Screening for novel essential genes of Saccharomyces cerevisiae involved in protein secretion

Abstract

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.

A Novel High-Throughput Screen Reveals Yeast Genes That Increase Secretion of Heterologous Proteins

“…Yeast gene libraries that allow either overexpression or deletion of each of the approximately 6,000 yeast gene products are available and could allow secretion analysis on a genomewide scale that could prove beneficial for discovering multiple gene products that improve the secretory processing of proteins (4,43). However, identification of improved yeast secretion strains requires quantitative measurement of secreted pro-teins by methods such as Western blotting or enzyme-linked immunosorbent assay (ELISA) that tend to be prohibitive on a genome-wide scale.…”

Characterization of GPI14/YJR013w mutation that induces the cell wall integrity signalling pathway and results in increased protein production in Saccharomyces cerevisiae

“…In a screen for new essential yeast genes involved in protein secretion (Davydenko et al , 2004), a strain was uncovered in which enhanced secretion of Hsp150p was observed. The screen used the strains in which a doxycyclin‐regulated tetO 7 ‐CYC1 promoter is placed in front of the novel essential genes, which ensures basal expression in the absence of doxycyclin, while increasing amounts of the drug should lower the gene expression.…”

“…In order to search for new genes potentially involved in protein secretion and intracellular transport we screened a set of novel essential yeast genes for their role in protein secretion (Davydenko et al , 2004). In the strains used for the screen, the expression of the novel essential genes is under a doxycyclin‐regulated promoter, which results in downregulation of the gene expression in the presence of doxycyclin.…”

Why are essential genes essential? - The essentiality of Saccharomyces genes

“…The challenge for essential genes is that death is the only “phenotype” in the absence of any essential gene. The generation of the tetracycline-regulatable alleles of essential genes 58 greatly facilitates large scale studies on essential genes, such as those involved in secretion 59, mitochondria 52 or G-protein signaling 60. These studies often identify the non-essential, or secondary functions of the essential genes, which may help further identify the essential functions.…”