1. Sporulation and aquaporins in Saccharomyces cerevisiae
Cecilia Geijer
Dept. of Cell and Molecular Biology
Göteborg University

2. Budding yeast Saccharomyces cerevisiae life cycle

3. Aquaporins
Aquaporins are channel proteins that enhance the permeability of cell membranes for water.
Orthodox aquaporins and aquaglyceroporins
Transport different small compounds: water, glycerol, urea, ammonia and CO2
Tetrameric membrane protein
Six transmembrane domains

4. Aqy1 is involved in sporulation
Northern Blot analysis of AQY1 in strain SK1. Low basal level of expression of AQY1 in vegetative cells. Upregulation of expression ~8 h after transfer to sporulation medium (1% KAc)
(The Saccharomyces cerevisiae aquaporin Aqy1 is involved in sporulation, Sidoux-Walter et al, PNAS 2004)

5. Expression of AQY1 in haploid cells
YPA = pre sporulation medium
KAc 1% = sporulation medium
YPA OD1 KAc 1h KAc 4h KAc 8h
1n 2n n 2n n 2n n n
AQY1
18S
AQY1 is also expressed in haploid cells experiencing nitrogen and glucose stress but in diploid cells expression is low until a specific time point during sporulation
Workpackage 1, 4

6. Haploid and diploid SK1 cultured in KAc 1% ±Glucose ±Nitrogen
SK1 1n
AQY1
18S
SK1 2n
AQY1
18S
AQY1 expression is repressed when glucose is added to medium. Only in KAc alone is the expression different between 1n and 2n cells.
Workpackage 1, 4

7. Haploid AQY1 expression in KAc (1%) + different carbon sources (2%)
KAc KAc Glucose KAc Raffinose KAc Galactose
1h h h h h h h h h h h h
AQY1
18S
KAc Ethanol KAc Glycerol
1h h h h h h
AQY1
18S
Fermentable carbon sources (Glucose and Raffinose) repress AQY1 expression.
Workpackage 1, 4

8. AQY1 promoter-lacZ plasmid
AQY1 promoter fused to the reporter gene lacZ on centromeric plasmid
Transform SK1 haploids and diploids, use β-galactosidase assay to measure promoter activity.
Transform knock out library, search for transcription factors regulating the AQY1 expression.

9. Future perspectives
Find transcription factors that regulate AQY1 expression using the AQY1 promoter-lacZ plasmid.
Elucidate the physiological role of Aqy1 in sporulation, why do spores need an aquaporin?
Order Aqy1 specific antibodies to study protein expression patterns.
Post-transcriptional regulation such as phosphorylation, internalisation and degradation?

10. Acknowledgements
The ”Hohmann group”, June 2006