Signal transduction by a nondissociable heterotrimeric yeast G protein

Abstract

Many signal transduction pathways involve heterotrimeric G proteins. The accepted model for activation of heterotrimeric G proteins states that the protein dissociates to the free G(alpha) (GTP)-bound subunit and free G(betagamma) dimer. On GTP hydrolysis, G(alpha) (GDP) then reassociates with G(betagamma) [Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649]. We reexamined this hypothesis, by using the mating G protein of the yeast Saccharomyces cerevisiae encoded by the genes GPA1, STE4, and STE18. In the absence of mating pheromone, the G(alpha) (Gpa1) subunit represses the mating pathway. On activation by binding of pheromone to a serpentine receptor, the G(betagamma) (Ste4, Ste18) dimer transmits the signal to a mitogen-activated protein kinase cascade, leading to gene activation, arrest in the G(1) stage of the cell cycle, production of shmoos (mating projections), and cell fusion. We found that a Ste4-Gpa1 fusion protein transmitted the pheromone signal and activated the mating pathway as effectively as when Ste4 (G(beta)) and Gpa1 (G(alpha)) were coexpressed as separate proteins. Hence, dissociation of this G protein is not required for its activation. Rather, a conformational change in the heterotrimeric complex is likely to be involved in signal transduction.

Figure 1

(a) The STE4-GPA1 fusion constructs, showing relevant restriction sites, and the sequences of the linkers inserted. (b) The known sites of α-β interactions and of α-effector (Upper) or β-effector interactions (Lower) do not overlap. The diagram shows the homology between mammalian G proteins and yeast Gpa1 and Ste4. Mammalian G_α interaction domains are drawn as in ref. . Additional data are from refs. and and references cited within. (c) Crystallographic structure of the G protein heterotrimer α_i1(GDP)β₁γ₂, showing the proximity of the carboxy terminus of the β₁ subunit to the amino terminus of the α_i1 subunit. Coordinates were from file 1GP2.pdb, and the figure was prepared with insightii biosym.

Figure 2

(a) Growth of transformants on glucose (Left) and galactose (Right) medium. On each plate, the SK1006 (ste4) derivatives are on the left and the SK1007 (ste4 gpa1) derivatives are on the right. Plasmids are, from top to bottom: pGT5 (vector), pG1501 (GPA1), pGT-STE4–1 (STE4), pSTE4-GPA1-a (STE4-GPA1-a), pSTE4-GPA1-b (STE4-GPA1-b). (b) Mating of transformants on glucose (Left) and galactose (Right) medium. Cells were patched onto a lawn of tester strain NKY102 (MATα ade8) on an SD-glucose or SD-galactose plate lacking uracil (10), and incubated at 30°C. The mixture was replicated to an SD-glucose plate with no supplements and incubated at 30°C, to select for diploids.

Figure 3

Halo assay for sensitivity to α-factor on galactose plates. Cells were pregrown for several hours in galactose medium, plated in a lawn of top agar on selective SD galactose plates, and 1 μg of α-factor (Sugen) was spotted on the solidified top agar, in 4-μl drops. The plates were incubated for 40 h at 30°C. Strains are (clockwise from top left): βwt carrying vector pGT5 (wild-type), and SK1007 (ste4Δ gpa1Δ) with pGT5, pSTE4-GPA1-a (STE4-GPA1-a), or pSTE4-GPA1-b (STE4-GPA1-b).

Figure 4

(a) Western blot showing detection by anti-Ste4 antibody of Ste4-Gpa1 fusions from cells grown in glucose (−) or galactose (+) medium. 40 μg total protein were loaded per lane. Transformants of strain SK1007 (ste4 gpa1), carry the following plasmids: 1, pGT5 (vector); 2, pGT-STE4–1 (STE4); 3, pSTE4-GPA1-a (STE4-GPA1-a); 4, pSTE4-GPA1-b (STE4-GPA1-b); 5, pG1501 (GPA1) and pGT-STE4–2 (STE4). Lanes 6–8 show specific blocking of the anti-Ste4 reactive bands with recombinant Ste4 protein. Lane 6, pSTE4-GPA1-a (STE4-GPA1-a). Lane 7, pSTE4-GPA1-b (STE4-GPA1-b). Lane 8, pG1501 (GPA1) and pGT-STE4–2 (STE4). A number of nonspecific bands are evident, the most prominent migrating at approximately 29, 47, 56, 65, and 110 kDa. The band at 47 kDa seems to be nonspecific, as it appears in the empty vector control, it appears in the absence of galactose, its strength actually decreases in samples grown in galactose, and it is blocked by recombinant Ste4 protein to a much lesser extent (<2-fold) than the bona fide anti-Ste4 reactive products (blocked >10-fold). (b) Western blot by using anti-Gpa1 antibody. Strains as in a. Both myristoylated (lower band) and nonmyristoylated (upper band) forms of Gpa1 are evident. The expected molecular weight of nonmyristoylated Gpa1 is 54 kDa. The band at 45 kDa is nonspecific.

Figure 5

(a) Induction of anti-Ste4 reactive protein on incubation in galactose. Cells were grown in selective glucose medium and transferred at time 0 h to galactose medium. Aliquots were removed at the indicated times and lysates prepared. Then, 20 μg total protein were loaded per lane. Nonspecific bands are as in Fig. 4a. Strain backgrounds: βwt (wild-type) or SK1007 (ste4 gpa1). Plasmids: pGT5 (vector), pSTE4-GPA1-b (STE4-GPA1-b), pG1501 (GPA1), pGT-STE4–2 (STE4). (b) The amounts of anti-Ste4 reactive protein were quantitated by using the National Institutes of Health image program. Panels were scanned from the same exposure. (c) Induction of mating ability on incubation in galactose. A total of 4 × 10⁷ exponentially growing washed cells were incubated at 30°C for the times indicated with a large excess of washed tester NKY102 (MATα ade8) cells. After washing, appropriate dilutions were plated on selective plates to score diploids, tester, and plasmid-carrying strain. Mating efficiencies represent the number of diploid cells per SK1007(pSTE4-GPA1-b) or SK1007 (pG1501 + pGT-STE4–2) cell.