Mechanisms of Dormancy and Mechanisms of Dormancy and Germination of The Baker’s Germination of The Baker’s Yeast Yeast S. cerevisiaeS. cerevisiae Spore Spore

Ivan PirkovIvan Pirkov

Dept. of Cell and Molecular BiologyDept. of Cell and Molecular BiologyGöteborg UniversityGöteborg University

Aim of This ProjectAim of This Project

• The aim of my project is to uncover how eukaryotic cells maintain dormant stages and how they are again reactivated

• We are using the ordinary baker’s yeast Saccharomyces cerevisiae as model organism

• S. cerevisiae produces a dormant stage in the form of the yeast spore

Spore GerminationSpore Germination

• Is most efficient when a readily fermentable carbon source is

present – e.g. glucose, fructose, galactose

– Only carbon source is essential for germination initiation

– Metabolism of the carbon source is necessary for germination, mere presence is

not enough

Herman and Rine (1997), EMBO J, 16:6171-6181

• RNA synthesis increases within minutes upon addition of

glucose and so does protein synthesis

Brengues et al (2002), JBC, 277:40505-40512

Spore GerminationSpore Germination

What is/are the environmental sensor(s) and the signal transduction pathway(s) involved in yeast spore germination?

• Spores of temperature-sensitive mutants of the Ras2/PKA-dependent signal-transduction pathway

(cdc25, ras2 or cyr1) are unable to germinate at the non-permissive temperature

• Overexpression of Ras2 protein increases germination rate several fold compared to control in

wild type cells, when spores are germinated on galactose

Herman and Rine (1997), EMBO J, 16:6171-6181

The purpose

• To investigate if the Ras2/PKA-pathway is involved in yeast spore germination

• To identify other metabolic pathways and specific genes that are associated with yeast spore germination if not Ras2/PKA

Microarray on Germinating Yeast Spores Microarray on Germinating Yeast Spores (WP2)(WP2)

Microarray on Germinating Yeast Spores Microarray on Germinating Yeast Spores (WP2)(WP2)

The experimental outline

• Diploid cells were sporulated in 1% KAc

• The spores were left resting at 4C for at least 14 days in 0.5% TritonX-100

solution

• Spores were then put to rich nutrient growth medium containing 2% glucose

• Samples for total RNA extraction were taken in a logarithmic time-fashion, 0, 4,

8, 16 min… etc.

• Resting spores were used as reference sample.

• The experiments were done in three independent replicates

The preliminary results for 0-32 min

Genes that are up-regulated or down-regulated after 4min

• Hexose metabolism (MIG1 PSK2 VID24)

• Protein folding and targeting to membrane (SSA1, SSA2, SSE1)

• Glycerol biosynthesis (RHR2, HOR2)

• Non significant

Genes that are up-regulated or down-regulated after 8min

• Hexose transport and metabolism (HXT1, MIG2, NRG1)

• Ribosome biogenesis and assembly (several genes)

• Transcription

• Meiosis (MSC1, ADY2)

• Pyruvate metabolism (PDC5, PGK1)

Microarray on Germinating Yeast Spores Microarray on Germinating Yeast Spores (WP2)(WP2)

The preliminary results for 0-32 min

Genes that are up-regulated or down-regulated after 16min

• Ribosome biogenesis and assembly

• mRNA processing

• Cell wall organization and biogenesis

• TCA-cycle, fatty acid oxidation, aerobic resp. (KGD1, POT1, SDH2)

• Sporulation (SPS100)

Genes that are up-regulated or down-regulated after 32min

• Ribosome biogenesis and assembly

• Amino acid and nucleotide metabolism

• Gluoneogenesis (HXK1, PCK1, FBP1)

• Fluid transport (AQY1, YFL054c)

Microarray on Germinating Yeast Spores Microarray on Germinating Yeast Spores (WP2)(WP2)

The Ras2/PKA-pathway in germination?

• No clear evidence but some indications

• RAS2 gene expression goes down from 0-32 min but not to the threshold-level (almost 2-fold after 32min)

• BCY1, the regulatory subunit of PKA is down-regulated >2-fold after 8 min and >4-fold after 16 min

• TFS1, a high copy suppressor of cdc25

– inhibits Ira2, which is an neg. regulator of Ras2 activity

– Is down-regulated >2 fold after 8 min and 4-fold after 16 and 32 min

– CDC25 expression was unchanged (0-32 min)

Microarray on Germinating Yeast Spores Microarray on Germinating Yeast Spores (WP2)(WP2)

The Ras2/PKA-pathway in germination?

• Adenylate cyclase (CYR1) expression is also down-regulated almost 2-fold after 8 min and >4-fold after 16 min

• Srv2, a subunit of adenylate cyclase complex

– N-terminus binds to Cyr1 and facilitates activation by Ras

– Is down-regulated >2-fold after 16 min

• Sdc25, homologous to Cdc25

– Expressed in poor nutrients and non-fermentable carbon sources

– Non-essential (in germination??)

– Is down-regulated >2-fold after 16 min

Microarray on Germinating Yeast Spores Microarray on Germinating Yeast Spores (WP2)(WP2)

Future PerspectivesFuture Perspectives

• Analyze the obtained microarray results in more detail (WP2)

• To do the microarray on the remaining time-samples (48-128 min) in order to follow the whole germination program (WP2)

• Compare gene expression, resting spore vs. growing vegetative cell to see which RNAs are present in resting spores and not in growing cells and vice versa (WP1, WP2)

• Screen the whole BY-strain deletion collection using the homozygote diploid strain, to identify mutants that are unable to germinate (WP2)

• Continue with the “long-term dormancy” experiment (WP1, WP2)

Thank You For Your Attention!