G-PROTEIN MEDIATED SIGNAL TRANSDUCTION IN SACCHAROMYCES CEREVISIAE

ABSTRACT V Yeast mating type locus gene alpha2 (MATa2), Yeast G protein complementing gene ( YGC1) and minichromosome maintenance gene (M C M 1) have been identified by isolation of plasmids that are able to complement or suppress a gpa1::HIS3 mutation. MATa2 and YGC1 rescue both MATa and M A Ta-gpa1::HIS3 haploid cell types whereas MCM1 complements only MATa gpal::HIS3 cell type. MATa2 is known to be a general repressor and a determinant of both haploid and diploid cell types. MCM1 is known to be a general transcriptional activator. YGC1 has not been characterised, hence its function or mode of action is not previously known. G protein alpha subunit (GPA1) is a yeast G protein a subunit that negatively controls the budding yeast pheromone signal transduction pathway. Disruption of GPA1 results in constitutive arrest of the signal pathway that leads to cell cycle arrest at the early G1 phase of the cell cycle. Both Southern analysis and sequencing showed that MATa2, YGC1, MCM 1 have no homology to GPA1. Disruption of MATa2 (that is mata2::URA3) leads to constitutive arrest of the cell cycle at the G 1 phase. MATa2 also has no sequence homology to G P A 2 , the other G protein a subunit in yeast, known to be involved in cAMP pathway in yeast. It has been shown here that MATa2 rescues gpal::HIS3 cells even in single copy, centromere plasmids. Mating efficiency is largely reduced in cells kept alive with MATa2. MATa2 does not have the pheromone response elements (PREs) common to the STE genes (whose disruption leads to insensitivity to mating factors). The plasmid TGC was also constructed and used in creation of the yeast haploid strains LG1 and LG2. This was an attempt to screen a mammalian cDNA library for possible analogs of GPA1. These strains were used to isolate two mammalian analogs that complement the gpa1::HIS3 mutation. The results indicate that MATa2, YGC1 and MCM1 are components or modulate component(s) of the signaling pathway. It also showed that MATa2 is even a more potent negative regulator of the signaling pathway than GPA1, since overexpression is not a prerequisite for negatively regulating the pathway. MATa2 does not belong to the G protein family since it has no GTP/GDP binding and/or exchange domains.