sporulation and aquaporins in saccharomyces cerevisiae cecilia geijer dept. of cell and molecular...
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Sporulation and aquaporins in Saccharomyces cerevisiae
Cecilia GeijerDept. of Cell and Molecular Biology
Göteborg University
2006-10-16
Budding yeast Saccharomyces cerevisiae life cycle
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
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)
Expression of AQY1 in haploid cells
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
YPA = pre sporulation
medium
KAc 1% = sporulation medium
Workpackage 1, 4
KAc KAc Glucose KAc (NH4)2SO4 KAc Glu (NH4)2SO4
1h 4h 8h 1 4 8 1 4 8 1 4 8
KAc KAc Glucose KAc (NH4)2SO4 KAc Glu (NH4)2SO4
1 4 8 1 4 8 1 4 8 1 4 8
SK1 1n
SK1 2n
AQY1
AQY1
Hap and dip SK1 cultured in KAc 1%, +-Glucose 2%, +-(NH4)2SO4 (5g/l)
18S
18S
Haploid and diploid SK1 cultured in KAc 1% ±Glucose ±Nitrogen
SK1 1nKAc KAc Glucose KAc (NH4)2SO4 KAc Glu (NH4)2SO4
1h 4h 8h 1 4 8 1 4 8 1 4 8
KAc KAc Glucose KAc (NH4)2SO4 KAc Glu (NH4)2SO4
1 4 8 1 4 8 1 4 8 1 4 8
SK1 1n
SK1 2n
AQY1
AQY1
Hap and dip SK1 cultured in KAc 1%, +-Glucose 2%, +-(NH4)2SO4 (5g/l)
18S
18S
SK1 2n
AQY1
18S
18S
AQY1
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
KAc KAc Glucose KAc Raffinose KAc Galactose
1h 4h 8h 1h 4h 8h 1h 4h 8h 1h 4h 8h
KAc Ethanol KAc Glycerol
1h 4h 8h 1h 4h 8h
AQY1
Haploid AQY1 expression in KAc (1%) + different carbon sources (2%)
18S
18S
AQY1
Fermentable carbon sources (Glucose and Raffinose) repress AQY1 expression.
Workpackage 1, 4
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.
www.yeastract.com
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?
Acknowledgements
The ”Hohmann group”, June 2006