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.

Cell division regulation by BIR1, a member of the inhibitor of apoptosis family in yeast.

The inhibitor of apoptosis (IAP) gene family comprises molecules that block the activity of pro-apoptotic caspase proteases. Paradoxically, yeasts contain IAP proteins but no caspases and no apoptotic program. To determine the function of these proteins in vivo, we disrupted the BIR1 gene, encoding the only known IAP in yeast Saccharomyces cerevisiae. Sporulation of heterozygous diploids yielded no viable mutant haploids, indicating that BIR1 is an essential gene. By flow cytometry, some heterozygous mutants were polyploid accumulating >4 N DNA content. These cells exhibited a 20-40% reduction in growth rate, which was rescued by plasmid-borne over-expression of BIR1 but not by its human counterpart, survivin. Deletion analysis revealed that the N-terminal domain of Bir1, containing the conserved baculovirus IAP repeat, was able to partially complement the cell growth defect caused by BIR1 deletion. Moreover, the full-length and truncated forms of Bir1 accelerated cell division in wild-type cells. Finally, BIR1 heterozygous mutants exhibited grossly altered cell morphology with misshapen or abnormally long buds connected to an unusually large mother cell. These findings identify a novel function of IAP proteins in the pleiotropic control of cell division, in addition to their role in the suppression of apoptosis.

Insertional inactivation of the Listeria monocytogenes cheYA operon abolishes response to oxygen gradients and reduces the number of flagella.

The nucleotide sequence of a region downstream of the Listeria monocytogenes flagellin gene, flaA, revealed two putative chemotaxis genes, cheY and cheA. These genes have been shown to be transcribed as a bicistronic unit. In this study Tn916 delta E mutagenesis was used to generate two mutants, PF10 and PF16, which contain transposon inserts in the promoter region of this operon. These mutants were motile in liquid, but had reduced flagellin expression and were unable to burrow or swarm on soft agar plates. Complementation of the single transposon-copy mutant PF16 with cloned cheY and cheA in trans partially restored microaerotaxis and swarming on soft agar. The complemented strain did not exhibit any increase in flagellin production. Both PF10 and PF16 appear deficient in their ability to attach to the mouse fibroblast cell line 3T3.