Transcript Yeast spores

Mechanisms of Dormancy and
Germination of The Baker’s
Yeast S. cerevisiae Spore
Ivan Pirkov
Dept. of Cell and Molecular Biology
Göteborg University
Aim 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 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 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
Microarray on Germinating Yeast Spores (WP2)
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 (WP2)
The experimental outline
• Diploid cells were sporulated in 1% KAc
• The spores were left resting at 4C 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 timefashion, 0, 4, 8, 16 min… etc.
• Resting spores were used as reference sample.
• The experiments were done in three independent replicates
Microarray on Germinating Yeast Spores (WP2)
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 (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 (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 (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
Future 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!