Aut7p, a Soluble Autophagic Factor, Participates in MultipleMembrane Trafficking Processes

Aster Legesse-Miller, Yuval Sagiv, Rina Gluzman, and Zvulun Elazar*Department of Biological Chemistry, The Weizmann Institute of Science,

Rehovot, 76100 Israel

Running title: Involvement of Aut7 in membrane trafficking

*Corresponding author: Zvulun ElazarDepartment of Biological ChemistryThe Weizmann Institute of ScienceRehovot, 76100 IsraelTel: 972-8-9343682Fax: 972-8-9344112E-mail: bmzevi@weizmann.weizmann.ac.il

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 2, 2000 as Manuscript M000917200 by guest on A

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Abstract

Aut7p, a protein recently implicated in autophagic events in the yeast

Saccharomyces cerevisiae, exhibits significant homology to a mammalian protein,

p16 (herein termed Golgi-associated ATPase Enhancer of 16 kD – GATE-16), a

novel intra-Golgi transport factor. Here we provide evidence for the

involvement of Aut7p in different membrane trafficking processes. Aut7p

largely substitutes for the activity of GATE-16 in mammalian intra-Golgi

transport in vitro. In vivo, AUT7 interacts genetically with ER-to-Golgi SNAREs,

specifically with BET1 and SEC22. Aut7p interacts physically with two v-

SNAREs: Bet1p, which is involved in ER-to-Golgi vesicular transport, and

Nyv1p, implicated in vacuolar inheritance. We suggest that, in addition to its

role in autophagocytosis, Aut7p has pleiotropic effects, and participates in at

least two membrane traffic events.

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Introduction

Membrane trafficking in eukaryotic cells is a highly regulated process which is

essential for secretion of macromolecules as well as for the maintenance of

distinct subcellular compartments (1,2). This process encompasses a series of

highly regulated events, including cargo selection and vesicle budding at the

donor membrane, followed by transport, docking, and fusion of the transport

vesicle with the target organelle. We previously identified a cytosolic factor,

GATE-16, which participates in intra-Golgi transport (3,4). However, the yeast

homologue of GATE-16, Aut7p, was recently shown to participate in autophagy

(5,6). In this study, we have questioned whether Aut7p plays a role in

constitutive protein transport in addition to its involvement in autophagy.

Vesicular transport between the ER and the Golgi apparatus in the yeast

Saccharomyces cerevisiae has been extensively studied. The first step in this

process, vesicle budding, involves the assembly of the COPII coat, composed of

the Sec13p/Sec31p complex (7-9), the Sec23p/Sec24p heterodimer (10), as well as

a small GTPase, Sar1p (11), and the multidomain protein Sec16p (12,13).

Docking of an ER-derived COPII vesicle with the cis-Golgi compartment takes

place just after, or concurrently with, a tethering event mediated by Uso1p (14),

the yeast homologue of p115 (15,16). It has been further suggested that docking

involves the interaction of ER to Golgi v-SNAREs - Bet1p, Bos1p, Sec22p, and

Ykt6p (17-20) - with Sed5, the cognate t-SNARE on the Golgi (21) to form the v/t-

SNARE complex. This complex binds the yeast SNAP (Sec17p) and NSF

(Sec18p), which in turn catalyze its disassembly (19) after a round of fusion, thus

allowing a new round to take place (22,23).

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Homotypic vacuolar fusion is the last step in yeast vacuole inheritance.

Like many membrane trafficking processes, it is mediated by a number of

membrane and soluble factors, including Vam3p (a t-SNARE), Nyv1p (a v-

SNARE), Ypt7p (a Rab protein), Sec17p, Sec18p, and a low-molecular-weight

factor, LMA1 (24-28). Vacuolar homotypic fusion has been divided into three

distinct subset reactions: priming, docking, and fusion. Priming of SNARE

molecules for a new fusion event is mediated by Sec17p, Sec18p, and LMA1

(22,29). Based on a cell-free system reconstituting vacuolar homotypic fusion, it

appears that the formation of the SNARE complex is only an intermediate step in

the overall fusion reaction (30). Accordingly, SNARE molecules are involved in

docking between donor and acceptor membranes, whereas another set of

proteins participates in subsequent stages of the fusion process. This model for

the course of events is supported by Peters and Mayer (31), who have suggested

that calmodulin and other yet-unidentified factors are involved in mediating late

stages of vacuolar fusion.

The yeast vacuole takes in membrane-bound traffic through at least five

different transport pathways: the carboxypeptidase Y (CPY) pathway, the

alkaline phosphatase I (ALPI) pathway, the endocytic pathway, autophagy, and

the cytoplasm to vacuole targeting (Cvt) pathway (32). Each of these pathways

has different cargo, transport intermediates and genetic requirements.

Autophagy is a bulk protein degradation process by which cytoplasmic

components, including organelles, become enclosed in double membrane

structures (autophagosomes), which are then delivered to the vacuole for

degradation (33). Recent studies have revealed that the autophagic process in

the budding yeast S. cerevisiae is similar to that of higher eukaryotes (34-36).

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Autophagy in yeast may be dissected into a series of subreactions: starvation

signaling, formation of autophagosomes, targeting of autophagosomes to the

vacuole, docking and fusion with the vacuolar membrane, and degradation of

the autophagosome body within the vacuole (37,38). Transport of

autophagosomes to lysosomes or vacuoles should therefore be regarded as a

membrane traffic process. The Cvt pathway is a constitutive biosynthetic process

which shares many common transport components with autophagy (39,40). Both

Cvt and the autophagy pathways involve at least two membrane fusion events,

which are dependent on Sec18p and on the vacuolar t-SNARE Vam3p; the latter

acts as a multispecific receptor for heterotypic membrane docking and fusion

reactions (24). Additionally, Tlg2p, a member of the syntaxin family of t-SNARE

proteins, and Vps45p, a Sec1p homologue, are reported to be required for the

constitutive Cvt pathway (41).

In this study, we demonstrate that Aut7p can largely replace GATE-16

activity in vitro, indicating that the two proteins share a similar, conserved

function. Aut7p is a peripheral membrane protein localized predominantly on

the Golgi complex and vacuolar membrane. It interacts genetically and

physically with Bet1p, a v-SNARE involved in ER to Golgi protein transport.

Aut7p also interacts physically with the vacuolar v-SNARE Nyv1p. We

hypothesize that Aut7p participates in several transport steps by interacting with

the docking and fusion machinery.

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Experimental Procedures

Antibodies - Antisera against Bet1p, Sec22p and Bos1p were a generous gift from

R. Schekman and S. Ferro-Novick. Anti-carboxypeptidase Y (CPY) and anti-

glycophospholipid-anchored surface glycoprotein (Gas1p) were obtained from

H. Riezman. Anti-Ufe1p antibodies were obtained from H. Pelham. Affinity-

purified anti-Sed5p were obtained from D. Gallwitz. Anti-3-phosphoglycerate

kinase (PKG) and anti-dolichol phosphate mannose synthase (Dpm1p)

antibodies were purchased from Molecular Probes, Inc. HRP conjugated

secondary antibodies were obtained from Bio-Rad Labs.

Strains and Media - The yeast strains used in this study are listed in Table I.

Yeast strains were grown either in complete medium (YPD: 1% yeast extract, 2%

peptone, and 2% glucose), in synthetic complete medium (SC: 2% glucose, 0.67%

yeast nitrogen base without amino acids, supplemented with amino acids and

nutrients), or in synthetic minimal medium (SD; 2% glucose, 0.67% yeast

nitrogen base without amino acids, supplemented with the appropriate

auxotrophic nutrients). Transformation of S. cerevisiae was done by the lithium

acetate method (42). Standard yeast techniques for sporulation, tetrad analysis

and gene disruption were employed as described (43). E. coli transformations

were done as previously described (44).

Plasmids - Constructs containing the AUT7 open reading frame were generated

by PCR amplification of yeast genomic DNA using Vent polymerase (BioLabs)

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and subsequent ligation into the vectors pRS426 or pYEP50 (URA3, 2µ and CEN-

based, respectively), behind the AUT7, ADH or GAL1 inducible promoters.

To produce His6-Aut7p fusion protein, an EcoRI-BamHI fragment

containing the AUT7 gene generated by PCR was introduced into pQE30

(Quiagen) and expressed in E. coli XL1-blue strain. After inducing protein

expression for 2 h with 1 mM isopropyl-β-D-galactopyranoside, the fusion

protein (His6-Aut7p) was affinity-purified on a Ni2+-NTA-agarose beads

(Quiagen) and further purified by Mono S (cation exchange) column.

Antibody Production and Purification - Rabbit polyclonal antisera were prepared

against recombinant His6-Aut7p. Pure His6-Aut7p, emulsified in Freund’s

complete adjuvant, was injected subcutaneously into two rabbits (0.6 mg/rabbit).

Polyclonal antibodies were affinity-purified on nitrocellulose strips containing

pure His6-Aut7p.

Intra-Golgi Transport Assay - The standard assay mixture (25 µl) contained 0.4

µCi UDP-[3H] N-acetylglucosamine (America Radiolabeled Chemical), 5 µl of 1:1

mixture of donor and acceptor CHO Golgi membrane, and crude bovine brain

cytosol. Transport reactions were incubated at 30ºC for 2 h and the incorporation

of [3H] N-acetylglucosamine into VSV-G protein was determined as previously

described (45). The GATE-16-dependent assay was performed as previously

described (3). Briefly, each assay contained 0.4 µCi UDP-[3H] N-

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acetylglucosamine, 5 µl of 1:1 mixture of donor and acceptor CHO Golgi

membrane, 100 µg β (a crude cytosolic fraction obtained by anion exchange

chromatography), 0.5 µg p115, 5 ng His6-NSF, 60 ng His6-SNAP, 10 mM

palmitoyl-Coenzyme A, and ATP and UTP regeneration systems.

Disruption of AUT7 - A fragment of 1700 bp containing full-length genomic

AUT7 was PCR amplified, using oligonucleotides containing BamHI and KpnI

sites and yeast genomic DNA as a template. The resultant PCR product was

subcloned into the BamHI and KpnI sites of the pSK+ cloning vector to yield pSK-

AUT71.7. Next, LoxP-KANR-LoxP module selection marker was inserted into the

AccI and HpaI sites of pSK-AUT71.7, removing ~350 bp from the open reading

frame of AUT7. AUT7 disruption strain in a wild type background was

constructed by transforming the PvuII fragment containing the aut7::KAN

disruption into the diploid yeast strain W303. Disruption of one of the AUT7 loci

was verified by Southern analysis and PCR analysis. Upon sporulation of this

diploid strain (ZE1), the resulting tetrads were dissected to yield haploid aut7

cells (ZE14 and ZE15). ZE15 cells were then crossed to bet1-1 and sec18-1 mutants

to give diploid strains which sporulated and dissected to yield haploid yeast

bearing both mutations (Table 1).

Secretion of Media Proteins and Pulse–chase Analysis - Secretion of proteins into the

medium was assessed using the method described by Gaynor and Emr (46).

Intracellular protein processing was monitored by pulse-chase analysis with

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[35S]methionine (Amersham), using anti-CPY and anti-Gas1p antibodies in

immunoprecipitation reactions as described (47,48). Autoradiography was

performed with a fluorescence enhancer.

Subcellular Fractionation and Gradient Analysis – Cellular fractionation of yeast

cells was performed as described (49) with minor modifications. Briefly, cells (25

OD600 units) were harvested during log phase and lysed with glass beads in 500

µl of buffer 88 (20 mM Hepes, pH 7.0, 150 mM KOAc, 5 mM Mg(OAC)2, 1 mM

DTT, 0.1 µg/ml PMSF, 5 mM phenanthroline, 2 µg/ml aprotonin and 2 µg/ml

leupeptine). After lysis, extracts were spun at 1,000 g for 5 min at 4˚C to remove

intact cells and cell wall debris. The total yeast lysate (S1) was centrifuged at

13,000 g for 10 min at 4˚C to generate supernatant (S13) and pellet (P13) fractions.

The obtained S13 fraction was layered over a 14% sucrose cushion (in buffer 88)

and centrifuged at 200,000 g for 30 min to yield supernatant (S200) and pellet

(P200) fractions.

For experiments involving membrane treatments, the S1 fraction was

centrifuged at 200,000 g for 30 min and the pellets were suspended with either

buffer 88 or in one of the three extraction solutions (1% Triton X-100 in buffer 88,

1 M NaCl in buffer 88, or 100 mM Na2CO3 in H2O). Following 30 min incubation

on ice, the extracts were layered over 14% sucrose cushion adjusted to the final

concentration of the extraction solution, and centrifuged at 200,000 g for 30 min

to produce supernatant and pellet fractions. For sucrose gradient centrifugation,

0.3 ml of P200 was layered on top of a 5 ml linear sucrose gradient (20-60%)

made up in 10 mM Hepes, pH 7.4. The gradient was subjected to centrifugation

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at 4˚C in a SW50.1 rotor (Beckman) at 100,000 g for 17 h. Fractions of 300 µl were

collected from the top of the gradient, diluted 1:1 with 10 mM Hepes, pH 7.4, and

centrifuged at 200,000 g for 30 min at 4˚C. The pellets were resuspended with

sample buffer and analyzed by immunoblotting.

Membrane Extract Preparation and Immunoprecipitation - For co-precipitation

experiments, the S1 fraction was centrifuged at 200,000 g for 30 min, and the

pellet was resuspended in solublization buffer (1% Triton X-100, 10 mM

Hepes/KOH, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 2 µg/ml

leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotinin) and then incubated on ice for 2

h. Unsolublized material was removed by centrifugation (10 min at 200,000 g).

Proteins (150 µg) were diluted to a final Triton X-100 concentration of 0.4%, and

incubated with the indicated antibodies and 20 µl protein A beads at 4˚C

overnight. The beads were reisolated by centrifugation at 1000 g and washed

five times with 0.5 ml of solublization buffer containing 0.4% Triton X-100.

Proteins were eluted from the beads by adding 2% SDS and heating to 95˚C for 5

min. SDS sample buffer was added to the eluted proteins followed by heating to

95˚C for 3 min. Proteins were resolved by SDS-PAGE on 13% polyacrylamide

gels, transferred to nitrocellulose, and immunoblotted with the indicated

antibodies.

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Results

Aut7p substitutes for GATE-16 activity in intra-Golgi transport in vitro

Aut7p is a conserved yeast protein exhibiting high homology to proteins found

in eukaryotes, with no other homologues in S. cerevisiae. Caenorhabditis elegans

has two AUT7 related genes, Arabidopsis thaliana has at least five different AUT7

related genes, and mammals have three different AUT7 homologues. We have

recently isolated one of the mammalian homologues of Aut7p, GATE-16, which

exhibits 56% amino acid sequence identity and 75% similarity, and demonstrated

that it participates in intra-Golgi protein transport (3,4).

To determine whether Aut7p plays a role in membrane traffic, we have

performed a series of biochemical and genetic experiments. Aut7p is a 117

amino acid protein, constitutively expressed in S. cerevisiae. Anti-Aut7p

polyclonal antibodies specifically recognized on Western blot a 14 kD

polypeptide corresponding to Aut7p in a wild type strain, and absent in an aut7

null strain (Fig. 1A). These antibodies also recognized GATE-16 (the mammalian

homologue of Aut7p) in bovine brain cytosol (Fig. 1A) and recombinant GATE-

16 expressed in E. coli (data not shown). When added to a cell-free intra-Golgi

transport assay, affinity purified anti-Aut7p antibodies specifically inhibited

transport (Fig. 1B), whereas, antibodies preincubated with recombinant Aut7p

did not. Clearly, in addition to their sequence homology, Aut7p and GATE-16

share immunogenic determinants which are recognizable by anti-Aut7

antibodies. To test whether recombinant Aut7p could stimulate intra-Golgi

transport, similar to GATE-16, we purified recombinant His6-Aut7p from E. coli

on a Ni2+-NTA-agarose column followed by Mono-S chromatography. When

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added to the GATE-16-dependent transport assay (see Experimental Procedures),

Aut7p significantly stimulated transport, although to a somewhat lesser extent

than its mammalian homologue (Fig. 1C). These experiments suggest that Aut7p

and GATE-16 share a similar function in mediating membrane trafficking.

Aut7p is a peripheral membrane protein

Many soluble proteins involved in vesicular transport are associated with the

membrane. To determine the subcellular distribution of Aut7p, we subjected

yeast homogenate to differential centrifugation and obtained a soluble fraction,

S200 (representing the cytosol), and two membrane fractions, P13 (pelleted at

13,000 x g) and P200 (pelleted at 200,000 x g). With this series of differential

centrifugation steps, all organelles of the secretory pathway are present in one or

more of the pellet fractions: the ER and vacuole are located primarily in the P13

fraction, whereas the Golgi partitions in the P200 fraction. Using anti-Aut7p

antibodies, Western blot analysis revealed that Aut7p was distributed between

the membrane fraction and the cytosol (Fig. 2A). The membrane-associated

Aut7p co-fractionated with the cis-Golgi marker Sed5p, found in the P200 pool,

and to a somewhat lesser extent with the ER marker Dpm1p, found in the P13

pool. Phosphoglycerate kinase (PGK), serving as a control cytosolic factor,

exclusively fractionated to the S200 fraction (Fig. 2A). We then further

fractionated the P200 fraction, loading it on top of a 20-60% sucrose gradient that

was ultra-centrifuged at 100,000 g for 17 h. Fractions collected from this gradient

were separated by SDS-PAGE and analyzed by Western blotting using different

antibodies. Aut7p was found in the 30-49% sucrose fraction, co-fractionating

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with Sed5p (Fig. 2B), thus corroborating the differential centrifugation

experiments.

To find whether Aut7p is a peripheral membrane protein, we attempted to

extract the protein from the membrane using a detergent, high salt, or high pH.

Although integral, lumenal and peripheral membrane proteins are extractable by

detergents that solubilize membranes, a peripheral membrane protein is

characteristically extracted by high salt or high pH, conditions which often

disrupt protein-protein interactions. Most of the Aut7p associated with the

membrane was extracted with Triton X-100, 1M NaCl, or with 100 mM sodium

carbonate, pH 11.5 (Fig. 2C). A similar extraction pattern was observed for

Sec17p, serving as a control for peripheral membrane proteins, but not for

Sec22p, an integral membrane protein. Evidently, Aut7p is peripherally

associated with membranes probably by interaction with other proteins, whose

identity remains to be elucidated.

AUT7 interacts genetically with BET1 and SEC22

Considering the involvement of Aut7p in autophagocytosis under

starvation and in protein transport processes under normal conditions, we

analyzed systematically the genetic interaction of AUT7 with genes involved in

membrane traffic. The ability of Aut7p to largely replace the activity of its

mammalian homologue, GATE-16 in intra-Golgi transport, suggested

participation in early steps of secretory pathway. We therefore examined

whether the AUT7 could act as a multicopy-suppressor of the temperature-

sensitive (ts) growth phenotype in mutants defective in these transport steps.

Table II summarizes a multicopy-suppression experiment performed on a

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number of ts secretion mutants. It appears that overexpression of Aut7p

specifically suppresses the temperature sensitivity of bet1-1 and sec22-2 mutants

(Fig. 3A), whose gene products are ER-to-Golgi v-SNAREs. The suppression

effect of Aut7p on these mutants was accompanied by a complete recovery of

total protein secretion under non-permissive conditions (Fig. 3B). Thus, Aut7p

interacts genetically with ER to Golgi v-SNAREs by restoring the transport

function of their ts alleles.

Aut7p and Bet1p form a protein complex

To determine whether the protein products of AUT7 and BET1 interact

physically, total yeast membrane extracts were prepared and subjected to

immunoprecipitation with anti-Aut7p antibodies. Sepharose protein A beads

coupled to anti-Aut7p antibodies were mixed with Triton X-100 membrane

extracts, then washed, and the eluted material was subjected to Western blot

analysis with various antibodies. Anti-Aut7p antibodies specifically precipitated

Aut7p and significant amounts of Bet1p (Fig. 3C). Similarly, when anti-Bet1p

antibodies were used to precipitate Bet1p from the membrane extract, Aut7p co-

immunoprecipitated. Other proteins, such as Ufe1p, Sec17p and Bos1p, did not

precipitate with the anti-Aut7p antibodies (Fig. 3D). The immunoprecipitation

observed in these experiments was specific; no Bet1p was precipitated with the

anti-Aut7p antibodies when the aut7 null strain ( aut7) was used as a source for

the membrane extract (Fig. 3C). These experiments indicate that Aut7p and

Bet1p form a protein complex. Using anti-Aut7p and anti-Sec22p antibodies in

immunoprecipitation experiments, we obtained inconclusive results and we

therefore cannot rule out a physical interaction between these proteins.

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aut7/bet1-1 double mutant exhibits a synthetic delay in ER to Golgi transport

To test the relevance of the physical interaction between Aut7p and Bet1p for

protein transport in vivo, we generated four haploid strains: wild type, aut7,

bet1-1, and a aut7/bet1-1 double mutant. Like the other haploid strains, the

double-mutant strain exhibited normal growth at the permissive temperature.

However, we found a significant decrease in the rate of ER-to-Golgi transport for

both CPY (a vacuolar protein) and Gas1p (a periplasmic protein) markers at the

permissive temperature (Fig. 4). The maturation of these markers, as reflected by

changes in their mobility on SDS-PAGE, was delayed after a short chase,

whereas after a long period of chase the difference between the wild type and the

double mutant strain was less apparent. We next tested for growth defects in the

aut7/bet1-1 double mutant strain in different temperatures. Cells were replica

plated on selective medium and grown for overnight at the indicated

temperatures (Fig. 4C). It appears that deletion of AUT7 along with the bet1-1

background inhibit growth of these cells at temperatures normally permissive for

growth of bet1-1 mutant (35˚C and 36˚C). This synthetic lethality phenotype can

be suppressed by over expression of Aut7p. The data presented here suggest

that Aut7p increases the efficiency of ER-Golgi transport, at least in part through

interaction with Bet1p.

Aut7p forms a complex with Nyv1p

During this study, Aut7p/Apg8 was reported to be essential for

autophagocytosis (5,6). We have deleted the AUT7 gene by replacing the coding

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region of Aut7p with a KAN gene cassette. In agreement with the

aforementioned studies (5,6), the null haploid mutant, aut7, is viable and shows

normal growth on YPD medium, but a diploid aut7 strain is unable to sporulate

(data not shown). We also found that upon starvation, which triggers

autophagocytosis, this mutant strain shows lower rates of survival and protein

degradation (data not shown), consistent with reduced autophagocytosis. In

addition, the level of Aut7p was dramatically elevated, reaching a peak 5 h after

cells were transferred into a starvation medium (data not shown). These data

strongly suggest that upon starvation, Aut7p is an essential and possibly rate-

limiting factor for autophagocytosis.

During autophagocytosis, autophagosomes containing cytosol fuse with

the vacuolar membrane. We have tested whether Aut7p is in fact present on the

vacuolar membrane. For that purpose, vacuolar membranes were isolated by

flotation through a discontinuous Ficoll step gradient as described by Conradt et

al. (50). The Ficoll interphase (0-4%) was collected and tested by Western

analysis using specific antibodies. Aut7p was found in this fraction together

with the vacuolar markers Vam3p and Nyv1p (Fig. 5A); the Golgi marker, Sed5p,

and the ER marker, Dpm1p, were absent.

As we have shown that Aut7p interacts with Bet1p, an ER-to-Golgi v-

SNARE protein (Fig. 3), we tested by co-immunoprecipitation whether Aut7p

also interacts with a vacuolar SNARE. As shown in Fig. 5B, anti-Aut7p

antibodies specifically precipitated Aut7p from yeast membrane extracts,

together with Nyv1p, a vacuolar v-SNARE. Only a small fraction of the vacuolar

t-SNARE Vam3p co-precipitated with Aut7p. When the yeast membrane extract

was incubated with anti-Nyv1p antibodies, significant levels of Aut7p were

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found in the pellet, this time together with higher amounts of Vam3p (Fig. 5B).

Aut7p does not interact with v-SNARE such as Snc1p and Snc2p involved in

Golgi to plasma membrane transport (data not shown). No precipitation of

Nyv1p with anti-Aut7p antibodies was detected when a aut7 mutant strain was

used (Fig. 5B), confirming the specificity of the interaction.

SNARE molecules can be found either as free monomers or assembled

into a v- t-SNARE complex. It has been demonstrated that NSF/Sec18p together

with SNAP/Sec17p disassembles these complexes in the presence of ATP (30,51).

This reaction is accompanied by dissociation of Sec17p from the membrane (22).

To test whether Aut7p interacts with the Nyv1p/Vam3 complex or with a free

Nyv1p, membranes were isolated and resuspended in the presence or absence of

1 mM MgATP. After incubation for 10 min at 25˚C the membrane proteins were

extracted with detergent and immunoprecipitated with the anti-Aut7p

antibodies. The resulting immunoblot indicates that while Sec17p was removed

from the membranes in the presence of ATP (Fig. 5C), no significant change in

the overall amount of Nyv1p which co-immunoprecipitated with Aut7p (Fig.

5D). Furthermore, in both cases, only a small level (less than 0.5%) of Vam3p co-

immunoprecipitated with Aut7p. In addition, anti-Vam3p antibodies failed to

co-precipitate Aut7p from membrane extracts (data not shown). These results

indicate that that Aut7p primarily interact with Nyv1p and to a lesser extend

with the Nyv1p/Vam3p complex. The association of Aut7p-Nyv1p implies a

function of Aut7p at the docking stage.

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Discussion

Aut7p, previously identified as an autophagic factor (5,6), is shown here to take

part in multiple intracellular membrane trafficking processes. We have

demonstrated that Aut7p specifically interacts with different v-SNARE

molecules involved in ER-to-Golgi transport and in vacuolar fusion. Our

findings imply that in addition to its involvement in transport to the vacuole,

Aut7p also participates in membrane fusion events that take place in the early

secretory pathway.

That Aut7p is essential for autophagocytosis (5,6) is consistent with the

notion that it participates in membrane traffic. Lang and co-workers (5)

proposed that Aut7p and Aut2p are involved in the delivery of autophagosomes

to the vacuole along microtubules. Kirisako and co-workers (6) suggested that

Aut7p/Apg8p plays an important role in autophagosome formation. We

propose that the formation of autophagosomes and/or their fusion with the

vacuolar membrane are SNARE dependent, and that Aut7p is essential for the

fusion process. The t-SNARE Vam3p – a protein known to participate along with

other vacuolar protein sorting (VPS) gene products in directing endosomes to

vacuole transport – has also been shown to be involved in autophagy (24).

Vam3p interacts primarily with Nyv1p, the vacuolar v-SNARE involved in

homotypic fusion (25). As we show here, Aut7p and Nyv1p form a complex,

suggesting that Aut7p may play a role in the fusion between autophagosomes

and vacuoles by interacting with the docking/fusion machinery. It was reported

that Nyv1p is not involved in transport of AP1 to the vacuole; in contrast, Vti1p,

a v-SNARE found in Golgi-derived vesicles and known to be involved in many

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transport events, is required for this transport step (52). Additionally, Tlg2p, a

member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p

homologue, are required in the constitutive Cvt pathway, but not in inducible

macroautophagy (41). It is therefore likely that Aut7p may interact with an as

yet unidentified related v-SNARE molecule that mediates fusion between

autophagosomes and the vacuole. Defining the origin of the donor membrane

required for the formation of autophagic vesicle, and isolation of these vesicles

using Aut7p as a marker, will allow a better understanding of the roles of the

various proteins involved in this pathway.

It appears that Aut7p interacts with v-SNAREs and thereby affects their

activity. Several regulatory proteins that interact with SNAREs have been

reported. Sec1p in yeast and its homologues nSec1p, Munc18, and unc18 in

higher eukaryotes have been shown to regulate exocytosis (53-58). This protein

family acts directly on the syntaxin t-SNAREs (54,59-61). Sec1p also inhibits the

assembly of a v- t- SNARE complex in vitro and dissociates from syntaxin upon

formation of the v/t-complex (54,56,60). Furthermore, experiments in vivo in

Drosophila melanogaster and C. elegans showed that over-expression of Sec1p

homologues inhibits neurotransmitter release (62,63). It has therefore been

suggested that these proteins act as negative regulators of fusion. Vsm1p,

another factor that interacts with v-SNARE (Snc2p), was recently suggested to

regulate Snc2p entry to the SNARE complex (64). Finally, LMA1, a low-

molecular-weight heterodimer composed of thioredoxin and the protease B

inhibitor IB2 - originally identified as a protein required for in vitro vacuolar

homotypic fusion (28,65) as well as for ER to Golgi transport (14,66) - was shown

to interact with the vacuolar t-SNARE Vam3p in a Sec18p-dependent manner

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(67). Possibly, Aut7p acts on v-SNAREs in a similar manner to that by which

LMA1 acts on the t-SNARE Vam3p.

Bet1p, Bos1p, and Sec22p, three v-SNAREs, have been implicated in

transport between the ER and the Golgi. It have been recently proposed that

Bos1p participates exclusively in anterograde transport from the ER to the Golgi,

Sec22p is involved in retrograde transport from the Golgi to the ER, whereas

Bet1p acts in both directions (68). We show here that Aut7p specifically interacts

with Bet1p but not with Bos1p, suggesting that it is not involved in anterograde

transport but rather in retrograde transport from the Golgi to the ER. This is

supported by the suppression effect of AUT7 on the retrograde mutant sec22-2.

The viability of the aut7 null strain and the lack of a detectable transport defect in

this strain suggest that Aut7p plays a regulatory role in this process, or that other

factors may substitute its function. Since under starvation conditions Aut7p is

essential for autophagy, we speculate that Bet1p or Sec22p may participate in the

formation of the autophagic membrane, or that under starvation Aut7p interacts

with other SNARE molecules mediating autophagy.

We have recently found that GATE-16, the mammalian homologue of

Aut7p, interacts specifically with a Golgi v-SNARE in an NSF- and SNAP-

dependent manner (4). Although the precise mechanism for the function of

Aut7p in membrane traffic is uncertain, we propose that it may act as a positive

regulator of v-SNAREs. The data presented in the present study support the

notion that the function of Aut7p is closely related to the activity of SNAREs.

Our findings are summarized by the model described in Fig. 6.

Accordingly, under normal growth conditions Aut7p functions in early steps of

the secretory pathway by interacting with v-SNARE molecules such as Bet1p.

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This Aut7p activity can probably be replaced by other unidentified factor(s).

Under constitutive steady-state conditions, Aut7p is essential for the Cvt

pathway. Upon nitrogen starvation, Aut7p expression levels are significantly

increased and it becomes involved in autophagy. Based on the fact that Aut7p

interacts with the vacuolar v-SNARE, Nyv1p, we propose that Aut7p may be

involved in vacuolar fusion, or that Nyv1p is involved in autophagy triggered

upon nitrogen starvation. Further experiments are required to resolve this issue.

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Figure legends:

Fig. 1. Aut7p is a functional homologue of mammalian GATE-16. A. GATE-16

is recognized by anti-Aut7p antibodies. Bovine brain cytosol (100 µg) or yeast

cytosol (10 µg) were separated on 13% SDS PAGE and blotted onto nitrocellulose

membranes. Western blots were probed with affinity-purified anti-Aut7p

antibodies. B. Affinity-purified anti-Aut7p antibodies inhibit mammalian in

vitro intra-Golgi transport. Increasing amounts of affinity-purified antibodies

directed against Aut7p were added to a standard cell-free intra-Golgi transport

assay. For the control assay, the antibodies were incubated with purified

recombinant Aut7p prior to the transport assay. The transport reactions were

incubated at 30ºC for 2 h. [3H] N-acetylglucosamine incorporated into VSVG-G

protein was determined. C. Recombinant Aut7p partially substitutes for

mammalian GATE-16 in the transport assay. Recombinant GATE-16 or Aut7p

(100 ng) were added to the GATE-16-dependent intra-Golgi transport assay (see

Experimental Procedures).

Fig. 2. Aut7 is a peripheral membrane protein, predominantly localized on the

Golgi. A. Aut7p partitions into soluble and membrane-associated pools.

Lysate obtained from wild type strain was differentially centrifuged as described

in Experimental Procedures. The obtained fractions were subjected to Western

blot analysis using antibodies to Aut7p, Sed5p (a cis-Golgi marker), Dpm1p (an

ER marker) and PGK (a cytosolic marker). B. Sucrose density centrifugation of

the P200 fraction (see Experimental Procedures). Fractions obtained from the

sucrose gradient were subjected to Western blot analysis. C. Aut7p is a

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peripheral membrane protein. The S1 supernatant was centrifuged at 200,000 g

to obtain P200-total membrane pellet. The P200-total was incubated for 30 min

on ice with buffer 88, 1% Triton X-100, 1 M NaCl, or 100 mM Na2CO3, pH, 11.5,

centrifuged at 200,000 g to separate into supernatant (right panel) and pellet (left

panel) fractions.

Fig. 3. Overexpression of Aut7p suppresses the temperature-sensitive

phenotype of bet1-1 and sec22-2. A. Suppression of the temperature-sensitive

phenotype of bet1-1 and sec22-2 mutants by Aut7p. Mutant strains were

transformed with multicopy plasmid [pRS426-AUT7 behind the AUT7 promoter

(+) or vector only (-)] and grown on selective medium. Ten µl of cell suspension

(1X105 cells) was spotted on a replica plate and incubated at 25°C and 37°C. B.

Secreted media proteins precipitated from bet1-1 and sec22-2 mutants, carrying

either AUT7 plasmid (+) or vector only (-), were pulse-chase labeled with

[35S]methionine at 37˚C. Molecular mass is given in kilodalton (kD). An asterisk

designates those proteins routinely identified in the culture medium of wild type

cells. C. Aut7p co-immunoprecipitated with Bet1p. Total membrane extract was

prepared from wild type (WT) and from aut7 strain (as a control). Extracts were

incubated with anti-Aut7p or anti-Bet1p antibodies and protein A agarose beads

overnight at 4˚C. Beads were washed five times and the precipitates were eluted

and loaded on 13% SDS-PAGE and analyzed by Western blot. Ten percent of the

input detergent extract was shown as total 10%. IB= Immunoblot, IP=

Immunoprecipitation. Densitometric quantification of the western blots revealed

that 8% (± 0.9%) of total Bet1p was precipitated with the anti-Aut7p antibodies

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and 7% (± 0.7%) of total Aut7p was precipitated with the anti-Bet1p antibodies.

D. Total membrane extracts prepared from the WT strain were

immunoprecipitated with anti-Aut7p antibodies. Immunoprecipitation and

detection were essentially as in C.

Fig. 4. Synthetic delay of ER to Golgi transport in bet1-1/ aut7 mutants.

Intracellular processing of CPY (A) and Gas1p (B) was followed in WT, bet1-1,

aut7 and bet1-1/ aut7 cells that were maintained at 25˚C by pulse-chase analysis.

Cells were labeled with [35S]methionine for 5 min (pulse) followed by the

addition of fresh medium containing unlabeled excess methionine and cysteine

(chase). At various time points, samples were removed, and cellular proteins

were extracted, immunoprecipitated with the indicated antibodies,

electrophoresed, and quantified by densitometry. The positions of the three CPY

variants, the ER precursor (p1), the Golgi-modified precursor (p2), and the

mature vacuolar enzyme (m) are indicated. The migration of the immature, (i)

(105 kD) and mature (m) (125 kD) forms of Gas1p are shown. The percentages of

the mature forms of CPY (%mCPY) and of Gas1p (%Gas1p) are given below. C.

Deletion of aut7 lowers the temperature cutoff of bet1-1. The indicated strains

were grown overnight in selective medium and 1 X 104 cells were spotted on

replica plats and incubated for 24 h at the different temperatures.

Fig. 5. Aut7p co-immunoprecipitates with Nyv1p. A. Aut7p is found in

isolated vacuolar membranes. Vacuolar membrane was isolated according to

Conradt et al. (1992). Spheroplases were prepared from wild type strain and

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carefully resuspended in cold 15% Ficoll buffer containing DEAE-dextran. The

vacuoles were isolated by flotation through a discontinuous Ficoll step gradient.

The Ficoll interphase (0-4%) was collected (right lane) and tested by Western blot

using specific antibodies as indicated. The left lane contains total lysate. B.

Nyv1p interacts with Aut7p. Total membrane extract was prepared from wild

type (WT) and from aut7 strain (as a control). Extracts were incubated with

anti-Aut7p or anti-Nyv1p antibodies and protein A agarose beads overnight at

4˚C. Beads were washed five times and the precipitates were eluted and loaded

on 13% SDS-PAGE and analyzed by Western blot. Ten percent of the input

extract was shown as total 10%. IB= Immunoblot, IP= Immunoprecipitation.

Densitometric quantification of the western blots revealed that 0.8% (± 0.6%) of

total Vam3p was precipitated with anti-Aut7p antibodies while 10% (± 1.4%) of

total Nyv1p was precipitated with anti-Aut7p antibodies. In reciprocal

experiments 7% (± 0.5%) of total Aut7p was precipitated with anti-Nyv1p

antibodies. C. Total membranes were re-isolated, incubated for 10 min at 25˚C

with buffer 88 containing 3 mM MgCl2 in the presence or absence of 1mM ATP.

Membranes were re-isolated by centrifugation, resuspended with SDS-sample

buffer and analyzed by Western bolts with anti-Sec17p antibodies and anti-

Nyv1p antibodies. D. After ATP treatment the membrane proteins were

extracted with detergent buffer, immunoprecipitated with anti-Aut7p antibodies,

and analyzed by Western bolt.

Fig. 6. A model for trafficking pathways involving Aut7p. We propose that

under normal growth conditions, Aut7p interacts with Bet1p and Sec22p and

participates in ER to Golgi transport. Aut7p activity in this process, however,

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can be bypassed in vivo. Aut7p is required for the constitutive Cvt pathway to

the vacuole and under nitrogen starvation conditions, Aut7p is essential for

transport of autophagosomes to the vacuole, as previously described (5,6). Aut7p

interacts with Nyv1p, which is required for vacuolar fusion, but the functional

relevance of this finding is not yet known. PVC stands for pre vacuole

compartment.

Acknowledgments

The authors are grateful to J. E. Gerst, O. Giladi and S. Fishburn for helpful

advice and providing the reagents. We thank R. Schekman, H. Riezman, D.

Gallwitz, A. Mayer, S. Ferro-Novick, H. Pelham, J. Lewis, C. Barlowe, for

providing strains and antibodies. Special thanks to Hedva Later and Frida

Shimron for technical help. We are grateful to the members of Dr. Elazar’s group

for stimulating discussion. Z. E. is incumbent of the Shloimo and Michla

Tomarin Career Development Chair of Membrane Physiology. This work was

supported in part by the Israeli Science Foundation (ISF) and the Weizmann

Institute Minerva Center.

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Table I. Yeast strains used in this study

Strain Genotype Source

W303-ab Mat a/α ade2 his3-11,15 leu2,3-112 trp1-1 ura3-1 J. Gerst

W303-1a Mat a ade2 his3-11,15 leu2,3-112 trp1-1 ura3-1 J. Gerst

W303-1b α ade2 his3-11,15 leu2,3-112 trp1-1 ura3-1 J. Gerst

RSY944 Mat a lys2-801 ura3-52 bet1-1 R. Schekman

RSY271 Mat a his4-619 ura3-52 sec18-1 R. SchekmanRSY976 Mat a ura3-52 ypt1-3 R. Schekman

RSY271 Mat a his4-619 ura3-52 sec18-1 R. SchekmanRSY979 Mat ura3-52 sec7-4 R. Schekman

RSY941 Mat ura3-52 leu2,3-112 sec12-1 R. Schekman

RSY314 Mat a ura3-52 sec13-3 R. SchekmanRSY1010 Mat a lue2-3,-112 ura3-52 sec21-1 R. SchekmanRSY324 Mat a ura3-52 sec22-2 R. SchekmanRSY641 Mat leu2-3,–112 ura3-52 sec23-2 R. Schekman

CBY265 Mat a trp1 ura3 leu2 lys2 sed5-1 C. BarloweRSY955 Mat leu2-3, -112 sec32-1 R. Schekman

RSY952 Mat a lue2,3 ura3-52 sec31-1 R. Schekmansec20-1 Mat a sec20-1 ura3-52 his4-619 J. LewisMLY101 Mat a trp1-1 ade2-1 can1-100 leu2-3,-112 his3-11,-15 ura3-52

ufe1::TRP1 + (CEN LUE ufe1-1)J. Lewis

RSY955 Mat leu2-3, -112 sec32-1 R. Schekman

ZE1 Mat a/ α ade2 leu2 trp1 ura3 aut7::KAN This study

ZE14 Mat a ade2 leu2 trp1 ura3 his3 aut7::KAN This studyZE15 Mat ade2 leu2 trp1 ura3 his3 aut7::KAN This study

ZE15-bet1 Mat a ade2 leu2 trp1 ura3 aut7::KAN bet1-1 This studyZE15-18 Mat a ade2 trp1 his3 ura3 aut7::KAN sec18-1 This study

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Table I. Aut7p suppresses the temperature-sensitive phenotype of ER to Golgisecretion mutants.

Mutant Function of gene product

Suppression:bet1-1 ER-Golgi v-SNAREsec22-2 ER-Golgi v-SNARE

No suppression:sec18-1 NSFusol-1 p115

sec21-1 COPI subunitsec12-1 COPII subunitsec13-1 COPII subunitsec31-1 COPII subunit

ypt1-3 ER-Golgi docking/fusion regulatorsly1-1 ER-Golgi SNARE-associated proteintip20-1 ER-Golgi transport factorsec7-1 ARF exchang factor

bos1-1 ER-Golgi v-SNAREsed5-1 ER-Golgi t-SNAREufe1-1 ER t-SNAREsec9-1 PM t-SNARE

AUT7 gene was tested for its ability to suppress the temperature-sensitive (ts)

growth defect of different mutants. Mutants were transformed with multicopy

plasmid containing the AUT7 gene and incubated at 37°C. PM stands for plasma

membrane.

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Aster Legesse-Miller, Yuval Sagiv, Rina Gluzman and Zvulun ElazarProcesses

Aut7p, a Soluble Autophagic Factor, Participates in Multiple Membrane Trafficking

published online June 2, 2000J. Biol. Chem. 

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