Screening for novel essential genes of Saccharomyces cerevisiae involved in protein secretion.
We describe here a screening procedure devised for searching new genes involved in protein secretion in Saccharomyces cerevisiae. The screening procedure takes advantage of yeast strains constructed within the EUROFAN project, in which the promoters of the novel essential genes were replaced by the doxycycline-regulated tetO(7)-CYC1 promoter. This promoter is active in normal growth medium but results in downregulation of the gene in the presence of doxycycline. The yeast cells were grown in the presence or absence of doxycycline, and both the growth and secretion of the heat shock protein, Hsp150p, into the culture medium were determined. In seven strains there was a specific effect on protein secretion. In a strain in which the RPN5 gene was downregulated, the level of secreted Hsp150p was increased compared to the control culture. When RER2 was downregulated, cells secreted Hsp150p that was not of the mature size. In five strains, secretion was more severely reduced than cell growth. One of these downregulated genes, YGL098w, was recently reported to encode an ER-located t-SNARE, USE1. Four of the genes detected, NOG2, NOP15, RRP40 and SDA1, encode proteins involved in ribosome assembly, suggesting a possible new signalling pathway between ribosome biogenesis and production of secreted proteins. The results obtained here indicate that the present screen could be successfully used in larger scale to identify novel secretion-related genes.
Davydenko
Svetlana G
SG
VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Finland.
Juselius
Jukka K
JK
Munder
Thomas
T
Bogengruber
Edith
E
Jäntti
Jussi
J
Keränen
Sirkka
S
D015153:Blotting, Western; D004318:Doxycycline / Q000494:pharmacology; D020043:Genes, Essential / Q000187:drug effects / Q000235:genetics*; D005800:Genes, Fungal / Q000187:drug effects / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
D006023:Glycoproteins; C074229:HSP150 protein, S cerevisiae; D006360:Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; D004318:Doxycycline