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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.

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Sporulation and aquaporins in Saccharomyces cerevisiae

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  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 strainSK1. 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 β-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

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