Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

Abstract

Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing Ϸ600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na ؉ ͞K ؉ homeostasis system constitute major downstream targets of the yeast PKA͞RAS pathway and point to a possible connection between the early secretory pathway and the cells' response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies.caffeine ͉ paraquat ͉ salt tolerance ͉ yeast

High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae

“…We observed that most yeast membrane protein-GFP fusions expressed to higher levels under the GAL1 promoter than under the TEF promoter. Because the majority of membrane proteins when constitutively expressed retard cell growth (15), an inducible promoter such as GAL1 may be a better choice for overproduction in yeast. Moreover, most membrane protein-GFP fusions expressed better in the pep4 deletion strain FGY217, suggesting that some overexpressed proteins may be susceptible to vacuolar proteolysis.…”

“…Previously, we estimated overexpression levels for 553 cloned S. cerevisiae polytopic membrane proteins by whole-cell Western blot analysis (15). From this collection, 20 proteins with expression levels ranging from 77 to 500 arbitrary units (the maximal expression recorded in the collection was 1,000 arbitrary units) were selected for the current study, the majority being transporters of various kinds [supporting information (SI) Table 1, top half].…”

Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N in –C out and N out –C in transmembrane helices

“…Preparation of whole-cell lysates was done as described in ref. 28. Whole-cell lysates were subjected to SDS/PAGE and Western blotting with an HA antiserum.…”

Functional importance of individual rRNA 2′-O-ribose methylations revealed by high-resolution phenotyping

“…We have earlier introduced a phenotyping approach for the quantitative extraction of these three physiological parameters from Saccharomyces cerevisiae deletion strains in environmental microculture arrays . The precision of the method allows not only the resolution of phenotypes into effects of different aspects of cellular physiology but also the quantification of marginal phenotypes not detectable by standard, more qualitative approaches Osterberg et al 2006).…”