Sporulation and aquaporins in Saccharomyces cerevisiae

1
Sporulation and aquaporins in Saccharomyces
cerevisiae

• Cecilia Geijer
• Dept. of Cell and Molecular Biology
• Göteborg University
• 2006-10-16

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
1n 2n 1n 2n 1n 2n
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 4h 8h 1h 4h
8h 1h 4h 8h 1h
4h 8h
AQY1
18S
KAc Ethanol KAc Glycerol
1h 4h 8h 1h
4h 8h
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
  ß-galactosidase assay to measure promoter
  activity.
• Transform knock out library, search for
  transcription factors regulating the AQY1
  expression.

www.yeastract.com
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