Transcript Sporulation and aquaporins in Saccharomyces cerevisiae

Sporulation and aquaporins in
Saccharomyces cerevisiae
Cecilia Geijer
Dept. 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, SidouxWalter et al, PNAS 2004)
Expression of AQY1 in haploid
cells
YPA = pre
sporulation
medium
KAc 1% = sporulation
medium
YPA OD1 KAc 1h
KAc 4h
KAc 8h
1n
1n
1n
2n
1n
2n
2n
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
Haploid and diploid SK1 cultured
dip
cultured
1%,
Hapand
and
dipSK1
SK1
culturedin
inKAc
KAc
1%,++in Hap
KAc
1%
±Glucose
±Nitrogen
Glucose
Glucose2%,
2%,+-(NH4)2SO4
+-(NH4)2SO4(5g/l)
(5g/l)
SK1
SK11n
1n
SK1
1n
KAc
KAc
KAc
KAcGlucose
Glucose
1h
1h 4h
4h 8h
8h 11 44
KAc
KAc(NH4)2SO4
(NH4)2SO4 KAc
KAcGlu
Glu(NH4)2SO4
(NH4)2SO4
88 11
44 88
11 44
88
AQY1
AQY1
AQY1
18S
18S
18S
SK1
SK1
2n
SK12n
2n
KAc
KAc
11
KAc
KAcGlucose
Glucose
44
88 11 44
KAc
KAc(NH4)2SO4
(NH4)2SO4 KAc
KAcGlu
Glu(NH4)2SO4
(NH4)2SO4
88 11
44 88
11 44
88
AQY1
AQY1
AQY1
18S
18S
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
Haploid AQY1 expression in KAc (1%)
+ different carbon sources (2%)
KAc
1h
KAc Glucose
4h
8h
1h
4h
KAc Raffinose
8h
1h
4h
KAc Galactose
8h
1h
4h
8h
AQY1
18S
KAc Ethanol
1h
4h
8h
KAc Glycerol
1h
4h
8h
AQY1
18S
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