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 approximately 600 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 PKA/RAS 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.

Fig. 1.

Quantification of protein expression. (A Left) Increasing amounts (arbitrary units) of purified Pga3p-HA-His-8-Suc2-His4C were loaded onto 6.25% SDS/PAGE and visualized by Western blotting with anti-HA antibody. (A Right) Quantified band intensities versus amount of protein loaded (r² = 0.997). (B) Relative expression levels for 553 cloned S. cerevisiae proteins. The dashed line indicates an expression level of ≈0.5 mg/liter as estimated from GFP-fusions (see Results and Discussion).

Fig. 2.

Phenotypic consequences of membrane protein overexpression. (A) Growth curves in synthetic defined medium (Upper) and under oxidative stress (Lower) for the reference strain transformed with empty vector (wt) and strains overexpressing Erp1p and Ycl045cp. (B) Distribution of phenotypes (LSC) during no stress (open bars) and stress (filled bars). Negative LSC values indicate reduced fitness. (C) Phenotypes (LPI values) for the top 20 most stress tolerant (P < 0.001) overexpression strains.

Fig. 3.

Correlation between overexpression levels and degree of growth defects. Average growth defect (LSC) of membrane protein overexpression strains were divided into equally sized bins according to level of protein expression (I, <14; II, 14–34; III, 34–70; IV, 70–120; V, >120 units relative expression level). Asterisks indicate significant (P < 0.001) deviation from the mean growth defect of all investigated strains.

Fig. 4.

Hierarchical clustering of overexpression phenotypes recorded in synthetic defined medium (basal conditions; LSC values) and different stress conditions (NaCl, caffeine, and paraquat; LPI values). Green indicates a reduced growth rate, and red indicates an increased growth rate relative to the reference STY50 strain.