Yeast seminars - hu-berlin.de
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[email protected] Towards a holistic understanding of the Eukaryotic cell and a Eukaryotic whole cell model Marcus Krantz [email protected] Yeast seminars http://www.devbio.biology.gatech.edu/wp- content/uploads/2011/04/yeast-microbe1.jpg Towards a holistic understanding of the Eukaryotic cell and a Eukaryotic whole cell model Marcus Krantz [email protected]
Transcript of Yeast seminars - hu-berlin.de
Towards a holistic understanding of the Eukaryotic cell and a Eukaryotic whole cell model
Marcus Krantz [email protected]
Yeast seminars
http://www.devbio.biology.gatech.edu/wp-content/uploads/2011/04/yeast-microbe1.jpg
Towards a holistic understanding of the Eukaryotic cell and a Eukaryotic whole cell model
Marcus Krantz [email protected]
The whole cell perspective
Transcription
Replication
Ribosome assembly
Morphology
Life cycle
CDC
Mitochondria
Peroxisomes
Vacuoles
Growth
Protein Synthesis
Sensing & Signalling
Growth & Proliferation: - Metabolism
- Catabolism - Anabolism
- Growth - Division
The central dogma: - DNA -> RNA -> Protein
Signalling: - Available nutrients - Pheromones & Stress
Life decisions: - Sporulation & Mating
Cell structure: - Compartments - Morphology
Environmental Cues
Metabolism, growth and division
Growth
Metabolism
Nutrients
Cell Division
Cycle G1/S
G2/M
Uptake
The cell division cycle
M
Spindle formation
Nuclear migration
Chromosome segragation Nuclear division
Cytokinesis
Growth START
Bud emergence
G2
S
G1
http://mpf.biol.vt.edu/research/budding_yeast_model/pp/intro.php
DNA replication
I. The fundamental mechanism of proliferation. II. Conserved in all Eukaryotic cells. III. Driven by ”Cyclin dependent kinases” (CDKs) and
cyclins, and monitored by check points. IV. Cyclins build up during cell cycle phases until they
trigger a transition and their own destruction
Growth
Protein
Glycogen
Trehalose
Mannan
Other carbohydrates
RNA
DNA
Free amino acids
Lipid
Ash
Slow
Fast
doi: 10.1099/00221287-143-1-203 Microbiology January 1997 vol. 143 no. 1 203-218
• In a rapidly growing yeast cell: • 60% of total transcription is
devoted to ribosomal RNA • 50% of RNA polymerase II
transcription and • 90% of mRNA splicing are devoted
to ribosomal proteins (RPs) (Warner, JR; 1999)
• Growth is targetted, depending on cell
cycle phase:
Moseley and Goode 2006
Nutritional requirements
Energy/carbon source - Fermentative carbon sources;
- Glucose/Fructose/Mannose (Glucose repression) - Galactose/Sucrose/Raffinose (Glucose repressed)
- Non-fermentative carbon sources; - Ethanol, Glycerol
Nitrogen source - Ammonium, amino acids - Priority; Nitrogen Catabolite Repression (NCR)
Minerals and vitamins (e.g. YNB) - Phosphate, Sulphate, etc.
Amino acids (auxotrophic strains)
Anaerobic specific requirements: - Sterol (Ergosterol) - Unsaturated fatty acids (Tween 80)
Lindegren 1949
Biotin 2 μg Calcium pantothenate 400 μg Folic acid 2 μg Inositol 2000 μg Niacin 400 μg p-Aminobenzoic acid 200 μg Pyridoxine hydrochloride 400 μg Riboflavin 200 μg Thiamine hydrochloride 400 μg Boric acid 500 μg Copper sulfate 40 μg Potassium iodide 100 μg Ferric chloride 200 μg Manganese sulfate 400 μg Sodium molybdate 200 μg Zinc sulfate 400 μg Potassium phosphate monobasic 1 g Magnesium sulfate 500 mg Sodium chloride 100 mg Calcium chloride 100 mg
YNB
Metabolism
Regulation by: - Allosteric regulation
(substrate/products)
- Covalent modification (signalling)
- Expression (transcription)
Slower
Metabolism: Carbon
Lindegren 1949
Glucose
Fructose
Sucrose Raffinose
x1/2
x1/2 x1/3
Maltose
Galactose
Glycerol
Ethanol
Glycolysis: - Redox neutral fermentation - Anaerobic growth possible:
- Biomass balanced by glycerol production (NADH reoxidation)
- Fermentation even under aerobic conditions -> EtOH Varela et al. 2012
Metabolism: Carbon
Lindegren 1949
The TCA cycle: - Produce reducing power for oxidative
phosphorylation (NADH; FADH) - Produce intermediates for biomass
production - Surplus NADH can be reoxidised via
glycerol biosynthesis (anaerobically) Varela et al. 2012
Metabolism: Nitrogen
Ljungdahl & Daignan-Fornier 2O12
Nitrogen metabolism NH4
+ fixation via Glutamate and Glutamine biosynthesis
De novo synthesis of other Amino acids and Nucleotides use amine groups from Glutamine or Glutamate
Quality differences among nitrogen sources; cell utilises good N-source first: Catabolism of good N-sources
yield carbon skeletons that can be metabolised
while the carbon skeletons of bad N-sources are converted into growth inhibitory fusel oils.
Metabolism: Nitrogen
Nitrogen metabolism Amino acid biosynthesis
start from glycolytic or TCA cycle intermediates
Glycolysis and TCA needed for biogenesis – not only energy production
TCA cycle needed even when cells grow fermentatively or even anaerobically
Biomass production is not redox neutral – accumulation of reducing equivalents (NADH).
Leads to redox imbalance during anaerobic growth -> solved via production and export of glycerol. Ljungdahl & Daignan-Fornier 2O12
Metabolism: Sulfur assimilation
Sulfur metabolism Sulphate (SO4
2-) uptake via Sul1 and Sul2.
Reduction to Sulfide (S2-). Incorporation into
homocysteine. Methyl cycle: Methionine
and SAM. Transsulfuration: Cystein. Cystein + Glutamate +
Glycin = Gluthathione; redoxbuffer.
Met4 TF master regulator of the sulfur assimilation pathway(s).
In turn inhibited by Cystein. Ljungdahl & Daignan-Fornier 2O12
Uptake
Channels Symporters Antiporters Pumps Diffusion
Facilitated diffusion Along gradient only (electrochemical)
Multiple affinities
General vs. specific channels
Consumption maintains gradient
E.g.; Hxt1-11, Hxt13, Hxt15-17, Gal2 Agp1, Agp2, Gap1,… Fps1 (gated)
Secondary active transport Import/export linked to co-factor
Uses combined gradient
Pumps may create initial gradient
E.g.; Nha1 (H+) Pho84 (H+), Pho89 (Na+)
Primary active transport Import/export consumes ATP
E.g.; Pma1/Pma2 Ena1
Passive diffusion Along gradient only
E.g., Weak acids (protonated) Ethanol
Substrate-specific transmembrane transporter activity [301 gene products] alcohol [5 gene products] amide [1 gene products] amine [46 gene products] carbohydrate [30 gene products] coenzyme A [1 gene products] FAD [4 gene products] ion [166 gene products] macromolecule [20 gene products] (ER/mitoch.) nicotinamide mononucleotide [1 gene products] nucleobase, nucleoside, nucleotide and nucleic acid [16 gene products] Oligopeptide [3 gene products] organic acid [59 gene products] (includes aa) substrate-specific channel activity [15 gene products] Thiamine [2 gene products] water [3 gene products]
The membrane potential
Pma1/2 Ena1-5
H+
H+
Na+
Na+
Nha1 Trk1/2
K+
K+
Li+
Li+
Na+ H+
Na+ H+
Li+
Pho84
H+ Pi
H+ Pi
Pho89
Na+ Pi
Na+ Pi Li+
Stl1
H+ Gly
H+ Gly
H+ driven nutrient uptake and detoxification Gradient generation & buffering
H+ independent backup systems
Cells invest ATP to extrude protons: >20% (possibly more than >50%) of cellular energy consumption.
pH gradient; electrochemical force
K+ influx balances the electrical potential; increased chemical gradient
Chemical gradient used for charge neutral symport and antiport
Problems in Alkaline environment – difficult/impossible to maintain a sufficient delta-pH: switch to alternative systems
Two coupled forms of cellular energy: ATP and the proton gradient (over PM, Vacuolar membrane and Inner mitochondrial membrane).
Metabolism driven uptake
AAP
aa
aa
Passive diffusion along the concentration gradient
Gradient is maintained steep by rapid depletion of intracellular metabolite, e.g. by sugar phosphoryaltion
Multiple permease affinities to optimise uptake
Repression of general permeases in presence of preferred substrates – minimising loss through leakage?
Mep1-3
NH4+
NH4+
Sul1/2
S
S
Gal2
Gal
Gal
Hxt1-7
Glc
Glc
Hxk
GlcP
Gal1
GalP
Environmental Cues
The central dogma
Growth
Metabolism
Nutrients
Cell Division
Cycle G1/S
G2/M
Transcription/Translation
Uptake Gradients
• Sixteen in yeast • Basic structural element in the nucleus • Chromatin structure in interphase cells
heterogeneous – reversible modifications of DNA and
histones. – results in altered physical properties:
• local chromatin folding • three-dimensional organisation of the
genome
• Interacts with the stable structural elements:
– the nuclear envelope: • embedded SPB(s) • tethers for heterochromatin • tethers for ribosomal DNA
– the nuclear pores – the nucleolus
The Chromosomes
Llorente et al. 2000
DNA replication
Replication initiation Requires licensed origins (assembled in the absence of Cdk1 activity). Cdk1-Clb5/Cdk1-Clb6 phosphorylates multiple components to fire the licensed origins. Dissociation of key components prevent firing before licensing in the next G1 – prevents multiple cycles of DNA
replication in the same cell division cycle.
Enserink and Kolodner 2010
Transcription
Tata-box containing promters (20%)
Requires TBP; often SAGA
Highly regulated
”Stress” response genes
Tata-less promoters (80%)
Requires TBP; often TFIID
Nucleosome free promoters
Stable expression
”Housekeeping” genes
Components: UAS/URS
TFs Mediator
Core Promoter SAGA TFIID
Uffenbeck and Krebs 2006
Packing & organisation
Regulation:
Sequence default site
Chromatin remodellers
Swi/Snf
Rsc
ISWI
Histone modifications
HATs/HDACs
Transcription: Chromatin
Jansen and Verstrepen 2011
Cell Cycle Dependent Transcription
Consecutive waves of TF activity -> directionality in the Cell division cycle.
Enserink and Kolodner 2010
Cokus et al. 2006
Transcription: TF network
Topology from ChIP-Chip Network includes 115 TFs (102 dispensable)
Posttranscriptional regulation
Warringer et al. 2010
Splicing (rare in S. cerevisiae)
mRNA stability
Translation rate
Protein stability
Posttranslational regulation (signalling modifications)
Ubiquitin based signalling:
Degradation (via the proteasome)
Endocytosis (internalisation of receptors)
Finley et al. 2012
Environmental Cues
Signalling & Life decisions
Growth
Metabolism
Nutrients
Cell Division
Cycle G1/S
G2/M
Signal Transduction
Transcription/Translation
Stress Pheromone
Uptake Gradients Receptors
Redox
Nutrient signalling
Protein Kinase A (PKA) • Signal via cyclic AMP (cAMP) • Active during growth • Inhibits the general stress response (Msn2/Msn4) • Essential for cell cycle progression • Conserved to human
Snf3/Rgt2 • Sugar sensors • Sugar transporters with long c-terminal tails • Regulates hexose transporter expression • Similar system for nitrogen: Ssy1/Ptr3/Ssy5
Snf1 • Inactive on glucose (glucose repression) • Activates use of alternative carbon sources (inhibits Mig1) • Essential for growth on sucrose, ethanol,
galactose, etc. • Conserved to human (AMPK); implicated in
diabetes
Target of Rapamycin (TOR) • TORC1 signals abundant nitrogen (Tor1 or Tor2) • Regulates e.g. translation • Activation mechanism unknown • Inhibited by Rapamycin
• TORC2 is involved in cell polarity (Tor2)
• Essential • Conserved to human (mTOR)
MAPK signalling
• Conserved core of three kinases: MAPKKK -> MAPKK -> MAPK
• Stimuli and output related to cell
morphology
• Upstream components include cell polarity machinery (Cdc42, Rho1)
• MAPK shuttles between cytoplasm
and nucleus
• MAPK localises to promoters and take active part in transcriptional activation
• Conserved to human
Mating pathway (MAT) • Responds to pheromone; triggers cell cycle arrest and partner search (schmooing) Pseudohyphal differentiation (PHD) • Triggered by nutrient depletion, triggers foraging behaviour High osmolarity (glycerol; HOG) • Responds to dehydration (hyper-osmotic stress)
Protein kinase C (PKC) • Responds to cell wall stress; e.g. hypo-osmolarity, heat, calcofluor
Regulating the cell division cycle
Six cyclin dependent kinases: Cdc28
necessary and sufficient to drive the cell cycle
Cln1-3 (G1/S) Clb1-6
Pho85
Involved in the earlier phases of the cell cycle
Pcl1-2, 9 (G1/S) Phosphatate metabolism
Kin28 Ssn3 Ctk1 Bur1
Cak1: a distant, highly diverged CDK family member, Cak1, which is involved in activation of several CDKs.
Cell Division
Cycle G1/S
G2/M
Sic1 Far1
Cdc28/ Cln
Cdc28/ Clb
Swe1 Mih1
Hog Mating
Growth?
Environmental Cues
Cell structure
Growth
Metabolism
Nutrients
Cell Division
Cycle G1/S
G2/M Polarity/Morphology
Signal Transduction
Transcription/Translation
Stress Pheromone
Uptake Gradients Receptors
Redox
Cell Cycle and Morphology
G1: Oval cells G1/S: Polarisation S: Polarised growth; bud G2: Isotropic growth; bud M: Neck polarisation;
cytokinesis
Howell and Lew 2012
Moseley and Goode 2006
Cell structures
Larabell and Nugent 2010
Nucleus
Nucleolus
Mitochondria Vacuoles
Lipid bodies
• Harbours the genetic material • Ensures that the genetic material is
properly: – expressed – duplicated – repaired – segregated
• And that RNA is properly: – processed – exported
The Nucleus
Utsugi et al. 1999
The Mitochondria
Oxelmark et al. 2000
• The cellular power plant – Harbours the respiratory chain – Respiration is optional (mutants viable)
• Essential for anabolism – TCA cycle provides building blocks – Mitochondrial biogenesis mutants lethal
• Dynamic: – Responsive to carbon source – Regulated fusion and fission – Active migration to daughter cells
• Mitochondrial DNA – 28 ORFs (6575) – 17 verified ORFs (5036) – 2 uncharacterised ORFs (761) – 9 dubious ORFs (778) – 24 tRNA (275) – 2 rRNA (25) – 1 ncRNA (14) – Total: 55 features; 85.779 bp
Peroxisomes
• No β-oxidation in yeast mitochondria • β-oxidation occurs exclusively in
peroxisomes • Peroxisomes are massively induced during
growth on fatty acids as sole carbon source
• β-oxidation 1. CoA activation 2. Oxidation 3. Hydration/Dehydrogenation 4. Thiolytical cleavage Produces Acetyl-CoA -> Mitochondria for oxidation
• The glyoxylate cycle – Converts 2 Acetyl-CoA into Succinate – Replenish the TCA cycle – Essential for growth on oleate or 2C
substrates (Ethanol; Acetate) Kohlwein et al. 2013
The course
Date Theme Topic PMID 13/4 Lecture I Introduction
Yeast as a model system N/A
20/4 Lecture II Overview of yeast cell biology N/A
27/4 Transcription Transcriptional regulation Chromatin dynamics
22084422 21646431
4/5 Quality control RNA Degradation The ubiquitin-proteasome system
22785621 23028185
11/5 Cellular building blocks
Amino acid, nucleotide, and phosphate metabolism Metabolism & Regulation of Glycerolipids
22419079 22345606
18/5 Nutritional control
Nutritional Control of Growth and Development Target of Rapamycin (TOR)
22964838 22174183
1/6 The life cycle Sporulation & Mating 22084423 20066086
merge with 18/5
8/6 The cell division cycle
Cdk1-controlled targets and processes Mitotic Exit
20465793 23212898
merge with 15/6
15/6 Morphology Morphogenesis and the Cell Cycle Cell Polarization and Cytokinesis
22219508 22701052
22/6 Signalling Response to hyperosmotic stress Regulation of Cell Wall Biogenesis
23028184 22174182
29/6 Organelles Mitochondrial assembly Lipid Droplets and Peroxisomes
23212899 23275493
6/7 Nuclear function
The Yeast Nuclear Pore Complex Structure and function in the nucleus
22419078 22964839
merge with 13/7
13/7 Conclusion Summary, discussion & evaluation N/A
The course
Date Theme Topic PMID 13/4 Lecture I Introduction
Yeast as a model system N/A
20/4 Lecture II Overview of yeast cell biology N/A
27/4 Transcription Transcriptional regulation Chromatin dynamics
22084422 21646431
Rodrigo, Jorin Benjamin, Kora
4/5 Quality control RNA Degradation The ubiquitin-proteasome system
22785621 23028185
Tom, André Olga, Cornelus
11/5 Cellular building blocks
Amino acid, nucleotide, and phosphate metabolism Metabolism & Regulation of Glycerolipids
22419079 22345606
Alexander, Avan
18/5 Nutritional control & The life cycle
Nutritional Control of Growth and Development Sporulation & Mating
22964838 22084423 20066086
Pascal, Melanie Laura, Mareike
15/6 The cell division cycle
Cdk1-controlled targets and processes Morphogenesis and the Cell Cycle
20465793 22219508
Mareike, Laura Sarah, Deborah
22/6 Signalling Response to hyperosmotic stress Regulation of Cell Wall Biogenesis
23028184 22174182
Jorin, Rodrigo Olga, Cornelius
29/6 Organelles Mitochondrial assembly Lipid Droplets and Peroxisomes
23212899 23275493
Kora, Benjamin
13/7 Nuclear function Conclusion
The Yeast Nuclear Pore Complex Structure and function in the nucleus Summary, discussion & evaluation
22419078 22964839
N/A
