Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.

Abstract

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.

Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

“…Their analysis of the localization of prenylation-and thioacylationdefective mutants of Ste18p led them to conclude that lipid modifications on Ste18p are required for plasma membrane localization of G ␤␥ (Hirschman and Jenness, 1999). However, these findings are inconsistent with previous data showing that nonprenylated Ste18p functionally couples to the plasma membrane-bound receptor Ste2p in in vitro assays (Grishin et al, 1994a). This suggests that nonlipidated Ste18p is localized at the plasma membrane at least transiently and suggests that the signaling defect caused by lack of thioacylation is associated with defective interactions with downstream components of the pathway or maintenance of G ␤␥ on the plasma membrane.…”

“…Therefore, we sought to determine whether Ste18p is thioacylated and how this modification impacts Ste18p function. Attempts to isolate epitope-tagged Ste18p expressed in yeast were unsuccessful because of low levels of protein expression (Grishin et al, 1994a). However, we were able to detect expression of GFP fused to full-length Ste18p using a linker of 18 amino acids, including an HA epitope, between GFP and the N terminus of Ste18p.…”

“…Yeast cells (DY150) carrying pVT-HA-Ste18 (Finegold et al, 1990), pAG3STE4 (Grishin et al, 1994a), and wild-type or mutated pCMGpa1-HA were grown to midlogarithmic phase (A 600 , 0.5-0.7) in sucrose (2% wt/vol) synthetic medium at 30°C. Gpa1p and Ste4p were induced by adding CuSO 4 (final concentration, 100 M) and galactose (final concentration, 2% wt/vol), respectively.…”

“…GFP vectors (pVTU-GFP-HA-Ste18p WT, C106S, or C107S) were transformed into yeast strain SWY518 along with pAG3STE4 (Grishin et al, 1994a). Early logarithmic cells (A 600 , 0.3-0.5) were treated with a final concentration of 25 M cerulenin (Sigma, St. Louis, MO) and 2% (final concentration) galactose for 105 min.…”

“…GFP vectors (pVTU-GFP-HA-Ste18p WT, C106S, or C107S) were transformed into yeast strain SWY518 along with pAG3STE4 (Grishin et al, 1994a After radiolabeling, yeast were collected by centrifugation, washed in sterile water, and suspended in lysis buffer (50 mM Tris, pH 8, 10% glycerol, 0.1 M NaCl, 11 mM EDTA, 1 mM EGTA, and protease inhibitors [2.1 g/ml aprotinin, 1 mM benzamidine hydrochloride, 1 g/ml chymostatin, 8.2 g/ml leupeptin, 3.2 g/ml lima bean trypsin inhibitor, 1 mM Pefabloc SC (Boehringer Mannheim), and 1 g/ml pepstatin A]). SDS was added to a final concentration of 2%, and the cells were lysed by vortexing with 0.5-mm glass beads (Biospec Products, Bartlesville, OK), eight times for 1 min each.…”