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Ursula Heins Marroquín

Journal Club22.03.17

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Saccharomyces cerevisiae (Budding yeast)

unicellular eukaryotic organism small genome comprising about 6000 genes, which has been completely sequenced

molecular genetic tools

Cost effective model

Easy to manipulate

High throughput screening

31% of yeast gene has a human homolog

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Introduction

S. cerevisiae

Aged yeast cells- Mitochondria fragmentation- Increase ROS- Loss of mitochondrial DNA

AGEING

Chronological life span

Replicative life spanS. cerevisiae

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Introduction

S. cerevisiae

Aged yeast cells- Mitochondria fragmentation- Increase ROS- Loss of mitochondrial DNA

AGEING

Chronological life span

Replicative life spanS. cerevisiae

Number of times an individual cell divides

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Introduction: Classical RLS

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Method: Mother enrichment program (MEP)

Mother Cell

Cell division

Daughter Cell

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Method: Mother enrichment program (MEP)

Daughter Cell

the transcription factor Ace2

- estr

adiol Sequester in the cytoplasm

+ estradiol Transported into the nucleus

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Method: Mother enrichment program (MEP)

Cre-EBD78

SUMO-conjugating enzyme

Activator of the anaphase-promoting complex (APC)

Arrest in M-phase

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Results: Mitochondrial dysfunction in aged yeast

Calcofluor staining of the bud scar

TOM70-GFPMitochondrial marker

Nr. Cell division

Characterization of age-associated mitochondrial phenotypes

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Results: Decreased of mitochondrial function

DiOC6 : ΔΨ-dependent mitochondrial fluorescent dye

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Mitochondrial structure in young cells

250 genes1)

2) Overexpression of the 250 genes

MEP strain expressing Tom70-GFP

Delayed to the onset of altered mitochondrial morphology in aged cells

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Results: VAM1 and VPH2

VAM1 VPH2

A Catalytic subunit of V-ATPase

V-ATPase assemblyV-ATPase : Acidification of the vacuole

Overexpression of VMA suppressed mitochondrial dysfunction in 93 %

Overexpression of VPH2 suppressed mitochondrial dysfunction in 77 %

Suppression by VMA1 or VPH2 overexpression required V-ATPase activity

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Results: Vacuolar and mitochondrial function are linked

• Overexpression of VMA1 and VPH2 increase both media and maximal replicative lifespan.

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How vpH affects mitochondrial function?

vacuolar protein degradation?

Deletion of PEP4

No alteration of mitochondrial structure

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How vpH affects mitochondrial function?

Import of vacuolar metabolite?

Overexpression of vacuolar importers

+ConcA

ATV1 Neutral amino acid transporterPrevent mitochondrial dysfunction

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Results: ATV1 prevents mitochondrial fragmentation

Overexpression of ATV1

• Prevent alteration of mitochondrial structure• No change in vacuolar acidification

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Results: ATV1 as aging modulator

Deletion of ATV1

Development of age-induced mitochondrial dysfunction

Overexpression = Extending RLS

Deletion = Shortening of RLS

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Conclusion

o Yeast is a good model for agingo Link between vacuolar pH and mitochondrial functiono Link between metabolite transport and agingo Vacuole functionalion as aging modulatoro Reestablishment of acidification in the vacuole in the

daughter cells

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Thank you