Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.
Genetic analysis of cell-cell signaling in Saccharomyces cerevisiae has led to the identification of a novel factor, known as Sst2p, that promotes recovery after pheromone-induced growth arrest (R. K. Chan and C. A. Otte, Mol. Cell. Biol. 2:11-20, 1982). Loss-of-function mutations lead to increased pheromone sensitivity, but this phenotype is partially suppressed by overexpression of the G protein ␣ subunit gene (GPA1). Suppression is allele specific, however, suggesting that there is direct interaction between the two gene products. To test this model directly, we isolated and characterized several dominant gain-of-function mutants of SST2. These mutations block the normal pheromone response, including a loss of pheromone-stimulated gene transcription, cell cycle growth arrest, and G protein myristoylation. Although the SST2 mutations confer a pheromoneresistant phenotype, they do not prevent downstream activation by overexpression of G (STE4), a constitutively active G mutation (STE4 Hpl ), or a disruption of GPA1. None of the SST2 alleles affects the expression or stability of G␣. These results point to the G protein ␣ subunit as being the direct target of Sst2p action and underscore the importance of this novel desensitization factor in G-protein-mediated signaling.The yeast Saccharomyces cerevisiae is a unicellular eukaryote that can grow vegetatively either as a diploid or as one of two haploid cell types, known as the mating types a and ␣. Haploid cells secrete specific peptide pheromones (a and ␣ mating factors) that act on cells of the opposite type to promote cell fusion, leading to the formation of an a/␣ diploid (28, 46).Pheromone signaling is mediated through receptors of the seven-transmembrane-segment class (other examples include the -adrenergic receptor and rhodopsin) and is coupled to downstream events via a guanine nucleotide-binding regulatory protein (G protein) (28). Agonist binding to these receptors promotes exchange of GDP for GTP on the G-protein ␣ subunit and dissociation of G␣ from the G protein  and ␥ subunits (4, 6, 11). In yeast cells, it is the /␥ moiety that activates downstream signaling events leading to mating, which include alterations in gene transcription, morphological and cytoskeletal changes, and growth arrest in the G 1 phase of the cell cycle. Indeed, overexpression of the  subunit or loss of the ␣ subunit leads to mating even in the absence of pheromone or receptor (13,17,25,36,39,53).A property of signal-response systems in general, and of G-protein-coupled receptors in particular, is that prolonged stimulation often results in a loss of responsiveness over time (desensitization) (23). The molecular basis for this phenomenon has been extensively characterized for vertebrate G-protein-coupled receptors (2, 32) and has also been described for S. cerevisiae (3, 8-10, 12, 16, 19, 27, 33, 37, 40, 41, 47). Despite the profound physiological and morphological changes that yeast cells undergo in preparation for mating, cells that fail to mate become desensitized to the...
3-Methylcrotonyl-coenzyme A carboxylase (MCCase) is a mitochondrial biotin-containing enzyme whose metabolic function is not well understood in plants. In soybean (Glycine max) seedlings the organ-specific and developmentally induced changes in MCCase expression are regulated by mechanisms that control the accumulation of MCCase mRNA and the activity of the enzyme. During soybean cotyledon development, when seed-storage proteins are degraded, leucine (Leu) accumulation peaks transiently at 8 d after planting. The coincidence between peak MCCase expression and the decline in Leu content provides correlative evidence that MCCase is involved in the mitochondrial catabolism of Leu. Direct evidence for this conclusion was obtained from radiotracer metabolic studies using extracts from isolated mitochondria. These experiments traced the metabolic fate of [U-14 C]Leu and NaH 14 CO 3 , the latter of which was incorporated into methylglutaconyl-coenzyme A (CoA) via MCCase. These studies directly demonstrate that plant mitochondria can catabolize Leu via the following scheme: Leu 3 ␣-ketoisocaproate 3 isovaleryl-CoA 3 3-methylcrotonyl-CoA 3 3-methylglutaconylCoA 3 3-hydroxy-3-methylglutaryl-CoA 3 acetoacetate ؉ acetylCoA. These findings demonstrate for the first time, to our knowledge, that the enzymes responsible for Leu catabolism are present in plant mitochondria. We conclude that a primary metabolic role of MCCase in plants is the catabolism of Leu.
Initiation of the mating process in yeast Saccharomyces cerevisiae requires the action of secreted pheromones and G protein-coupled receptors. As in other eukaryotes, the yeast G protein ␣ subunit undergoes N-myristoylation (GPA1 gene product, Gpa1p). This modification appears to be essential for function, since a myristoylation site mutation exhibits the null phenotype in vivo (gpa1 G2A). Here we examine how myristoylation affects Gpa1p activity in vitro. We show that the G2A mutant of Gpa1p, when fused with glutathione Stransferase, can still form a complex with the G protein ␥ subunits. The complex is stabilized by GDP and is dissociated upon treatment with guanosine 5-O-(thiotriphosphate). In addition, there is no apparent difference in the relative binding affinity of G ␥ for mutant and wild-type Gpa1p. Using sucrose density gradient fractionation of cell membranes, Gpa1p associates normally with the plasma membrane whereas Gpa1pG2A is mislocalized to a microsomal membrane fraction. A portion of G ␥ is also mislocalized in these cells, as it is in a gpa1⌬ strain. In contrast, wild-type Gpa1p reaches the plasma membrane in cells that do not express G ␥ or cell surface receptors. These findings indicate that mislocalization of Gpa1p G2A is not caused by a redistribution of G ␥ , nor is it the result of any difference in G ␥ binding affinity. These data suggest that myristoylation is required for specific targeting of Gpa1p to the plasma membrane, where it is needed to interact with the receptor and to regulate the release of G ␥ .
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