes

(Kohlwein et al. 2001, Schneiter et al. 1996, Schneiter et al. 2004, Leonard et al.

2002). FA function and category are determined mostly by the length of the

carbon chain: short FAs range from 2 to 6 carbons, medium FAs from 8 to 12

carbons, long chain FAs from 14 to 18, and very long chain fatty acids (VLCFA)

from 20 carbons.

In yeast, VLCFA also serve as building blocks for much more complex

molecules but are not taken up as nutrients, which makes their endogenous

synthesis through the elongation pathway an essential process for the cell. The

protein complex catalyzing fatty acid chain elongation is located in the

endoplasmic reticulum (ER) membrane (Nugteren 1965) and uses long chain FAs

(C16–C18) generated by the cytosolic fatty acids synthase as substrates.

In this work, the yeast S. cerevisiae was used as a model organism to

characterize cotton fiber genes involved in the VLCFA elongation pathway. The

disturbed VLCFA profile of yeast deletion mutants lacking the elongase genes

was restored to wild type when the corresponding cotton homologues were

expressed in these mutant cells.

Prior to the initiation of this study, three of the four enzymes of the elongation

cycle in yeast were known. The dehydratase, however, had not been identified

(Han et al. 2002).

N-linked glycosylation is the main protein modification in eukaryotes and it

takes place on nascent proteins in the ER lumen on asparagine side chains in the

sequence Asn-X-Ser/Thr, unless X is a proline (Welply et al. 1983). The added

glycan increases the mass and solubility of the nascent peptide, prevents the

aggregation of hydrophobic patches, and makes proteins more resistant to

denaturation and proteolysis (Helenius & Aebi 2004). Glycan synthesis is

initiated on the cytosolic side of the ER membrane and proceeds until a structure

composed of a dolichol pyrophosphate, two N-acetylglucosamine and five

mannose residues (DolPP-GlcNac2-Man5) is formed (Bugg & Brandish 1994). A

key step in the glycan synthesis is the translocation of the lipid linked

oligosaccharide (LLO) across the ER membrane in an Rft1p-dependent process

17

(Helenius et al. 2002). The glycan facing the ER lumen will be further elongated,

by the addition of four mannose and three glucose moieties, up to DolPP-

GlcNac2-Man9-Glc3 and thereafter transferred to the nascent peptide.

In this study, a chimeric protein engineered to introduce redundancy in the

dehydratase step of the VLCFA elongation pathway suppressed the reported yeast

rft1Δ lethal phenotype (Helenius et al. 2002). The data demonstrated that the

suppression was due to unspecific LLO flipping. Our observation that inactivation

of the ER associated protein degradation pathway (ERAD) rescues the growth of

the S. cerevisiae rft1Δ highlights the importance of the malfolded protein response

for the specificity of the LLO flipping and membrane integrity in general. The

data obtained through functional complementation of the yeast rft1Δ mutant

demonstrated that the human homologue was capable of substituting for the yeast

Rft1p in flipping (Haeuptle et al. 2008). Furthermore, the results also showed that

a human RFT1 variant isolated from a patient diagnosed with a glycosylation

defect, was unable to complement the yeast rft1Δ N-glycosylation phenotype.

This is the first case of a glycosylation deficiency caused by a loss-of-function

mutation mapped to hRft1p.

18

2 Review of the literature

2.1 Sphingolipids

The eukaryotic membrane is a lipid bilayer consisting of three major types of

lipids: glycerolipids, sphingolipids (SLs), and sterols. Studies on these

compounds have elucidated their crucial role in membranes and their involvement

in turn-over in signaling pathways (Lahiri & Futerman 2007).

SLs are essential compounds in eukaryotes and account for approximately

10% of the total mass of the cellular lipid species (Smith & Lester 1974). The

backbone of the SLs is a sphingoid long chain base (LCB). The most common

LCBs are sphiganine and sphingosine, but the structure of the carbon chain varies

depending on the functional requirements (Lahiri & Futerman 2007). In the yeast

S. cerevisiae, the LCB is usually phytosphingosine (Dickson 1998). The

hydrophobic part of SLs is almost exclusively composed of a hexacosanoic acid

or α-hydroxyhexacosanoic acid (Lester & Dickson 1993), and a long chain base

(LCB) linked to the VLCFA via an amide linkage (fig. 1.).

Fig. 1. General chemical structure of the sphingosine class of sphingolipids. “R” can

be either hydrogen in the case of ceramides; phosphocholine in sphingomyelin;

phosphoinositol in inositolphosphorylceramide-C; or sugars in glycosphingolipids.

Ceramides are the simplest SLs; they are the key hub for the elaboration of more

complicated SLs by the attachment of different head groups (fig. 1.) (Lahiri &

Futerman 2007). Glycosphingolipids (GSL) comprise the class of SLs that

presents the highest degree of structural variety; more than five hundred variants

have been described (Schwarz & Futerman 1996, Kolter et al. 2002). To date, the

reasons for such a variety are not fully understood. Thus it is not yet possible to

determine a priori for any individual case if a particular structure can serve a

19

unique purpose, or if the aggregation and distribution of the GSLs over the

membrane is key to the biological function. Hence, the role of any one variant in

the cellular context has to be determined individually (Lahiri & Futerman 2007).

2.1.1 Simple sphingolipids

Like many other membrane lipids of the cell, SLs have more than just a structural

role. SLs have been shown to be involved in intracellular signaling pathways

controlling cell proliferation, differentiation, growth, senescence, and apoptosis.

Ceramide and sphingosine-1-phosphate have been proposed to play opposite roles

in these pathways (Lahiri & Futerman 2007). They are also involved in other

cellular bioactivities like cell adhesion and recognition (Schneiter 1999, Wells &

Lester 1983, Hanada et al. 1992, Hakomori & Igarashi 1995). Furthermore, they

are essential components in many processes involved in the transfer of material in

and out of cells, as well as in cellular communication (Eisenkolb et al. 2002,

Dupré & Haguenauer-Tsapis 2003, Boukh-Viner et al. 2005, Simons & Ikonen

1997).

In apoptosis, a large number of external stimuli induce an increase in the

ceramide concentration that accompanies the events leading to cell death. Heat

shock, radiation, oxidative stress, progesterone, tumor necrosis factor α,

interleukine-1α (IL) and interferon-γ can trigger this increase in ceramide

concentration (Mathias et al. 1998, Gulbins & Li 2006). There is a direct

correlation between the administration of ceramides or their analogues and the

induction of apoptosis in cancer cells and cancer cell lines, suggesting an anti-

carcinogenic activity (López-Marure et al. 2002, Samsel et al. 2004, Struckhoff et

al. 2004, Granot et al. 2006, Senkal et al. 2006).

2.1.2 Glycosphingolipids

GSLs (see fig. 1.) are involved in brain development via a change in their

concentration between the embryonic and the adult stage (Yates 1986). In adults,

they also appear to play a role in germ cell transformations: male spermatogenesis

is affected if the GSL concentration is too low (Takamiya et al. 1998).

Some of the GSLs are able to bind a certain class of immune T cells, the

invariant natural killer T cells (Hayakawa et al. 2004). The activation of these

cells upon GSL binding induces the secretion of IL-4, interferon-γ, and the

transactivation of various cells of the innate and adaptive immune systems (Fujii

20

et al. 2004). Based on these findings, several studies concerned with the

therapeutic use of GSLs in the treatment of autoimmune diseases (Van Kaer 2005),

type 1 diabetes (Eldor et al. 2005), multiple sclerosis (Jahng et al. 2001) and

rheumatoid arthritis (Elewaut 2005) have been carried out. Unfortunately,

preliminary results from these studies in mice indicate an exacerbation of the

symptoms and some severe side effect (Van Kaer 2005).

In yeast, the phenotype of sphingolipid-deficient mutants can be suppressed

by adding glycerophospholipids containing VLCFAs to the culture medium.

Presumably, this is achieved by mimicking the sphingolipid structure (Lester et al.

1993), thus underlining the key role of VLCFAs in sphingolipid functions.

2.2 Very long chain fatty acids

VLCFAs are molecules with a chain length from 20 to 36 carbons (Leonard et al.

2004) that play an important role in the structure, function, permeability and

fluidity of biological membranes by imparting unique physical properties to the

lipids in which they are incorporated (Schneiter et al. 1996, Han et al. 2002, Ho et

al. 1995, Millar et al. 1998, McMahon et al. 2007, Vasireddy et al. 2007). For

instance, VLCFAs are required to stabilize the highly curved membrane at the

nuclear pore complexes (Schneiter et al. 1996, Schneiter et al. 2004).

VLCFAs exhibit different chain length abundance profiles across species and

tissues, and these differences provide the basis for functional specialization and

diversity. For example in S. cerevisiae, VLCFAs with 22 carbons are sufficient to

support essential functions, but can neither compensate for all the VLCFA roles in

membrane-based processes and GPI lipid anchors, nor for the role played in the

trafficking of proteins in the secretory pathway (Dickson et al. 2006, Schneiter &

Toulmay 2007).

Alterations of the VLCFA elongation pathway suppress the Ca2+ sensitive

phenotype of a csg2Δ mutant by preventing the accumulation of

inositolphosphoceramide due to the absence of mannosylation by Csg2p

(Kohlwein et al. 2001, Zhao et al. 1994, Beeler et al. 1998, Dunn et al. 2000).

In higher eukaryotes, the VLCFA acid composition varies in different tissues.

For instance, the brain contains mainly saturated or monounsaturated C24-C26,

whereas in the kidney and testis the α-hydroxylated forms are predominant. In

testis, epidermis and retina, the VLCFAs are mostly polyunsaturated with a chain

length of up to 34 carbons (Han et al. 2002). Apart from their structural role,

VLCFAs also have bioactivities that regulate various cellular processes. VLCFAs

21

and their derivatives can act as signaling molecules (Leonard et al. 2002), i.e.

long-chain acyl-CoA and unsaturated VLCFAs act as ligands for nuclear

transcription factors that modulate target gene expression (Hertz et al. 1998, de

Urquiza et al. 2000). In both human and Chinese hamster cells, VLCFAs serve as

extracellular signals that activate specific G-protein complexes (Briscoe et al.

2003, Itoh et al. 2003).

2.3 Very long chain fatty acid synthesis

The majority of cellular fatty acids have 16 or 18 carbons and are synthesized by

the cytosolic soluble multifunctional enzyme fatty acid synthase I (FAS I)

complex (Jenni et al. 2007, Lomakin et al. 2007). These FAS I products serve as

substrates for the VLCFA elongation pathway (Kohlwein et al. 2001), and a great

deal of effort has been invested in understanding this pathway. It has been known

for some time that the enzymes responsible for VLCFA elongation are ER

membrane-bound and only soluble in detergent (Cinti et al. 1992).

In yeast, VLCFA de novo elongation partially overlaps with the activity of

FAS I (Rössler et al. 2003), and is required for the normal biogenesis of

piecemeal microautophagy of the nucleus (PMN), which is used for degradation

of nonessential nuclear material. Alteration of the elongation pathway leads to

smaller PMN blebs and vesicles (Kvam et al. 2005). The ER fatty acid chain

elongation process requires four reactions identical to the process catalyzed by

cytosolic FAS I: condensation, reduction, dehydration, and a final reduction, but

the substrates are acyl-CoA instead of acyl-ACP. All evidence points towards

a: ”one peptide – one function” system in this pathway (Han et al. 2002, Denic &

Weissman 2007) (fig. 2.) rather than multifunctional enzymes. Each member of

the complex has several transmembrane domains and a very basic pI value (>9)

(Han et al. 2002, Denic & Weissman 2007).

22

Fig. 2. -The pathway of fatty acid chain elongation and LCB synthesis in yeast.

Generation of FAs up to palmitic acid takes place in the cytosol and is carried out by

the soluble FAS type I multifunctional enzyme. Then palmitoyl-CoA is elongated to a

C26 VLCFA by a membrane-associated fatty acid elongating system. Each cycle of

elongation requires four successive reactions; condensation of malonyl-CoA with the

acyl-CoA substrate, reduction of the 3-ketoacyl-CoA, dehydration of the

3-hydroxyacyl-CoA, and reduction of the trans-2-enoyl-CoA.

23

2.3.1 The ketoacyl-synthase step

In the condensation reaction, two carbons from a malonyl-CoA are added to an

existing long/very long carbon chain (Kohlwein et al. 2001, Schneiter &

Kohlwein 1997). This step has been proposed to be the slowest and rate limiting

reaction in the VLCFA elongation pathway (Han et al. 2002). The reaction is

catalyzed by proteins, some of which belong to a large family of elongase

proteins (Elops) (Jakobsson et al. 2006, Moon et al. 2001, Paul et al. 2006,

Westerberg et al. 2006).

The results of complementation and mutational studies in yeast led to the

conclusion that this class of proteins is responsible for chain length selection and

specificity across organisms and cell types (Leonard et al. 2004, Denic &

Weissman 2007, Jakobsson et al. 2006).

In yeast, Elo1p catalyzes the condensation of medium chain FA, whereas the

VLCFA condensation can be catalyzed by Elo2p and Elo3p to form products up

to a length of twenty four or twenty six carbon atoms, respectively (Denic &

Weissman 2007). This conclusion is supported by the study of VLCFA profiles in

haploid yeast mutants with single ELO gene deletions: the disruption of ELO2 in

yeast results in a severe decrease of C26:0 (down to 20% of the wild-type level)

and no C20 are detectable (Oh et al. 1997). On the other hand, when ELO3 is

disrupted in yeast, C26:0 and HO-26:0 are no longer detectable, whereas the

amount of shorter fatty acids (C20 and C22) is increased by a factor 10. The

hydroxy C16-C24 levels are also elevated compared to wild-type.

Hence Elo2p displays specificity towards elongation to up to twenty- four

carbon atoms (Oh et al. 1997). In contrast, Elo3p appears to be more specific for

the last condensation reaction that extends the carbon chain length to 26 atoms

(Oh et al. 1997), which is the class of VLCFAs most abundant in the budding

yeast cell (Lester & Dickson 1993, Nagiec et al. 1993).

Both Elo2p and Elo3p are ER membrane-integrated proteins with five

predicted transmembrane domains (Oh et al. 1997). The catalytic sites of the

fungal enzymes are predicted to face the cytosol or at its direct proximity,

allowing the utilization of malonyl-CoA which is synthesized by the cytosolic

acetyl-CoA carboxylase (Ivessa et al. 1997). Furthermore, complementation of

the gene deletion phenotype in yeast by plant homologues, known to have their

active site in the cytosol, supports the model for this type of protein topology

(Paul et al. 2006).

24

Unlike the FAS I domain that shows condensing activity, Elo2p and Elo3p do

not harbor a conserved cysteine residue (Paul et al. 2006), which may be the

reason why the FAS inhibitor cerulenin is not effective against the VLCFA

elongation pathway. According to a study by Ferrer et al., the HXXHH motif

found in Elops is typical for proteins binding the phosphate moieties of CoA

(Ferrer et al. 1999). Moreover, this CoA binding motif does not seem to be critical

for the global folding of the proteins, since a condensing enzyme mutated in the

conserved HXXHH domain can still be part of the elongation complex. The motif,

however, does play a role in the enzymatic function. A very well conserved lysine

residue can be found thirteen amino acids upstream of the CoA binding motif. It

was demonstrated in extensive studies that this lysine plays a key role in the

determination of the final length of the elongation cycle product (Denic &

Weissman 2007). This specificity depends on the presence and location of this

lysine residue in the second to last transmembrane domain of the protein, which is

part of a hydrophobic pocket in Elo proteins. The distance between the cytosol

and this particular lysine would be a physical “caliper” for the elongation: the

deeper the lysine sits in the pocket, the longer the synthesized VLCFAs will be. If

this amino acid is closer to the cytosol, elongation will stop at an early stage (fig.

3.) (Denic & Weissman 2007).

Fig. 3. Control of VLCFA chain length by Elops by a molecular caliper mechanism. The

second-to-last transmembrane domain (an α-helix) in the hydrophobic pocket and the

key lysine are highlighted in the representation of Elo3p. (A) In the wild-type elongase,

the final product is C26. (B) Introduction of a lysine at an earlier helix turn will lead to

a final product with a shorter carbon chain length (modified from Denic et al. 2007).

25

The results of studies on the in vitro reconstituted complex showed that the

fatty acid is neither released during the elongation process, nor taken back into the

complex if released prematurely. This strongly suggests that Elo2p and Elo3p are

never present in the same complex and efficiently terminate specific processive

elongation starting from the same long chain fatty acid substrate (Denic &

Weissman 2007).

2.3.2 The 3-Ketoreductase step

In yeast, the 3-ketoreductase activity is catalyzed by Ybr159p, which is required

for the elongation of saturated and polyunsaturated FAs (Beaudoin et al. 2002).

The protein is localized at the nuclear-ER interface, where it associates in a

complex with Elo3p and Tsc13p (Han et al. 2002).

A ybr159w deletion strain is viable, although it grows poorly, due to a

redundancy in the 3-ketoreductase activity. The best candidate for the redundant

3-ketoreductase is Ayr1p (Han et al. 2002), which has been reported previously to

be a 1-acyldehydroxyacetone-phosphate reductase (Athenstaedt & Daum 2000).

The ybr159w mutation exhibits synthetic lethality in conjunction with elo2Δ

(Han et al. 2002). In addition, when a fas2 mutation was present or when a

ybr159wΔ strain was grown in the presence of cerulenin, synthetic lethality was

observed, indicating a link to cytoplasmic FAS (Rössler et al. 2003).

Like the elongase mutants, the ybr159wΔ yeast strain exhibits accumulation

of free LCBs, phytosphingosine (PHS) and dihydrosphingosine without an

increase of the enzymatic activities responsible for the synthesis of these

compounds (Kohlwein et al. 2001, Han et al. 2002). The presence of the Ybr159p

in the protein complex was shown to be required for the full dehydratase activity

in the VLCFA elongation pathway (Han et al. 2002).

2.3.3 The dehydratase step

The very recently discovered 3-hydroxyacyl-CoA dehydratase has been shown to

play an essential role in sphingolipid metabolism (Schuldiner et al. 2005). The

enzyme, encoded by PHS1, has been proposed to contain six transmembrane

domains (Denic & Weissman 2007).

As is the case for other members of the pathway, yeast cells expressing

decreased levels of this protein accumulated LCBs and failed to grow on media

containing myristic acid (C14) when the cytosolic FAS I was chemically

26

inactivated by cerulenin (Rössler et al. 2003, Denic & Weissman 2007).

Furthermore, in the Denic et al. study, Phs1p has been shown to

coimmunoprecipitate with the other members of the VLCFA elongation

machinery. In addition a phs1Δ strain only accumulates 3-hydroxyacyl-CoA; the

first two elongation steps (condensation and first reduction) are not affected by

the absence of the dehydratase.

Phs1p does not show any homology with the known hydratases, but several

studies on close homologs of Phs1p have reported an induction of tumorigenesis

in plants upon alteration of the enzyme function (Bellec et al. 2002, Da Costa et

al. 2006). In a canine model, alteration of the gene encoding the Phs1

homologous enzyme by tRNA insertion leads to muscle myopathy, providing a

model for human disease (Pelé et al. 2005).

2.3.4 The enoylreductase step

This last step of the VLCFA elongation pathway is catalyzed in yeast by Tsc13p,

which is a protein essential for cell viability (Tuller et al. 1999). It catalyzes the

reduction of a double bond at the α,β position, but unlike the condensing enzyme,

it does not exhibit chain length preference. The protein has been predicted to

contain six transmembrane domains, with a cytosolic orientation of both N and C-

termini (Paul et al. 2007).

The protein is evolutionarily conserved in eukaryotes and displays amino acid

sequence similarity in its carboxy-terminal part to the steroid-5,α-reductase

enzyme (Kohlwein et al. 2001).

Tsc13p has been shown to co-immunoprecipitate with Elo2p and Elo3p and is

also found in the nucleus-vacuole (NV) junctions, which are sites of physical

contact between the nucleus and the vacuole (Kohlwein et al. 2001, Kvam et al.

2005, Pan et al. 2000). Moreover, the local concentration of Tsc13p in NV

junctions and in PMN increases during growth. The localization of this protein in

the NV junction is dependent upon the presence of VLCFAs. Alteration of

VLCFA production alone is sufficient to block protein targeting to given

membrane domains. The increase of Tsc13p is directly correlated with the

increase of the size of NV, which is controlled by the expression level of Nvj1p, a

protein that is up-regulated in response to nutrient depletion or nitrogen starvation

(Kvam et al. 2005, Gasch et al. 2000, Roberts et al. 2003).

27

2.4 Protein glycosylation

Protein glycosylation is a membrane associated process important in protein

maturation. Glycoproteins display a very broad diversity in structures and

functions. This is especially significant in mammals, where the number of ORFs

encoding proteins is lower than expected for a normal functioning organism.

Toward this end, protein glycosylation allows for a higher degree of diversity.

In mammals, it has been estimated that over a thousand different

carbohydrate structures are present (Vigerust & Shepherd 2007). To date, 41

variants of glycosylation have been referenced. They involve 13 different glycans

which can be linked to eight different amino acids (Spiro 2002). Asparagine-

linked glycosylation (NLG) is the most common (Nalivaeva & Turner 2001).

2.4.1 Overview of the asparagine-linked glycosylation pathway

The events required for protein glycosylation take place in several different cell

compartments. The polypeptides subject to glycosylation are synthesized on

ribosomes attached to the cytosolic side of the ER and translocated into the ER

lumen.

The initial steps of core glycan synthesis occur on the cytosolic side of the

ER, while subsequent additions of sugars take place in the ER lumen. During

these reactions, the nascent glycan is anchored to the ER membrane via a dolichol

moiety (see below). Attachment of the glycans to the polypeptide is carried out in

the ER lumen. Once the protein is properly folded, it is transported to the Golgi

apparatus where the N-glycans are further trimmed and modified.

Dolichol synthesis

Dolichol, the longest lipid synthesized in mammalian cells (fig. 4.), is an

isoprenoid essential for N- and O-glycosylation (Krag 1998, Burda & Aebi 1999,

Schenk et al. 2001). In these pathways, it anchors glycans to the ER membrane.

Yeast cells unable to produce dolichol have an abnormal accumulation of ER

and Golgi membranes (Sato et al. 1999).

28

Fig. 4. Dolichol phosphate structure. In the yeast S. cerevisiae, n=10–04 when the cis-

prenyl transfer reaction is catalyzed by Rer2p, and n=14–43 when Srt1p carries out the

reaction.

The first steps of dolichol synthesis occur in the cytosol, and are shared with the

sterol and ubiquinone synthesis pathways (Jung & Tanner 1973, Adair &

Cafmeyer 1987). Divergence occurs after the farnesyl diphosphate (FPP)

synthesis step (fig. 5.). The prenyltransferase, Erg20p in yeast, is an essential

enzyme with orthologs in all organisms (Song & Poulter 1994) and is responsible

for the synthesis of FPP, which is a precursor of a large variety of isoprenoid

compounds. The regulation of the synthesis of the isoprenoid is achieved by

Erg20p feedback inhibition (Grabińska & Palamarczyk 2002).

In dolichol synthesis, regulation via FPP accumulation and its derivatives

occurs through a rapid degradation of the 3-hydroxy-3-methylglutaryl-coenzyme

A reductase (HMGR) by Hrd1p, Hrd2p and Hrd3p, which are proteins involved in

the ER associated protein degradation machinery. The regulation via protein

degradation occurs in the ER by ubiquitination of HMGR (Gardner & Hampton

1999, Hampton et al. 1996, Hampton & Bhakta 1997, Correll et al. 1994, Meigs

et al. 1996).

A decrease in the dolichol pool leads to a diminution of the N-linked

glycosylation rate (Carlberg et al. 1996, Wang et al. 1999). Several studies

indicate that dolichol availability may be a limiting factor in protein glycosylation,

as supplementation with external dolichol phosphate can increase the level of N-

linked glycosylation in mammalian cell culture. According to another study,

however, this effect is cell type dependent (Grant & Lennarz 1983, Pan & Elbein

1990, Kousvelari et al. 1983, Carson et al. 1981, Yuk & Wang 2002).

29

Fig. 5. Dolichol synthesis in yeast.

30

The asparagine-linked oligosaccharide synthesis pathway

NLG is the most ubiquitous protein co-translational modification in the ER lumen

(Welply et al. 1983). It is an essential and highly conserved protein modification

pathway in eukaryotic cells (fig. 6.), where N-glycosylation is a determinant for

specific molecular recognition, protein folding and stability, and facilitates the

proper protein orientation towards a membrane (Helenius & Aebi 2004, Dempski

& Imperiali 2002).

Fig. 6. The asparagine-linked oligosaccharide synthesis pathway in S. cerevisiae.

OSTs is the oligosaccharyl transferase protein complex.

Eukaryotic glycoproteins are involved in a broad variety of cellular processes like

the immune response, intercellular recognition, signal transduction, cell adhesion,

31

receptor activation and endocytosis (Varki 1993, Cabral et al. 2001, Vigerust &

Shepherd 2007).

Some viruses can take advantage of the host glycosylation pathway in order

to ensure the maturation and modification of their proteins by using the host

cellular chaperones and folding factors. Glycosylation of the viral proteins allows

the virus to escape identification by the immune system, increases recognition by

cell receptors, and thus enhances infection. The viruses causing AIDS, influenza

and hepatitis, among others are known to exploit host cells in this way (Meunier et al.

1999, Land & Braakman 2001, Slater-Handshy et al. 2004, Vigerust & Shepherd

2007).

When a peptide enters the ER lumen, its sequence is scanned by the

oligosaccharyltransferase (OST) protein complex for asparagines in the Asn-X-

Ser/Thr motif, where X can be any amino acid except proline (Welply et al. 1983,

Silberstein & Gilmore 1996). According to Petrescu and co-workers (Petrescu et

al. 2004), the asparagine is more likely to be glycosylated when the threonine is

present.

The presence of such a sequence is the signal for a co-translational or post-

translational modification by the addition of a preassembled glycan (fig. 7.),

which increases the hydrophilicity of the nascent peptide and thus inhibits the

possible aggregation of unstructured peptides through interaction of their

hydrophobic patches (Ruddock & Molinari 2006, Ruiz-Canada et al. 2009). It

was shown by Bosques and co-workers that the prion peptide tends to form fibrils

when the glycosylation site Asn81 is free (Bosques & Imperiali 2003).

32

Fig. 7. The oligosaccharide structure added to a nascent eukaryotic peptide. The

oligosaccharide has three branches (A,B,C) and each mannose and glucose are

numbered. Mannoses 1–1 are assembled on the cytosolic side of the ER membrane,

mannose 6–6, and glucose 1–1 are added in the ER lumen. Mannoses 2, 4, 5 and

glucose 1 mediate the interaction of the mono-glucosylated glycans with lectins.

Peptide N-glycosylation, via the generation of an N-glycosidic bond, occurs co-

translationally, indicating that tertiary structure is not important for glycosylation

site recognition. It has been demonstrated, however, that the secondary structure

and physical properties surrounding the glycosylation site can be very important

for the glycan transfer reaction (O'Connor & Imperiali 1997, O'Connor &

Imperiali 1998, Petrescu et al. 2004). Glycan transfer requires the formation of a

temporary loop in the substrate peptide in which the hydroxyl group of the serine

or threonine interacts with the amide group of the asparagine to make it more

nucleophilic (Bause 1983, Imperiali et al. 1992). This physical property explains

33

why a proline cannot be glycosylated, and why a folded polypeptide is a poor

substrate.

Furthermore, not all the potential glycosylation sites of a peptide are always

glycosylated, and they can play different roles in protein maturation by

introducing a local conformation; often a β-turn, infrequently a β-strand and

rarely an α-helix (O'Connor & Imperiali 1996, Bosques et al. 2004).

A statistical study on protein crystal structures listed in the protein data bank

(PDB) showed that glycans are often found in a position where the secondary

structure changes. The presence of the glycan favors the reorientation of the

peptide chain by stabilizing a particular local fold. Petrescu and associates have

proposed that glycans can play a role during the protein folding process by

stabilizing the conformation of intermediates (Petrescu et al. 2004). The majority

of glycans can have few contacts with the protein surface (Wormald et al. 2002),

showing that the glycans and the protein are relatively independent of each other.

This independence allows glycan modification without a noticeable effect on the

protein (Rudd et al. 1999). Furthermore, the modification can serve as a signal for

the physiological state of the cell (cell cycle, differentiation, infection) (Lowe &

Marth 2003).

The glycans attached to the protein are recognized by proteins called lectins

(Weis & Drickamer 1996, Lis & Sharon 1998). The alteration of the glycan

properties (number, conformation) will change the binding to the lectin (Weis &

Drickamer 1996), and ultimately the destiny of the glycoprotein.

The last glucose (glucose 3 on branch A in fig. 7.) added to the nascent

glycan plays a crucial role in the recognition of the mature lipid-linked-

oligosaccharide (LLO) before it is transferred to the polypeptide (Burda & Aebi

1998, Burda et al. 1999, Spiro 2000a).

Flipping of lipid-linked oligosaccharides

Oligosaccharide synthesis starts on the cytosolic side of the ER and then

continues on the luminal side. The LLO intermediates need to be aided in the

flipping process across the ER membrane, since the nascent glycan is hydrophilic

and too big to be translocated spontaneously (Hanover & Lennarz 1979,

McCloskey & Troy 1980a).

In S. cerevisiae, the translocation of the GlcNAc2Man5 requires the presence

of Rft1p (Helenius et al. 2002). Rft1p has been suggested to act as a flippase and

is an ER-integrated membrane protein predicted to have 12 transmembrane

34

domains split into two groups connected by a hydrophilic linker facing the cytosol

(fig. 8.) (Results obtained with the TMHMM program). The protein does not

belong to the ABC family of translocators, as ATP binding motifs are absent.

Homologues of the yeast protein can be found encoded in all higher eukaryote

genomes that have been sequenced so far (Helenius & Aebi 2002).

35

36

Fig. 8. Alignment of Rft1p from different eukaryotic species (A) and S. cerevisiae Rft1p

protein topology (B). The alignment (A) includes the Homo sapiens, Zebrafish,

C.elegans, and S. cerevisiae Rft1p amino acid sequences. (B) Rft1p protein

transmembrane domain prediction using the TMHMM prediction program

(unpublished data).

Oligosaccharyl transferase

The OST, in association with the translocon complex, transfers a fully assembled

glycan to the nascent polypeptide in a co-translational fashion. The OST is

thought to be present in excess relative to the active translocon (Ruiz-Canada et al.

2009). In yeast, the transfer is mediated by the catalytic subunit Stt3p (Burda et al.

1996, Wacker et al. 2002). In its C-terminal part, this enzyme harbors a WWDYG

sequence conserved in all the Stt3p homologues. Introduction of mutations in this

sequence lead to a loss of function (Wacker et al. 2002).

In S. cerevisiae, OSTs are complexes composed of two or three of the

following proteins: Ost1p, Ost2p, Wbp1, Swp1, Stt3p, Ost3p/Ost6p, Ost4p, and

Ost5p (Fig. 9.). All of them have multiple transmembrane domains and an active

site facing the ER lumen. Only the first five proteins listed are essential (Knauer

& Lehle 1999a). Ost3p and Ost6p are homologues which represent distinct

oligosaccharyl transferase isoforms (Knauer & Lehle 1999a, Karaoglu et al. 1995,

Karaoglu et al. 1997). With the exception of Ost4p and Ost5p, homologues for all

the OST proteins have been found in mammals (Dempski & Imperiali 2002,

Knauer & Lehle 1999b).

37

The OST complexes have been most thoroughly studied in S.cerevisiae,

where it exists in three protein associations: Ost1p-Ost5p, Ost2p-Sw1p-Wbp1p,

and Stt3p-Ost4p-Ost3/Ost6p (Dempski & Imperiali 2002, Yan & Lennarz 1999)

(fig. 9.).

Fig. 9. Model of the interrelationship of yeast oligosaccharide transferases. The

essential subunits (shown as ovals) are located within 12 Å from each other. Ost4p

and Ost5p interact only with a few subunits, Ost3p/Ost6p are present in the complex

alternatively. The solid arrows indicate the link between the proteins always present in

the complex. The dashed arrows show the interaction of the alternative members of

the protein complex (Modified from Yan & Lennarz, 2004).

In yeast, in vivo experiments have provided evidence that the synthesis of the

fully assembled Glc3-Man9-GlcNac2 is the limiting factor in the peptide

glycosylation reaction. Priority is given to the complete synthesis of an

oligosaccharide even at the risk of protein underglycosylation. If the fully

assembled moiety is not available, however, OSTs will transfer the incomplete

variant (Kelleher et al. 2001). In higher eukaryotes, in contrast, the dilemma

between site occupancy and transfer of fully assembled Glc3-Man9-GlcNac2 is

subject to regulation (Kelleher et al. 2003). In mammalian cells, two OST

complexes differ in selectivity toward the LLO. Stt3-A, which transfers most of

the glycan, has a higher affinity for the complete glycan, and therefore will

preferentially transfer it to the nascent polypeptide. In contrast, Stt3-B, which

exhibits a faster catalytic rate, can transfer incomplete glycans. Furthermore,

Ruiz-Canada and co-workers showed that the post-translational glycosylation of

the human blood coagulation factor VII is dependent upon the presence of Stt3-B

38

(Ruiz-Canada et al. 2009). The higher turnover rate of the Stt3-B isoform would

favor co-translational and post-translational modification of skipped glycosylation

site prior to protein folding.

It appears that the differences observed in the different cell types may reflect

a compromise between full peptide glycosylation and transfer of complete

glycans (Helenius & Aebi 2004). In Hela cells, the Stt3-A: Stt3-B ratio is

estimated to be roughly 60:40 (Ruiz-Canada et al. 2009).

Kinetics experiments done on the human blood coagulation factor VII

showed that some sites can only be glycosylated co-translationally by Stt3-Ap,

while other sites are post-translationally glycosylated by Stt3-Bp, indicating the

presence of a sequential scanning of the nascent polypeptide (Ruiz-Canada et al.

2009).

It was shown in a survey of glycoproteins from the SWISS-PROT database

and the PDB crystallographic database that about 2/3 of the glycosylation sites are

actually occupied by a glycan (Apweiler et al. 1999, Petrescu et al. 2004). Some

sites are ignored, some others are rarely glycosylated, and some are always

glycosylated. This finding highlights the influence of numerous factors in the

glycan transfer, among which the composition of the OST complex, the amino

acid composition and position of the sequon itself and the immediately adjacent

sequence, plus the availability of the glycans are the most important (Ben-Dor et

al. 2004, Helenius & Aebi 2004).

2.4.2 Physiological role of N-linked glycosylation

Work carried out during the last 15 years has produced clear evidence that the

sophisticated process of protein N-linked glycosylation with the addition of Glc3-

Man9-GlcNac2 on the nascent peptide is intimately linked to protein quality

control via its interaction with lectin-like proteins (Helenius 1994, Spiro 2000a,

Parodi 2000, Cabral et al. 2001, Lehrman 2001, Caramelo et al. 2004, Caramelo

& Parodi 2008). In all eukaryotic cells, malfolded and incompletely assembled

multisubunit proteins are destined to undergo ER associated protein degradation

(ERAD) (Klausner & Sitia 1990, Kopito 1997, Jarosch et al. 2003). Early quality

control during protein maturation is a major protective mechanism employed by

the cells to prevent improperly folded proteins or incomplete protein complexes

from arriving at their final destination where they may cause problems. Quality

control is achieved by modifying the glycan on the polypeptide in the ER, pre- or

cis-Golgi, by an α-glucosidase and an α-mannosidase, thus conferring to the

39

peptide a retention or a recycling signal in this calnexin cycle (Helenius 1994,

Burda & Aebi 1999, Herscovics 1999a, Parodi 2000, Spiro 2000a, Cabral et al.

2001, Lehrman 2001, Caramelo & Parodi 2008). The three glucose moieties

added on the A branch of the glycan play a crucial role (See fig. 7.) (Spiro 2000a).

A large body of evidence strongly suggests that an excision of the

oligosaccharide occurs before peptide degradation in the proteasome

(Karaivanova & Spiro 2000, Hirsch et al. 2003, Suzuki 2007). The

oligosaccharides released have been proposed to be early by-products of ERAD.

In S. cerevisiae, the vacuole α-mannosidase, Ams1p, plays a key role in the

degradation of free glycan (Chantret et al. 2003). In mammals, the degradation of

free glycans was reported to be a more complex mechanism (Moore 1999, Suzuki

& Funakoshi 2006).

The majority of the ER resident protein proteolysis is achieved through an

ubiquitin-proteasome system, following their retrotranslocation through the ER

membrane to the cytosol using the Sec61 protein-complex channel (Kopito 1997,

Pilon et al. 1997, Plemper & Wolf 1999, Jarosch et al. 2003). The implication of

the Sec61p arose from several studies which provided direct evidence of its

interaction with ERAD protein members. For instance, subunits of the Sec61

protein complex were found to bind various ERAD substrates en route to

degradation; mutations in SEC61 slow down the degradation of misfolded

proteins; and finaly Kalies and co-workers highlighted interactions between the

proteasome subunits and Sec61p (Pilon et al. 1997, Plemper et al. 1997, Kalies et

al. 2005). It is also noteworthy that, the required E3 ubiquitin ligases Doa10p and

Hrd1 in yeast, which are multi-spanning membrane proteins, could form or are

integral components of the retrotranslocation channel (Ravid et al. 2006, Kostova

et al. 2007). Recently, derlin-1p was proposed as a possible retrotranslocation

channel in mammals, as it co-precipitates with the receptor for non-glycosylated

BIP substrate (Schulze et al. 2005). It has also been postulated that large proteins

which are not completely unfolded could exit the ER through the formation of

lipid droplets (Ploegh 2007). The glycans attached to the peptide will be removed from the ERAD

candidates by the action of the cytosolic Png1p (Hirsch et al. 2003, Suzuki 2007).

The removal of glycan eases the peptide degradation by the proteasome, but the

presence of the glycan does not inhibits the proteasome action (Misaghi et al.

2004).

40

It has been reported, however, that some degradation can also occur without

the participation of proteasomes via the action of ER lumen resident proteases.

These proteases have been proposed to cut the loops between the transmembrane

domains of a membrane protein to facilitate its extraction before its degradation

(Heinemann & Ozols 1998, Urade et al. 1993, Loo & Clarke 1998, Moriyama et

al. 1998, Spiro & Spiro 2001).

Most ERAD substrates undergo ubiquitination before being degraded by the

proteasome. A prerequisite for the proteasome-dependent-degradation is the

action of three different enzymes: an ubiquitin-activating enzyme E1, an

ubiquitin-conjugating enzyme E2, and an ubiquitin ligase E3. In addition, an

ubiquitin-chain elongation E4 may be required in some cases to facilitate ERAD,

as the chain length may be a crucial criterion before the substrate is degraded

(Jarosch et al. 2002, Richly et al. 2005, Kohlmann et al. 2008, Nakatsukasa et al.

2008).

Role of protein N-glycosylation

It is important to understand the modifications that the Glc3-Man9-GlcNac2

undergo to evaluate the role that its intermediates play in protein maturation,

addressing and degradation.

The glucose present on the A branch (fig. 10.) of the glycan is removed by

glucosidase I, which is an integral membrane protein with its active site facing the

ER lumen. It rapidly removes the glucose 3 on the A branch, which is essential for

the OSTs for effective transfer of the complete oligosaccharide to the nascent

peptide. The trimming by the glucosidase I is co-translational (Chen et al. 1995).

The rate at which the glucose on the A branch is removed, along with the

inhability for the glucosidase I to remove the glucose from dolichol-PP-linked

glycan in vivo (but not in vitro) suggests the existence of a supercomplex with a

very precise orientation of all the components (Caramelo & Parodi 2008).

Then the two remaining glucose residues present on the same branch are

removed by the luminal glucosidase II (fig. 9.), which is a dimeric protein from

yeast to mammals (Spiro 2000b). The α-subunit harbors the catalytic activity

without an ER retention signal; the β-subunit displays a conserved ER retention

signal at its C-terminus from S. pombe to mammals (Wilkinson et al. 2006). The

protein dimer exhibits a higher activity towards glycans with intact mannose

branches than to glycans with a trimmed A branch (Grinna & Robbins 1980).

Recent studies demonstrated that the hydrolysis of Glc2-Man9-GlcNac2 by

41

glucosidase II was severely inhibited by the presence of Glc0-Man7-GlcNac2

glycan (Totani et al. 2006, Bosis et al. 2008). Bosis and co-workers also showed

that the glucosidase II can act on the M8B and M7 glycans which ensures that the

folding of the glycoproteins in the calnexin/calreticulin cycle would not be

delayed as a result of the trimming of the mannose residues (Bosis et al. 2008).

Thus, the role of glucosidase II is to enable the initial binding of calnexin/

calreticulin to the glycoproteins and later to stop the interaction. In the meantime,

calnexin and calreticulin can cycle on and off the glycoproteins freely several

times.

It is expected that correctly folded glycoproteins are quickly secreted. Thus,

the ER lumen should mainly harbor Glc1-Man9-GlcNac2 - Glc0-Man9-GlcNac2 N-

glycans attached to glycoproteins. In cells under folding-stress however, the ER

would exhibit glycans with trimmed mannose structure, belonging to misfolded

glycoproteins that are retained in the ER. A relatively low concentration of Glc0-

Man7-GlcNac2 glycan can severely slow down the hydrolysis of Glc2-Man9-

GlcNac2 to Glc1-Man9-GlcNac2, which causes a delay in the association of the

glycan with calnexin/calreticulin. Given the inhibitory nature of the

nonglucosylated glycans, these end product glycans may play a role in regulating

the entry of newly synthesized glycoproteins into the calnexin/calreticulin cycle

under folding stress conditions (Caramelo & Parodi 2008, Bosis et al. 2008).

Alternatively, endomannosidase can remove the last glucose and the last

mannose on the A branch (fig. 10.), ensuring that the biologically important

complex oligosaccharides of higher organisms can be formed (Moore & Spiro

1990).

In addition to glucose trimming, mannose removal via mannosidase I and II

also occurs in the ER (Weng & Spiro 1993, Weng & Spiro 1996, Jakob et al. 2001,

Clerc et al. 2009). Mannosidase I removes the sugar present at the top of the B

branch, while mannosidase II removes the last sugar of the C branch (fig. 10.).

42

Fig. 10. Representation of the ER early trimming sites on the Glc3-Man9-GlcNac2. The

enzymes and their cutting sites are indicated, specific inhibitors of the enzymes are

indicated in brackets. CST: castanospermine GDMJ: glucose-α1,3-

deoxymannojinrimycin; DMJ: 1-deoxymannojinrimycin; KIF: kifunensine.

Lectin chaperones and glucose on the A branch

The chaperones calnexin and calreticulin have been found in a large variety of

eukaryotic cells. Even in S. cerevisiae, a single gene has been proposed to express

an isoform of these two chaperones with an identity of 24% to the canine calnexin

and 21% to the mouse calreticulin (Parlati et al. 1995).

In higher eukaryotes, calnexin is an ER integral membrane protein, while

calreticulin is located in the ER lumen, but it can be found also in intermediate

compartments (IC) and Golgi. Both chaperones are believed to interact with

distinct glycoproteins (Spiro et al. 1996, Helenius et al. 1997, Zuber et al. 2000).

The presence of two distinct chaperones would facilitate the recognition of the

glycans on the glycoprotein: oligosaccharides in the vicinity of the ER membrane

43

would be bound to the membrane-integrated calnexin, while glycans more

lumenally orientated would bind the soluble calreticulin (Spiro 2004). Calcium

depletion or heat shock can trigger conformational changes of both calreticulin

and calnexin that induce their oligomerization and increase their binding affinity

to non-glycosylated substrates (Rizvi et al. 2004, Thammavongsa et al. 2005).

Studies have shown that the lectins calnexin and calreticulin specifically bind

N-linked monoglucosylated oligosaccharides, while no interactions were found

with intact or glucosidase I-treated oligosaccharides. On the other hand, the action

of mannosidases on branch B and C did not affect the binding of the chaperones

(Ware et al. 1995, Spiro et al. 1996, Helenius et al. 1997).

The interaction between the glycoprotein and the complex formed by

calnexin/calreticulin and ERp57 starts with the binding to calnexin or calreticulin,

and then the glycoprotein interacts with ERp57, which is a thiol oxidoreductase of

the protein disulfide isomerase (PDI) family. ERp57 forms transient disulfide

bonds with the bound glycoprotein (Huppa & Ploegh 1998, Oliver et al. 1999,

Molinari & Helenius 1999).

In higher eukaryotes, there is a UDP-Glc:glycoprotein glucosyltranferase

which can add back one glucose residue to de-glucosylated N-glycans after it has

been transferred to the peptide and already trimmed by the glucosidases

(Trombetta & Parodi 1992). As a result, the retention time of the folding protein

in the ER is increased, thus allowing more opportunities for the peptide to fold

properly. On the other hand, prevention of the glucose trimming on the A branch

results in a faster degradation of the polypeptide (Moore & Spiro 1993, Kearse et

al. 1994, Keller et al. 1998), demonstrating the importance of the

deglucosylation-glucosylation cycle.

In yeast, no UDP-Glc:glycoprotein glucosyltransferase enzyme has been

found (Fernández et al. 1994, Jakob et al. 1998b), suggesting that the

deglucosylation-glucosylation cycle may not occur. Jakob and co-workers (Jakob

et al. 1998b) have suggested that glucosidase II may be able to discriminate

between folded and unfolded proteins, thus acting as a chaperone.

Protein degradation and Man8-GlcNac2 specific lectin

In mammals, several studies have provided evidence that malfolded proteins or

unassembled complexes move between the ER and the Golgi before entering the

ERAD pathway (Hammond & Helenius 1994, Caldwell et al. 2001, Vashist et al.

2001, Arvan et al. 2002, Haynes et al. 2002, Taxis et al. 2002, Elkabetz et al.

44

2003). The protein may then expose the improper glycopeptide to a new array of

glucosidases which can trim the glycans even further than in the ER (Hosokawa

et al. 2003, Kitzmüller et al. 2003).

Both in higher eukaryotes and yeast, the degradation of improperly folded

proteins requires mannosidase I activity which has been shown to act slowly in

vivo. This reaction removes the last mannose on the B branch (see fig. 10.) (Jakob

et al. 1998a, Cabral et al. 2001). Since the overexpression of this enzyme

accelerates ERAD (Hosokawa et al. 2003), it has been proposed that mannosidase

I acts as a “timer” of the ER protein residence (Cabral et al. 2001).

Special proteins, ER degradation enhancing α-mannosidase-like potein

(EDEMs) in mouse (Hosokawa et al. 2001), and Mnl1p/Htm1p in S. cerevisiae

(Jakob et al. 2001, Nakatsukasa et al. 2001), have been found to specifically

recognize the particular structure of the Man8B glycan whether the peptide is

properly folded or not (Spiro 2004). The mouse EDEMs have been reported to

interact with calnexin via its transmembrane domain, but not with the soluble

calreticulin (Oda et al. 2003, Molinari et al. 2003). These glycosidases that

generate the signal for ERAD are ER-resident members of the glycosyl hydrolase

47 family of α1,2-mannosidases (Ruddock & Molinari 2006).

The mouse EDEMs and the yeast Mnl1p/Htm1p proteins are homologues of

mannosidase I, and exhibit mannosidase activity (Hirao et al. 2006, Olivari et al.

2006, Clerc et al. 2009). Overexpression of the mouse proteins results in an

acceleration of malfolded peptide degradation (Oda et al. 2003, Molinari et al.

2003, Hirao et al. 2006, Olivari et al. 2006). Overexpression of the yeast Htm1p

however, does not increase the degradation rate, suggesting that the Htm1p

activity is not rate limiting in the degradation pathway (Clerc et al. 2009). In yeast,

absence of Mnl1p/Htm1p leads to the preservation of improperly folded proteins

(Jakob et al. 2001), and its specific activity was recently characterized by Clerc

and co-workers who demonstrated a mannosidase acitivity on the C-branch of the

glycan, resulting in a particular conformation which would serve as a signal of the

presence of misfolded protein (Clerc et al. 2009). The Htm1p mannosidase

activity is dependent upon the removal of the last mannose on the B branch by

MnsI.

Based on the analysis of the degradation of artificially created ERAD substrates

in yeast, Vashist and Ng proposed that the ERAD pathway is divided into two

distinct quality control systems (Vashist & Ng 2004). According to this model, the

site of the lesion is the key element for the selection of the degradation route. The

45

ERAD-Lumenal (ERAD-L) is responsible for controlling the folding quality of

ER-lumenal domains. Degradation of an ERAD-L substrate requires ER-to-Golgi

transport, Der1p, and the E3 ubiquitin ligase Hrd1p. More recent data indicate

that the complex formed around Hrd1p links the early and late event in ERAD

(that is recognition and retrotranslocation events, respectively) (Carvalho et al.

2006). In contrast, the ERAD-cytosolic (ERAD-C) monitors the folding quality of

the cytosolic domains of the proteins. Degradation of proteins with a cytosolic

misfolded domain will follow the ERAD-C pathway irrespective of the presence

of another misfolded domain which would trigger an ERAD-L pathway

degradation. ERAD-C protein degradation pathway only requires Doa10p, which

is another ER-associated E3 ubiquitin ligase (fig. 11, see fig. 12. for a summary of

the ERAD pathway)

Fig. 11. The two distinct pathway of the yeast ERAD. (See text).

There is a growing body of evidence that the proteasome is not the only place for

protein degradation and that degradation also occurs in the ER (Heinemann &

Ozols 1998, Urade et al. 1993, Loo & Clarke 1998, Moriyama et al. 1998, Spiro

46

& Spiro 2001, Cabral et al. 2000, Fayadat et al. 2000, Mancini et al. 2003). In

this particular case, deglycosylation of the peptide must occur in the ER lumen

(Kitzmüller et al. 2003, Kamhi-Nesher et al. 2001).

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47

48

2.4.3 Glycosylation defect and congenital disorders

The N-linked glycosylation pathway is highly conserved throughout evolution. In

recent years, several glycosylation defects have been characterized in humans.

They have all been grouped into the congenital disorders of glycosylation (CDG)

family. The main symptoms are skeletal and facial dysmorphism, and retardation

in psychomotoric development (fig. 13.). The CDGs are sorted into two different

classes according to the nature of the defect:

– CDG type I: failure of the assembly of the oligosaccharide substrate

– CDG type II: failure of the proper processing of the oligosaccharide after

transfer to the protein in the ER

Fig. 13. Correlation between the amount of N-glycans on proteins and clinical

symptoms in CDGs. The triangle symbolizes the quantity of normal protein

N-glycosylation. Some of the symptoms are listed on the top (Modified from Aebi et al.

2001).

2.5 Yeast as a model organism to study essential genes

The baker’s yeast S. cerevisiae is a unicellular eukaryote that humans have been

using for food processing for millennia. Many basic cellular functions are well

conserved between human and yeast, and its short generation time, economy of

maintenance and the ability to grow in a diploid or haploid form (thus facilitating

the study of recessive mutations) have made it a favorite study object and model

49

organism for geneticists, molecular biologists and biochemists. Yeast is an

organism which is relatively easy to manipulate: it was the first eukaryote to be

successfully transformed with a plasmid (Beggs 1978), and also for which precise

gene knock-outs were generated (Rothstein 1983). In 1996, S. cerevisiae was the

first eukaryote of which the complete genome sequence was available (Goffeau et

al. 1996). As outlined in the previous pages, the VLCFAs and the N-glycosylation

synthesis pathways are also highly conserved between yeast and mammals.

Several well-characterized systems for induction or repression of gene

expression exist in yeast. One of the best known examples is the galactose

induction – glucose repression regulatory system (Gancedo 1992). Genes under

the control of the Gal-inducible promoter are repressed in the presence of glucose,

de-repressed in the presence of other carbon sources, and induced up to 1000-fold

in the presence of galactose. This system can be exploited for the study of

essential genes. By expression of the gene in question from a Gal-regulated

promoter on an expression vector, the level of the protein can be manipulated.

The effect of overexpression or depletion of the protein on the cell and

biochemical activity can therefore be observed.

50

51

3 Aim of the study

VLCFAs are important molecules in cells, and increasing our understanding of

their synthesis was the original aim of the study. Using biochemical and yeast

genetic approaches as well as bioinformatics we aimed to identify the enzymes

that are members of the yeast VLCFA elongation pathway. The expertise gathered

during this work allowed us to identify and characterize several of the cotton

counterparts involved in plant VLCFA elongation. We focused on candidates for

the condensing enzymes and ketoacyl-CoA reductase. VLCFA elongation in

cotton is important for plant fiber development, and knowledge of the regulation

of cotton fiber length may have a significant industrial and economical impact.

A second aim was to identify the enzyme responsible for the dehydratation of

the hydroxyacyl-CoAs. A chimeric protein was generated to serve as a

microsomal dehydratase analogue and this construct was used in a genetic screen

in yeast to isolate mutants and ultimately to clone and characterize the gene

encoding the yeast hydroxyacyl-CoA dehydratase. The screen yielded mutants

that did not affect fatty acid synthesis, but for which viability nevertheless was

dependent on the presence of the chimeric protein. This artificial protein exerted

unexpected effects on the glycosylation pathway in the ER and more precisely on

the flipping of lipid-linked oligosaccharides across the ER membrane.

The project was then redirected toward understanding the flipping mechanism,

and how the chimeric protein could suppress the native flippase deletion

phenotype in yeast. Because the human Rft1p homologue had not been identified,

we extended the work to include the characterization of the wild type and disease-

related variant of the human protein.

52

53

4 Material and Methods

4.1 Yeast strains, culture media and plasmid construction

Detailed information on the yeast strains, culture media and methods used to

engineer the different constructs are given in the original articles.

4.2 Hydratase2 enzymatic assay

Hydratase2p (H2) activity was measured from the extracts containing the yeast

microsomal fraction as the extent of complex formation of NADH (at 340–085nm) or

3-ketoacyl-CoA with Mg2+ (at 303nm) using trans-2-decenoyl-CoA or trans-2-

hexadecenoyl-CoA as substrates and a recombinant yeast (3R)-hydroxyacyl-CoA

dehydrogenase as an auxiliary enzyme (Hiltunen et al. 1989, Hiltunen et al. 1992),

in buffer containing: 50 mM Tris-HCl pH 8, 50 mM KCl, with 50 μg/ml BSA.

4.3 Random mutagenesis

4.3.1 Chemical mutagenesis

The colony-sectoring screen has been described (Bender & Pringle 1991). Cells

were subjected to ethane methyl sulfonate (EMS) mutagenesis, plated on Petri

dishes containing YPGalD medium and incubated at 30°C for 4–4 days. The

killing rate ranged between 25 and 95%. After incubation, non-sectoring red

colonies were picked and streaked on YPGalD media plates for single colonies to

confirm the phenotype. To test dependency on the pTSV30 ELO3’H2 plasmid,

pYES2 ELO3’H2 was shuffled in and sectoring colonies were picked up for

further testing. The dependency of mutated yeast cells on the chimeric Elo3’H2p

was assessed by streaking on YP plates containing 4% glucose and testing the

inability of the candidates to grow on this medium. Mutants that passed these

tests were further analyzed.

54

4.3.2 Transposon mutagenesis

Transpososome assembly

The kan-MX-Mu Transposon (details will be published elsewhere: (Paatero et al.

2008)) was isolated and purified by anion exchange chromatography as described

(Haapa et al. 1999). The in vitro transpososome assembly was performed as

described previously (Lamberg et al. 2002). The assembly reaction contained the

transposon DNA fragment, MuAp, Tris-HCl pH 6.0, glycerol, Triton X-100, NaCl

and EDTA. The reaction was carried out at 30 °C for 5 h. The complex was

concentrated and desalted as described previously (Pajunen et al. 2005). The

assembly and concentration of transpososomes was monitored by

agarose/BSA/heparin gels as described previously (Lamberg et al. 2002).

Yeast electroporation

Yeast cells were grown to stationary phase at 30 °C, diluted with fresh medium,

and grown to an OD600 of 1.1. Cells were collected by centrifugation, resuspended

in LiAc/DTT/TE (0.1 M lithium acetate, 10 mM dithiotreitol, 10 mM Tris-HCl

pH 7.5, 1 mM EDTA), and incubated for 1h at room temperature. After

centrifugation, the cells were washed twice with ice-cold 1 M sorbitol. After re-

collection by centrifugation, the pellet was suspended in ice-cold 1 M sorbitol to

yield ~200-fold concentration of the original culture density. Electroporation was

done using 100 µl of fresh cell suspension. An aliquot (1.3 μl) of transpososome

preparation (containing 1 μg transposon DNA) or 1 μl of plasmid pHTH36 DNA

(20 ng) was added to the cell suspension. The plasmid served as a control for the

overall electroporation efficiency, effectively monitoring the competence status of

the cells. Following incubation on ice, the mixture was transferred into a chilled

Bio-Rad cuvette (0.2 cm electrode spacing), and electroporation was carried out

using a Genepulser II (Bio-Rad, CA, USA). Following electroporation, YPD

medium was added, and the cells were incubated at 30 °C. The cells were plated

on YPGalGlu plates containing geneticin for the selection of the transposon

marker gene.

In order to identify the gene that was inactivated via the transposon insertion,

the yeast mutant genomic DNA was extracted and digested using the restriction

enzymes BamHI and BglII (which do not cut in the transposon cassette). The

digested genomic DNA was then ligated into the YCplac111 vector and

55

transformed into highly electrocompetent E.coli (at least 109 transformants per

microgram of plasmid DNA).

4.4 Yeast genomic DNA library transformation

For the cloning of the mutations by complementation, three different libraries

were used in our attempt to identify the mutated gene.

– Yep13 library, containing DNA fragments from 5 to 20kb (ATCC #37323)

– YCp50 library, containing DNA fragments from 10 to 20kb (ATCC #37415)

– pRS426 library, containing DNA fragments up to 5kb (Kastaniotis et al.

2004).

High efficiency transformation of yeast was carried out following the method of

Gietz and Woods (Gietz & Woods 2006). Transformants were selected on

synthetic complete media containing glucose at a concentration of 4% and lacking

uracil (YCp50, pRS426 libraries) or leucine (YEp13 library).

4.5 Western blot

W1536 5B rft1Δ/pTSV30a ELO3H2 and W1536 5B were grown in YPGalD, then

re-inoculated into YPGalD or YPD containing 4% glucose, and grown over night.

The cells were harvested, washed in Extraction buffer (20mM Hepes pH 8.0,

10 mM MgCl2, 1 mM EDTA, 10% glycerol), resuspended in T0 buffer (20 mM

Hepes pH 8.0, 5 mM EDTA, 20% glycerol) and broken as described previously

(Carrico & Zitomer 1998). The protein concentrations of the cell extracts were

normalized and equal amounts subjected to SDS – PAGE (12%). Western analysis

was carried out using mouse IgG anti – CPY primary antibody (Invitrogen, CA,

USA) and goat-anti mouse IgG – HRP secondary antibody (Zymed, CA, USA).

CPY bands were visualized using the ECL chemiluminescence kit (Amersham

Biosciences, UK).

4.6 Pulse chase and LLO preparation

Cells were grown to a density of 0.5–4 ×107.ml−1. The cells were suspended in 200 ml

YPD 0.1% containing 250 μCi D-[2-3H]-mannose (555 GBq/mmol, Amersham

Biosciences) and incubated for 10 min. The extraction of lipid-linked

oligosaccharides was performed according to Lehle (Lehle 1980). Labeling was

56

stopped by the addition of 5 ml chloroform/ methanol 3:2 (CM) under continuous

vortexing. The solution was then centrifuged and the supernatant was discarded.

Acid-washed glass beads (425–500 µm) were added to the cell pellet, and after

vortexing the pellet was washed twice with CM. The cell pellet was then washed with

UP(+) [chloroform:methanol:water (CMW) containing MgC12] and once with UP(−)

(CMW, 3:48:47, v/v/v). Lipid-linked oligosaccharides were then extracted with CMW

(10:10:3, v/v/v), the extraction solutions were combined and dried under nitrogen at

37 °C. For hydrolysis, the dried lipid-linked oligosaccharides were suspended in iso-

propanol, followed by the addition of 20 mM HCl and incubated at 100 °C for 45 min.

To remove the lipids, the hydrolysate was extracted with CM, dried under nitrogen,

dissolved in H2O and filtered. The oligosaccharides were stored at −20 °C until

analysis by HPLC (Millipore ultrafree-MC 0.45 µm). The eluate from the column was

mixed continuously with scintillation fluid (FLO-Scint A, Packard, Meriden, CT,

USA) at a ratio of 1:1.5 (eluate:scintillation mix, v/v). Radioactivity was monitored

using a flow monitor (FLO-ONE A-525, Packard) equipped with a 0.5 ml flow cell.

The counting efficiency for 3H in the flow cell was 22% (Zufferey et al. 1995).

4.7 Semi-quantitative reverse transcription (RT)-PCR analysis

Total RNA was extracted from wild-type cotton roots, leaves, stems, and ovules,

together with fibers at –3, 0, +3, +5, +10, +15, +20 dpa (day post anthesis) or

from −3, 0, +10 dpa fuzzless-lintless (fl) mutant cotton ovules. Cotton cDNA was

reverse-transcribed from 5 μg total RNA. The cotton ubiquitin gene UBQ7

(accession no. AY189972) was used as an internal control in each reaction. To

analyze heterologous expression of GhCER6 in yeast mutant cells, RT-PCR was

performed with the same primer pairs. The yeast actin gene, ACT1 (accession

number NP_116614) was used as an internal control in parallel reactions.

4.8 Real time quantitative (QRT)-PCR analysis of GhCER6 expression in different cotton tissues

QRT-PCR was carried out using the SYBR green PCR kit (Applied Biosystems, CA,

USA) in a DNA Engine Opticon–Continuous Fluorescence Detection System (MJ

Research, MA, USA). All QRT-PCRs were performed with the same parameters

described previously (Qin et al. 2005). Samples were analyzed in triplicate using

independent RNA samples and were quantified by the comparative cycle threshold

method (Wittwer et al. 1997).

57

4.9 RNA in situ hybridization

Cotton ovules were collected from 2 dpa cotton flowers and fixed in formalin–acetic

acid fixing solution. The fixed ovules were processed according to the protocol

previously described (Ruan & Chourey 1998). The samples were infiltrated with

paraffin. Tissue sections, 10 µm thick, were cut and placed on dampened slides

precharged with polylysine. Digoxigenin–11 UTP was incorporated into antisense or

sense RNA probes according to the manufacturer’s instructions (Roche, Switzerland).

Hybridization and detection were carried out using the method described previously

(Ji et al. 2002).

4.10 Preparation of ER extracts from yeast cells

Yeast cells were grown to exponential phase (2–2 × 106 cells.ml−1) and then harvested

by centrifuging for 15 min at 1,300 g, 4 °C. The pellet was washed with sterile H2O,

and suspended in ice cold lysis buffer. The cells were disrupted with glass beads and

centrifuged for 15 min at 15,000 g at 4 °C to remove the cell debris. The supernatants

were centrifuged for 90 min at 85,000 g in a Sorval Ti70 rotor at 4 °C to generate

pellet (P85) and supernatant (S85) fractions. The P85 was suspended in lysis buffer

and used for the elongase assay. The protein concentrations were determined by the

method of Lowry and coworkers (Lowry et al. 1951), using bovine serum albumin as

the standard. Equivalent amounts of total lysate, P85 and S85 were precipitated by

adding trichloroacetic acid (TCA) to 10% final concentration and processed for

immunoblotting.

4.11 Elongase assays of cotton 3-ketoacyl-CoA reductases

overexpressed in yeast cells

Palmitoyl-CoA was used as primer in all fatty acid chain elongation assays. The

elongation assays were divided into two groups. One was supplemented with both

NADPH and NADH (Han et al. 2002, Gable et al. 2000), and another was

supplemented only with NADH. To initiate the elongation reaction, 0.05 mg ER

protein extracted from yeast cells was added. The reaction mixture was incubated at

30 °C for the indicated time. The reactions were stopped and saponified by adding

KOH-methanol, and then acidified by adding HCl-ethanol. Fatty acids were collected

using hexane. The extractions were dried under nitrogen, and separated by thin-layer

chromatography (TLC) using hexane/diethyl ether/acetic acid. The TLC plates were

58

exposed to a PhosphorImager screen, the resulting image was analyzed, and the lipids

were quantified using a Bio- Imaging Analyzer with 2D Image master software

(Amersham Biosciences).

4.12 Lentiviral mediated hRFT1 expression

HEK293T cells were transfected with pLenti6-hRFT1 and the packing plasmid

mix (Invitrogen) using calcium-phosphate precipitation. Eight hours after the

transfection, the medium was replaced with fresh DMEM containing 10% FCS.

The cell supernatant was collected and lentiviruses were harvested by

centrifugation and filtration (Schleicher & Schuell, Germany). CDG and healthy

control fibroblasts were infected with recombinant lentiviral particles including

the human RFT1 cDNA or the EGFP gene as control. Infected cells were selected

on blasticidin (Invitrogen) for 10 days.

4.13 Human patient mutation analysis

Total RNA and genomic DNA were isolated from fibroblasts and blood samples,

respectively, using the TRIzol LS reagent (Invitrogen) according to the

manufacturer’s instructions. The human RFT1 cDNA was prepared from RNA

using primer and Omniscript reverse transcriptase (Qiagen, CA, USA). The

reaction mixtures were incubated at 37 °C for 1h. The protein-coding region of

the human RFT1 cDNA was amplified by PCR. Exon 3 of the human RFT1 gene

was amplified by PCR from 50 ng of genomic DNA. The PCR products were

sequenced (Synergene Biotech, Switzerland) after removal of the unincorporated

nucleotides with QIAquick columns (Qiagen, CA, USA).

59

5 Results

5.1 Identification of cotton orthologs to yeast enzymes involved in

the VLCFA elongation pathway in cotton

5.1.1 Identification of cotton 3-ketoacyl-CoA reductases

VLCFAs are abundant in plants and serve as a major building block for the

elaboration of waxes (Kunst & Samuels 2003). Plants synthesize fatty acids

within plastids using a type II synthesis system consisting of distinct enzymes (Lu

et al. 2004). The synthesized FAs are exported to the ER where elongation to

VLCFAs occurs (Ohlrogge & Browse 1995). Qin and co-workers have described

an up-regulation of the known genes encoding the enzymes involved in the

VLCFA synthesis pathway during cotton fiber development (Qin et al. 2005).

Some orthologs of the yeast S.cerevisiae Ybr159p were found in Arabidopsis

thaliana and Brassica napus (Beaudoin et al. 2002, Puyaubert et al. 2005).The

two 3-ketoacyl-CoA reductases in cotton were identified via hybridization using

the AtYBR159w coding sequence as a probe at low stringency (Beaudoin et al.

2002). Colonies that hybridized to the probe from both ends were sequenced and

putative full-length GhKCR1 and GhKCR2 cDNAs were obtained. The relative

level of expression was found to be higher in the cotton ovules and their fibers 5

days after flowering, while the expression level remained constant in the roots,

stems, and leaves. When the cotton ORFs were expressed in the yeast ybr159wΔ

mutant, the activity of the microsomal elongation machinery was similar to the

wild type level in an in vitro assay and displayed NADPH-dependence.

Furthermore, sub-cellular localization indicated that the cotton proteins were

localized in the ER membrane like Ybr159p (Qin et al. 2005).

5.1.2 Identification of cotton tissue specific 3-ketoacyl-CoA synthase

GhCER6 was reported to be upregulated more than seven-fold at 10 dpa during

cotton fiber development. The GhCER6 mRNA was mainly detected during

cotton fiber elongation in the differentiated cells, but not in the fuzzless-lintless

mutant, highlighting the importance of VLCFAs during cotton fiber elongation.

The cotton GhCER6p exhibits a high degree of similarity with the A. thaliana

AtCER6p (86%) and a lower similarity with AtKCS1p (58%), AtFDH1p (54%),

60

and AtFAEp (52%). No sequence similarity can be detected with the yeast VLCFA

elongation condensing enzymes Elo2p and Elo3p.

When the galactose-induced GhCER6 gene was introduced into the yeast

elo3Δ mutant, growth was restored to the wild-type level. Furthermore,

expression of the cotton GhCER6 in the non-viable yeast double knock out

elo2Δelo3Δ (Oh et al. 1997) was sufficient to restore viability.

Fatty acid elongation profile analysis in the yeast W1536 elo2Δelo3Δ/pYTV

GhCER6 indicated lipids ranging from C20 to C26. This profile was similar to the

wild-type cells, indicating complementation of the elo2Δ/elo3Δ mutant defect by

the cotton condensing enzyme (Qin et al. 2007b).

5.2 A genetic screen designed to identify the 3-hydroxyacyl-CoA

dehydratase located in the yeast ER

One aim of this project was the identification and characterization of the yeast

VLCFA elongation dehydratase, which was unknown at the time the project was

initiated (Han et al. 2002). Very recent data from another laboratory demonstrated

that this protein was encoded by PHS1 (Denic & Weissman 2007).

Our strategy to identify the missing 3-hydroxyacyl-CoA dehydratase was to

introduce a plasmid borne redundancy in the dehydratase function and via random

mutagenesis ultimately to isolate mutants and clone the gene by mutant library

complementation. Hence, we constructed a chimeric dehydratase carrying an ER

anchoring sequence. We chose the 2-enoyl-CoA hydratase2 domain of Candida

tropicalis Mfe2p to provide the chimera with an enzymatically active domain.

MFE2p is involved in the peroxisomal fatty acid β-oxidation and this hydratase2

catalyzes both hydration and dehydration reactions, as this reaction is reversible.

Even though it prefers the short and medium chain CoA- esters of fatty acids, it

also accepts the longer substrates (Koski et al. 2003).

In order to anchor the catalytic domain to the ER membrane where elongation

takes place, we fused the first two predicted transmembrane domains of the

elongase Elo3p (Elo3’) to the N-terminus side of the H2. This Elo3’ fragment

does not include the conserved domain that has been proposed to contain the

active site of Elo3p. The expression of the chimeric construct was under the

control of the GAL1 promoter. As the 3-hydroxyacyl-CoA dehydratase we sought

to identify was predicted to be essential, we used this chimeric construct in a

screen for mutants unable to lose this plasmid (Fig. 14.).

61

Fig. 14. Plasmid map of the pTSV30a ELO3’H2 plasmid used in the genetic screen. On

the plasmid LEU2 is the yeast selective marker gene; ADE3 will allow the yeast to

accumulate a red pigment leading to the red color colony phenotype; pGAL1 serves

as promoter for the chimera; ELO3’ represents the coding region for the two

transmembrane domains used to anchor the Hydratase2p (H2) in the ER membrane.

Before starting the random mutagenesis, it was important to determine whether

the chimera exhibited the predicted enzymatic activity and was targeted to the

desired subcellular location. In order to investigate this point, we isolated the

microsomal fraction of these yeast transformants and compared the H2 activity to

the one present in the supernatant and the corresponding wild-type fraction. The

H2 activity was more pronounced (3.5 times higher) in the ER extracted from

cells expressing the chimera compared to the wild-type. This data indicated that

the chimera was enzymatically active and properly located.

5.2.1 Random mutagenesis and colony-color-sectoring screen for identification of mutants dependent on the chimera

To monitor the dependency of the yeast mutant upon the plasmid carrying the

ELO3’H2 chimeric ORF, a color-colony based approach was used. This method

takes advantage of the red colony color development in the yeast ade2 mutants

due to the accumulation of 5’-phosphoribosylaminoimidazole, a red pigment. A

strain carrying the ade2/ade3 double mutation is white, as the Ade3p acts

upstream of Ade2p. If this double mutant strain is transformed with a plasmid

carrying the wild-type ADE3, the yeast colonies will appear red when the

presence of the plasmid is essential for survival. If the plasmid is not required for

growth, however, the colonies will display a sectoring phenotype. Each sector

represents an irreversible plasmid loss event (Fig. 15.).

62

Fig. 15. Yeast colony-color phenotypes. (A) In the yeast ade2∆/ ade3∆ mutant, the

colonies are white. (B) The yeast colonies have red and white sectors due to the

random failure of transmission of the ADE3-carrying plasmid to the daughter cell. (C)

The colony is entirely red as the plasmid carrying the ADE3 gene cannot be lost

without compromising cell viability.

This feature can be used to screen for mutations in essential genes. Wild-type

cells will be able to lose the plasmid carrying a functionally complementing

construct, but cells with an inactivating mutation or deletion in the essential gene

are forced to maintain a plasmid carrying the wild-type-copy gene or a gene that

can substitute for the wild-type. Conversely, if the mutant carrying the plasmid

with the wild-type gene is transformed with a plasmid carrying a complementing

construct, it will be able to lose the wild-type plasmid and colonies appear

sectored, indicating the function of the new construct or library clone. The first set of random mutagenesis was carried out using EMS, which

introduces point mutations in the yeast genome. About 120,000 colonies were

screened for the inability to lose the plasmid carrying the chimeric construct on

rich media containing galactose as the main carbon source. Among those, close to

500 candidates were picked for further testing, but only 6 mutants passed all the

controls for chimeric construct dependency: i.e. inability to grow on 4% glucose

rich media and a colony color sectoring phenotype when the chimera was

introduced using a different plasmid backbone. They fell into two different

complementation groups, one of which was composed of 5 members.

5.2.2 Transposon random mutagenesis in yeast

The candidates obtained via EMS mutagenesis demonstrated that the approach

yielded specific mutants whose viability depended upon the expression of the

Elo3’H2p chimera. Unfortunately, the identification of the mutated gene using

three different libraries and screening more than 60 million transformants did not

succeed. From the multicopy yeast genomic library we isolated one region

63

centered on REX4 that allowed the cells to lose the Elo3’H2p chimera. When the

different ORFs of that isolated genomic region were cloned on single copy

plasmids under their own promoter, the complementation was lost, indicating that

we were dealing with a multicopy suppressor.

Because the identification of our mutants by library complementation was not

successful, a transposon mutagenesis technique was applied to induce random

mutations in the same original strain. This approach allows the identification of

the mutation without relying on yeast genomic DNA library complementation.

About 30,000 colonies were obtained after transposon mutagenesis, from

which about 1,100 were picked for further tests identical to the ones used for the

EMS mutant screen. From the candidates isolated, only one fulfilled the

requirements of encoding an essential gene for which the product was localized in

the ER: RFT1. Furthermore, this candidate was in the same complementation

group as the main group obtained after the EMS point mutant mutagenesis

(manuscript).

An explanation for the absence of RFT1 in the different libraries could be that

Rft1p expressed under its native promoter is toxic to E. coli, as we found out

subsequently. Unless a point mutation, which does not affect the complementation

in yeast, was present in the promoter region, the ORF could not be cloned.

5.2.3 Investigation of the chimera function

We investigated whether the microsomal fatty acid chain elongation process

would be affected by the chimera protein expression level. Therefore the profile

of elongated fatty acids was compared between the wild-type strain and the

rft1Δ/pTSV30 ELO3’H2 strain in which the chimera was either overexpressed, or

repressed. The results of the fatty acid profile of an in vitro microsomal fatty acid

elongation assay indicated no discernable differences, suggesting the absence of

an effect of the chimera on the microsomal FA elongation pathway.

Depletion of Rft1p in yeast had previously been shown to result in the

accumulation of hypoglycosylated forms of CPY (Helenius et al. 2002), i.e. the

CPY glycosylation level was linked to the expression level of Rft1p.

To investigate whether the enzymes involved in the VLCFA elongation

pathway could have an effect on the glycosylation pathway, expression of TSC13

was repressed in a tsc13Δ background. In addition, the putative effect of an ELO3

repression on CPY glycosylation was studied in an elo2Δ/elo3Δ genomic

64

background. Neither of these experiments indicated any changes in the

glycosylation profile of CPY.

In the view of these findings, the CPY glycosylation level can be correlated

with the Elo3’H2p expression level in the rft1Δ mutant: when the protein was

overexpressed, the CPY glycosylation profile was similar to the wild-type profile,

whereas when the chimera expression was repressed, the CPY glycosylation

profile was similar to the one obtained by Helenius and co-workers in their Rft1p

depletion experiment (Helenius et al. 2002).

These results indicated that our construct was suppressing the rft1Δ mutant

phenotype, and had no effects on the VLCFA elongation pathway.

Several constructs were generated to elucidate the mechanism by which Elo3’H2p

could suppress a deletion of a gene involved in protein glycosylation.

The enzymatic activity of the H2 was eliminated by introducing the point

mutations corresponding to D804A and H813Q in the C. tropicalis Mfe2p. These

point mutations, located in the catalytic site, inactivate the enzyme, but do not

affect the folding of the protein (Qin et al. 2000, Koski et al. 2004). Because the

enzymatically inactive variant of Elo3’H2p also rescued the glycosylation defect,

we investigated the potential role of the H2 folding structure in the rft1∆ strain.

The corresponding ORF was first replaced with a protein known to form

dimers in a similar way, with the N-termini pointing from the same side of the

protein dimer. Thus the H2 coding sequence was exchanged for the GST1

(Kursula et al. 2005) and FilaminC (Pudas et al. 2005) coding regions (Fig. 16.).

Fig. 16. Comparison between different protein dimers. The arrows point to the

N-terminus of each monomer where the Elo3p transmembrane domains have been

attached. (A) Representation of the hydratase2p dimer; (B) GST1p dimer; (C)

hFilaminCp dimer.

65

The suppression of the rft1Δ deletion also occurred in these cases, prompting

additional studies on the role of protein dimerization. For the first construct, we

fused GFPp which is usually monomeric to Elo3’p (Westermann & Neupert

2000). However, GFPp has been reported to form dimers when present at high

concentrations (Yang et al. 1996). To further rule out the role of dimerization of

our chimera in the suppression of the rft1Δ phenotype, mYFPp was used. The

mYFPp had previously been demonstrated to be truly monomeric (Zacharias et al.

2002). The non-monomeric YFPp control was reverse engineered by mutating the

lysine 206 back to alanine.

The results that any of the constructs mentioned above could functionally

substitute for Elo3’H2p and suppress the rft1Δ mutation suggested that the

transmembrane domains of Elo3p were the key element for the suppression, and

not the dimerization of the chimeric constructs. Furthermore, overexpression of

the full length Elo3p did not restore the rft1Δ strain viability, whereas the

construct made by duplication of only the transmembrane domains was sufficient

for rft1Δ mutant suppression (fig. 17.(A) and (B)). These results suggested the

hypothesis that the observed complementation was due to the transmembrane

domains of the chimeric construct mimicking the Rft1p structure (fig. 17.(C),

manuscript).

66

Fig. 17. Schematic representation of the construct with duplicated two first

transmembrane domains. (A) Scheme of the plasmid construct: pGAL1 is the GAL1

promoter; ELO3’ represents the sequence encoding the two first transmembrane

domains of Elo3p; URA3 is the yeast selection marker. (B) Putative topology of the

Elo3’p duplex on the ER membrane. The transmembrane domains are colored

according to their identity. (C) Scheme representing a putative topology of Rft1p

according to the transmembrane prediction program TMHMM. The transmembrane

domains are organized in 2 groups connected by a soluble linker region.

5.2.4 Lipid-linked oligosaccharide profiles

To investigate further the role of the chimera on oligosaccharide synthesis in the

rft1Δ strain, the LLO profile was analyzed in YCN1 rft1Δ / pYES ELO3’H2.

Cells harboring the chimera construct were grown under different conditions in

which the chimeric protein was either overexpressed using 2% galactose as the

carbon source or repressed by growing the cells in the presence of 4% glucose.

The LLOs were then extracted and analyzed.

The LLO profile from the cells grown under conditions where the chimera is

overexpressed was similar to the wild-type LLO profile. In contrast, an

67

accumulation of DolPP-GlcNAc2Man5 was observed when the rft1Δ strain

harboring the pTSV30 ELO3’H2 plasmid was grown under the repressive

conditions. The results demonstrated an accumulation of DolPP-GlcNAc2Man5

dependent on the expression level of the chimera, consistent with the suppression

of the LLO flipping defect in the presence of the artificial construct. Investigation

of the selectivity towards the glycan structure showed that unlike Rft1p,

Elo3’H2p flipping is not dependent on a specific glycan structure (manuscript, fig.

7.).

Furthermore, deletion of the ubiquitin conjugating enzyme Ubc7p (Ravid &

Hochstrasser 2007) in the S. cerevisiae rft1Δ strain resulting in the double

deletion rft1Δ/ubc7Δ, improved the growth to the wild-type level (manuscript).

The glycosylation profile of CPY in this double deletion strain was also enhanced

up to full glycosylation. This improvement was temperature dependent:

glycosylation was almost absent at low temperature (24○C); while at high

temperature (37○C) the growth rescue was almost complete. These results point to

a link between the ERAD pathway and a suppression of the mutant phenotype

(manuscript).

5.2.5 Analyzing human patient mutation and yeast complementation

A patient displaying neurological abnormalities, a significant delay in physical

growth and dysmorphism was diagnosed with a CDG disorder. The asparagine-

linked oligosaccharide profile of the patient indicated that the nascent peptide was

glycosylated with a complete glycan structure, implying that glycan synthesis was

not affected. On the other hand, the LLO profile indicated an abnormal

accumulation of the DolPP-GlcNAc2Man5 entity resembling the rft1Δ yeast

phenotype.

Mutation mapping revealed a recessive missense mutation in hRFT1 (R67C).

The point mutation introduced a new PstI restriction site in the gene. Introduction

of the wild-type hRFT1 in the mutant patient fibroblasts using recombinant

lentiviruses restored the LLO profile to normal.

The wild-type hRFT1 was cloned into a yeast expression plasmid and

transformed into the rft1Δ yeast strain harboring ScRFT1 on a plasmid which

induces a red-pigment accumulation in this particular strain background. The

colonies obtained after the transformation developed white sectors, indicating a

functional complementation of the hRft1p over the ScRft1p. This phenotypic

result was confirmed by the analysis of the CPY glycosylation level by western

68

blotting. The glycosylation profile of CPY in the presence of hRft1p was similar

to the profile when the native ScRft1p was present.

When the mutant variant of hRFT1[R67C] was introduced under the same

conditions as described above for wild-type hRFT1, the colony phenotype was

different: no white sectors could be seen, indicating an absence of functional

complementation by the mutated hRft1p. This result was confirmed by western

blot probing for CPY: an accumulation of underglycosylated species of CPY was

observed (Haeuptle et al. 2008).

69

6 Discussion

6.1 Identification and characterization of genes encoding NADPH-

dependent 3-ketoacyl-CoA reductase from developing cotton fibers

In yeast, the majority of the ketoacyl reductase activity of fatty acid chain

elongation is catalyzed by Ybr159p, since a deletion of this ORF leads to an

accumulation of 3-ketostearate in these cells (Han et al. 2002). Expression of the

cotton 3-ketoacyl-CoA reductase candidates GhKCR1 and GhKCR2 in ybr159wΔ

strain restored the VLCFA profile to the wild-type level (Qin et al. 2005).

Furthermore, in the absence of NADPH, the VLCFA profile obtained from the ER

membranes extracted from the yeast ybr159wΔ strain expressing the GhKCRs was

similar to the VLCFA profile obtained in the uncomplemented ybr159wΔ deletion

strain. These results indicate that the enzymes identified from developing cotton

fibers are NADPH dependent 3-ketoacyl-CoA reductases.

The hypothesis that GhKCR1 and GhKCR2 are two independent reductases is

supported by the following evidence:

– The genes exhibit different expression profiles during various stages of cotton

fiber development and display tissue specific protein expression that correlates

well with the previously published findings on other genes involved in the same

pathway (Ma et al. 1995, Ji et al. 2003, Feng et al. 2004). These results indicate

that the long-chain fatty acids and VLCFAs may play an important role during

the fast elongation period of the cell.

– When heterologously expressed in yeast, both of the GhKCRs restored the FA

profile to a degree similar to S. cerevisiae 3-ketoacyl-CoA reductase.

– Both GhKCRs are found in the microsomal fraction of the cells and improve

the VLCFA profile in the ybr159wΔ strain. GHKCR2 was located in the

microsomal membrane fraction even though it does not have a clear ER

retention sequence.

6.2 Identification of a cotton fiber-specific fatty acid elongation

condensing enzyme

According to Matthes and Böger (Matthes & Böger 2002) saturated VLCFAs

account for 20% of the total fatty acids present in the cucumber seedling plasma

70

membrane. The condensation step controls the amount and the length of the fatty

acids present, and is therefore a key step in the synthesis pathway.

The fatty acid synthesis and elongation pathways are up-regulated during

cotton fiber development. GhKCS6 was induced most strongly in cotton fibers at

10dpa. This result indicates a major role of the fatty acids in cotton fiber

development and is in agreement with the previously published data. The

information previously gathered indicates a clear up-regulation of cotton

condensing enzymes and a number of cotton lipid-transfer protein genes (Ma et al.

1995, Ji et al. 2003, Feng et al. 2004, Shi et al. 2006).

The cotton GhKCS6p does not exhibit sequence similarity with any of the

yeast Elops (Oh et al. 1997), but nevertheless it is able to suppress the yeast elo3Δ

phenotype or the lethality of the elo2Δelo3Δ double deletion. Analysis of the fatty

acid profile indicated that GhKCS6p was able to restore the yeast wild-type

VLCFA profile and to participate in the production of FAs with chain length from

C20 to C26, which have been shown to be essential for the normal growth of the

yeast cell.

Recently, Qin and coworkers (Qin et al. 2007a) demonstrated that the saturated

VLCFAs can induce cotton fiber cell elongation. According to their findings,

saturated C24 FA is the most efficient. They have postulated that VLCFAs could

control cell extension by controlling ethylene biosynthesis.

6.3 Investigation of the flipping mechanism of the lipid linked glycan in the yeast N-linked glycosylation pathway.

The viability of the S. cerevisiae rft1Δ strain depends upon the maintenance of a

plasmid carrying the chimeric protein Elo3’H2p. The chimera, which was

originally created to be used as a tool for the identification of a dehydratase, does

not show any amino acid sequence similarity to Rft1p. Furthermore, although no

concrete structural information is available, it is unlikely that the two proteins

Elo3’H2p and Rft1p share similarities other than the presence of transmembrane

domains.

To explain how the chimera could suppress the rft1Δ strain, several aspects

have to be considered:

– Rft1p does not require any energy to allow the translocation of the LLO

across the ER membrane.

71

– Transmembrane prediction programs show a clear sorting of the Rft1p

transmembrane regions into two distinct groups with a fairly conserved

stretch of amino acids as a linker (fig. 8.).

– The two transmembrane domains used to anchor the hydratase2 in the ER

membrane do not act like the full size Elo3p protein, and do not harbor any

enzymatic activity.

– The hydratase2 can be replaced by a variety of different soluble protein

domains without affecting the rft1Δ suppression phenotype, indicating that

the Elo3p transmembrane domains are the key element.

The exact role of Rft1p in flipping remains unclear; it has been shown to be an

essential protein for translocation of the dolPP-GlcNAc2Man5 across the ER

membrane (Helenius et al. 2002), but an in vitro assay could not confirm such a

proposition (Frank et al. 2008). Our data obtained in suppression studies in yeast

may point to a faulty approach in the study done by Frank and co-workers (Frank

et al. 2008).

Rft1p may act as a passive selective gate in the LLO synthesis pathway,

allowing the glycan DolPP- GlcNAc2Man5 to flip across the membrane by

creating locally the proper conditions. This implies that the protein exhibits a

particular structure that allows it to reject the glycans that are not ready for

translocation, as well as to prevent the glycan facing the ER lumen from retro-

translocating to the cytoplasm.

In an analogous manner, the chimeric protein may generate some local

changes in the membrane that allow the glycan to flip, but in contrast to the case

of the native protein, the flipping is unspecific. It is possible that the incompletely

assembled protein complex or aberrant protein could create a continuum from the

cytoplasmic side of the ER membrane to the luminal side (manuscript fig. 8.).

Ubc7p is important for targeting aberrant proteins or members of incomplete

protein complexes to the ERAD (Ravid & Hochstrasser 2007). The effect of

ubc7Δ on the viability of the rft1Δ strain indicates that the alteration of the

membrane integrity due to the lack of degradation machinery may be a key

element in the phenotypic recovery of the yeast cell. Furthermore, the

improvement of the glycosylation pattern in the yeast ubc7Δ strain was

temperature dependent. In contrast, the strain harboring the wild-type degradation

machinery did not show any significant increase in glycan flipping across the ER

membrane when shifted to a high temperature. The temperature dependent

suppression of the glycan flipping defect may be explained by a synergistic

72

interplay of aberrant membrane proteins and changes in membrane malleability.

At high temperatures, the membrane fluidity would increase along with the

number of proteins and complexes exhibiting maturation defects. The

combination of the temperature effects and the absence of a functional ERAD

would lead to an augmentation of the effect of the aberrant proteins or incomplete

protein complexes on the more fluid ER membrane, and thus facilitate the

unspecific flipping of lipid linked oligosaccharides (manuscript fig. 9.).

73

Fig. 18. Possible mechanism for flipping improvement when ERAD is disrupted (part

1). (A) Under wild-type conditions, a malfolded protein or an incomplete protein

complex is targeted for degradation via ubiquitination as soon as it appears on the

membrane. Under these conditions, the flipping is specific and controlled by Rft1p. (B)

When the degradation machinery is altered, the membrane integrity is compromized

by the accumulation of malfolded proteins and incomplete protein complexes. The

flipping is not exclusively specific under these conditions.

74

Fig. 18. Possible mechanism for flipping improvement when ERAD is disrupted (part

2). (C) In the absence of Rft1p and degradation machinery, the glycan flipping is

altered, due to compromized membrane integrity as a result of the accumulation of

malfolded proteins and/or incomplete protein complexes. The flipping is not specific

under these conditions.

6.4 Implication to the general membrane flipping process

Biomembranes consist of a lipid bilayer in which membrane proteins are

integrated. As phospholipid biosynthesis takes place on the cytosolic side of the

membrane, translocation across the lipid bilayer of these organic compounds is

necessary in order to allow for normal membrane expansion. Furthermore, the

presence of symmetry (ER) or asymmetry (plasma membrane) is important for

the membrane properties.

The rate of self-translocation of the glycerophospholipids (GPL) in the

membrane is too low to be compatible with cell viability, because the

thermodynamic barrier for such an event is quite elevated (Kornberg &

McConnell 1971, McCloskey & Troy 1980b). The presence of such a barrier

implies that proteins must mediate the translocation. But despite several decades

of investigation, a specific ER GPL flippase has yet to be identified and the

molecular basis for its action remains a mystery (Vishwakarma et al. 2005).

75

The LLOs assembled on the cytosolic side of the ER membrane are bigger with a

larger hydrophilic part than the GPLs, and yet they can be translocated across the ER

membrane at a rate sufficient for yeast cell viability (manuscript). The in vitro assay

clearly indicated that the flipping of GPLs is protein mediated (Vishwakarma et al.

2005), but the reconstitution of an artificial membrane with properly incorporated

protein and protein complexes might be the key to the results of the assay. In a wild-

type cell, ERAD controls membrane integrity by targeting for degradation the

malfolded proteins or incomplete protein complexes present in the membrane. ERAD

is absent from the in vitro assay and therefore the membrane properties may be fairly

different from the in vivo conditions. In vitro, this small difference may be sufficient

to allow the flipping of the tested compounds (Vishwakarma et al. 2005, Sanyal et al.

2008).

6.5 Identification and analysis of a human patient mutation in the N-linked glycosylation pathway

The yeast Rft1p and the human RFT1 do not share a high degree of identity, but

alteration of the protein function apparently leads to the same biochemical

phenotype in both organisms: accumulation of GlcNAc2Man5. The heterozygous

presence of the R67C mutant allele in the parental genomes, complementation

studies in human cell culture and the yeast rft1Δ mutant all indicate that this loss

of function mutation is responsible for the disease phenotype.

The human patient homozygous for the recessive mutation in hRFT1 exhibits

symptoms similar to those of the patients suffering from the CDG-Id (Körner et al.

1999) and CDG-Ie (Imbach et al. 2000, Kim et al. 2000), which are caused by

defects in ALG3 or DPM1 (See fig. 6.), respectively. A deficiency in RFT1

function clearly has a different impact on the protein glycosylation profile, since

in rare cases the glycan can be flipped, possibly due to unspecific translocation.

The alg3 mutant accumulates GlcNac2Man5 because the latter cannot be

elongated, while the dpm1 mutant also displays this phenotype because the

material needed for completion of the glycan cannot be flipped across the ER

membrane. Even if all of the above mentioned defects share an accumulation of

GlcNAc2Man5, proteins in the RFT1p-deficient patient receive a complete glycan

on the rare occasions when the flipping of the glycan does occur, in contrast to the

case of a defect in ALG3p or DPM1p, when only incomplete glycans are

transferred. The similarity observed between the clinical symptoms indicates that

76

the limitation in the amount of GlcNAc2Man9Glc3 glycans alone is responsible for

the clinical manifestations (Haeuptle et al. 2008).

77

7 Conclusions

In this thesis study, the genes encoding the cotton ketoacyl reductase have been

identified. The ketoacyl reductase deficient S. cerevisiae cells harboring the

cotton homolog genes have a VLCFA profile which is similar to the wild-type

profile when assayed in vitro. The proteins encoded by the two genes identified

are NADPH-dependent.

The fatty acid synthesis and elongation pathways are up-regulated during

cotton fiber development; GhKCS6 was found to be induced most strongly at

10dpa. The cotton protein does not have any sequence similarity with the

homologous Elops in yeast, but GhKCS6 can nevertheless suppress the

phenotypes of the different Elop deletions, indicating that it functions as a

condensing enzyme in the VLCFA elongation pathway.

A chimeric protein was generated to identify the dehydratase involved in the

VLCFA elongation pathway using a colony color sectoring assay. The purpose

was to introduce redundancy in the function and subsequently perform a random

mutagenesis. The outcome was unexpected, namely, due to the properties of the

chimera. The data obtained did not lead to the identification of a dehydratase

enzyme, but provided new insights to the essential flipping process during LLO

synthesis. The incomplete Elo3p could trigger unspecific flipping of glycan

precursors sufficient to restore the glycosylation of CPY close to a wild-type level.

The role of ERAD was also highlighted via its function in the maintenance of

membrane integrity by prevention of the accumulation of malfolded proteins or

incomplete protein complexes.

The data collected during the analysis of the glycan flipping mediated by the

chimera or the truncated proteins improve our understanding of the general

flipping process on the membrane by highlighting the role of the interface

between the transmembrane domains of a protein and the glycerophospholipids.

The functional conservation of RFT1 in eukaryotes was highlighted by the

successful expression and complementation of the hRFT1 in the yeast rft1Δ

mutant strain. Furthermore, expression in yeast of the hRFT1 variant isolated

from a patient suffering from severe protein underglycosylation could not rescue

the rft1Δ phenotype.

78

79

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OECONOMICA

EDITOR IN CHIEF

PUBLICATIONS EDITOR

Professor Mikko Siponen

University Lecturer Elise Kärkkäinen

Professor Hannu Heusala

Professor Olli Vuolteenaho

Senior Researcher Eila Estola

Information officer Tiina Pistokoski

University Lecturer Seppo Eriksson

Professor Olli Vuolteenaho

Publications Editor Kirsti Nurkkala

ISBN 978-951-42-9068-8 (Paperback)ISBN 978-951-42-9069-5 (PDF)ISSN 0355-3191 (Print)ISSN 1796-220X (Online)

U N I V E R S I TAT I S O U L U E N S I SACTAA

SCIENTIAE RERUM NATURALIUM

U N I V E R S I TAT I S O U L U E N S I SACTAA

SCIENTIAE RERUM NATURALIUM

OULU 2009

A 524

François Pujol

EXPERIMENTS ON FATTY ACIDS CHAIN ELONGATION AND GLYCAN FLIPPINGIN THE ER MEMBRANE

FACULTY OF SCIENCE,DEPARTMENT OF BIOCHEMISTRY,UNIVERSITY OF OULU;BIOCENTER OULU,UNIVERSITY OF OULU

A 524

ACTA

François Pujol