Genome-wide Responses toMitochondrial Dysfunction

Transcriptional changes due to disruption of mitochondrial function

Two different yeast strains:

S. cerevisiae lacking mtDNA ()….(petite).• Respiratory deficient, obligatory fermentative metabolism.

Wild-type S. cerevisiae (+). •Respiratory competent, partially oxidative metabolism.

Compared genome-wide expression with microarrays.

Compared with previous microarray experiment looking at genome-wide changes in expression in yeast cells undergoing diauxic shift.

overview

Aerobic metabolism:(oxidative metabolism)

Glycolysis TCA cycle oxidative phosphorylation

Anaerobic metabolism:(fermentation)

Glucose Acetyl CoA Ethanol

Diauxic shift:

Metabolic change as fermentable carbon source is used up from…

Glucose Fermentative (Glycolysis Ethanol)

Oxidative Metabolism(Ethanol TCA cycle)

to…

yeast carbon metabolism

c

UQUQ

1/2

O 2

H 2 O

II

III

IV

V

F 1

F o

NADH

NAD+

H+

H+

H+H+

ADP + Pi

ATP

= Nuclear Encoded

= Mitochondrial Encoded

succinate

fumarate

I II

Proteins typically encoded in mitochondrion

differential gene expression

Clusters A and B:Oxidative phosphorylationApparatus

in petite

No attempt by cells to upregulate oxidativemetabolism

in diauxic shift

Change to oxidativemetabolism in shift Cluster C:

Cytoplasmic ribosomalProteins

in diauxic shift

Shows slowing of growth rate upon change to oxidative metabolism

Clusters D and E:intermediary metabolism& small moleculetransport pathways

in and diauxic shift

Reflect metabolic changesto compensate for loss ofrespiration in petite cells.

metabolic remodelling

Metabolic pathways were altered in respiratory deficient cells

Intermediates of TCA cycle needed for synthesis of amino acids and nucleotides

Oxaloacetate (OAA) is not regenerated in petite () cells’ TCA cycle, so must be replenished another way.

Metabolic pathways altered:

OAA and Acetyl-CoA supply

Acetyl-CoA hydrolase

in enzymes involved in fluxand conversion of metabolites madeby fatty acid oxidation to TCA and glyoxylate cycle intermediates.

in nutrient and metabolite transporters.

in enzymes for reoxidation of NADH

petites reconfigure metabolism byrecruiting peroxisomal activities,small molecule transport systems and lipid, sugar and amino acid turnover to get more OAA and Acetyl-CoA.

effect of mitochondrial inhibitors

Different transcriptional responses due to different mitochondrial inhibitors.

Antimycin: effects are similar to petite cells.

CCCP: different response.

Oligomycin: different response.

Petite expression profile shows effects of loss of electron transport rather than loss of mitochondrial ATP synthesis.

peroxisome proliferation

In petites, peroxisome biogenesis is induced.

peroxisome targeted GFP used to trace peroxisomes in cells.

Antimycin also induced peroxisome biogenesis.

RTG genes

RTG genes important in retrograde regulation.

RTG1, 2 & 3 upregulate peroxisomal citrate synthase in petits.RTG genes increase expression of TCA cycle enzymes in petits.

Array profiling of cells with mutations in each RTG gene.

Cluster A:Genes induced in petits whose induction is dependent on RTG signalling pathway.

Cluster B:Genes requiring RTG genes for expression in petits.

Cluster C:Genes showing enhanced expression in petits without RTG genes.

Cluster D:Genes showing enhanced expression in petits independent of RTG genes, indicating novel retrograde regulation pathways.