Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast.

Mammalian phosducins are known to bind G protein betagamma subunits in vitro, and are postulated to regulate their signaling function in vivo. Here we describe two homologues of phosducin in yeast, called PLP1 and PLP2. Both gene products were cloned, expressed, and purified as glutathione S-transferase fusions. Of the two isoforms, Plp1 bound most preferentially to Gbetagamma. Binding was enhanced by pheromone stimulation and by the addition of GTPgammaS, conditions that favor dissociation of Gbetagamma from Galpha. Gene disruption mutants and gene overexpression plasmids were prepared and analyzed for changes in signaling and nonsignaling phenotypes. Haploid spore products bearing the plp2Delta mutant failed to grow, suggesting that PLP2 is an essential gene. Cell viability was not restored by a mutation in STE7 that blocks signaling downstream of the G protein. Haploid products bearing the plp1Delta mutant were viable and exhibited a 6-7% increase in pheromone-mediated gene induction. Cells overexpressing PLP1 or PLP2 exhibited a 70-80% decrease in gene induction but no change in pheromone-mediated growth arrest. These data indicate that phosducin can selectively regulate early signaling events following pheromone stimulation and has an essential role in cell growth independent of its regulatory role in cell signaling.

A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins.

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family alpha subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Galpha subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed mutagenesis of alphao and alphai1 was done. G184S alphao and G183S alphai1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G --> S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alphao and alphai1 to compete with fluorescein isothiocyanate-alphao binding to glutathione S-transferase-RGS4. The G --> S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in alphao and alphai1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.

Regulation of G protein signalling in yeast.

A common property of cell signaling systems is the ability to adapt to chronic stimulation. A genetic analysis of receptor/G protein signaling in yeast has led to the identification of a new class of regulators of G protein signaling (RGS proteins), as well as to new insights about the regulatory role of G protein modifications (myristoylation, palmitoylation). Similar modes of regulation are now known to exist in humans. These discoveries fill some important gaps in our understanding of signal transduction, and provide an instructive example of how model organisms, like yeast, can provide new insights relevant to signal regulation in higher eukaryotes.

Selective uncoupling of RGS action by a single point mutation in the G protein alpha-subunit.

Heterotrimeric G proteins function as molecular relays, shuttling between cell surface receptors and intracellular effectors that propagate a signal. G protein signaling is governed by the rates of GTP binding (catalyzed by the receptor) and GTP hydrolysis. RGS proteins (regulators of G protein signaling) were identified as potent negative regulators of G protein signaling pathways in simple eukaryotes and are now known to act as GTPase-activating proteins (GAPs) for G protein alpha-subunits in vitro. It is not known, however, if Galpha GAP activity is responsible for the regulatory action of RGS proteins in vivo. We describe here a Galpha mutant in yeast (gpa1(sst)) that phenotypically mimics the loss of its cognate RGS protein (SST2). The gpa1(sst) mutant is resistant to an activated allele of SST2 in vivo and is unresponsive to RGS GAP activity in vitro. The analogous mutation in a mammalian Gqalpha is also resistant to RGS action in transfected cells. These mutants demonstrate that RGS proteins act through Galpha and that RGS-GAP activity is responsible for their desensitizing activity in cells. The Galphasst mutant will be useful for uncoupling RGS-mediated regulation from other modes of signal regulation in whole cells and animals.

The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers.

The Broad-Complex (BR-C) is essential for metamorphosis in Drosophila melanogaster. This locus is coextensive with the 2B5 ecdysone-responsive early puff and is necessary for puffing and transcription of many subsequently activated late genes in the developing salivary gland. Mapping of 31 cDNA clones indicates that approximately 100 kb of the genome is devoted to the synthesis of many BR-C RNAs. Sequence analyses of these cDNA clones show that the BR-C encodes a family of related proteins characterized by a common core amino-terminal domain fused to alternate carboxy domains each containing a pair of zinc fingers. Most proteins also contain domains rich in distinctive amino acids located between the common core and zinc finger regions. BR-C mutant alleles resulting from chromosomal rearrangements at 2B5 are associated with deletions of 5'-untranslated sequences, separation of the core coding domain from the downstream zinc finger domains, or a P element insertional disruption of a zinc finger coding sequence. We infer that the BR-C directly regulates late gene expression by specifying the synthesis of a family of proteins with DNA binding potential.