The Saccharomyces cerevislae GPA1, STE4, and STE18 genes encode products homologous to mammalian G-protein a, ,, and y subunits, respectively. All three genes function in the transduction of the signal generated by mating pheromone in haploid cells. To characterize more completely the role of these genes in mating, we have conditionally overexpressed GPAI, STE4, and STE18, using the galactose-inducible GAL] promoter. Overexpression of STE4 alone, or STE4 together with STEJ8, generated a response in haploid cells suggestive of pheromone signal transduction: arrest in G1 of the cell cycle, formation of cellular projections, and induction of the pheromone-inducible transcript FUSI 25-to 70-fold. High-level STE18 expression alone had none of these effects, nor did overexpression of STE4 in a MATa!a diploid. However, STE18 was essential for the response, since overexpression of STE4 was unable to activate a response in a stel8 null strain. GPAI hyperexpression suppressed the phenotype of STE4 overexpression. In addition, cells that overexpressed GPAI were more resistant to pheromone and recovered more quickly from pheromone than did wild-type cells, which suggests that GPAI may function in an adaptation response to pheromone.G proteins function as molecular transducers of extracellular stimuli, coupling these stimuli to intracellular responses (reviewed in references 12 and 48). G-protein-mediated signal transduction has now been described in a number of eucaryotes as phylogenetically diverse as fungi and vertebrates. Elements from several of these systems have been biochemically characterized in some detail, and they appear to conserve many aspects of structure and function. In the best-understood pathways, in mammalian cells, G proteins transduce a signal generated by stimulation of a membrane receptor of the rhodopsin/lB-adrenergic family (10, 36). These G proteins, consisting of a, 1, and y subunits, exist as inactive heterotrimers with GDP bound to the a subunits. Receptor stimulation leads to exchange of GDP for GTP, activating the G protein, which then dissociates into a and P-y subunits. Both a and fly subunits are then capable of interacting with diverse intracellular effectors in different systems (reviewed in reference 37), including adenylate cyclase, cyclic GMP phosphodiesterase, and phospholipase A2. Hydrolysis of GTP to GDP inactivates the a subunit, which then reassociates with free P-y to return to the inactive aIly configuration.

No abstract

In a screen designed to identify novel mutations in the mating response pathway of Saccharomyces cerevisiae, we isolated conditional alleles of NMT1, the gene encoding N-myristoyl transferase. Genetic data indicate that Nmtl deficiency results in the activation of the pheromone response at the level of Gpal, the ot subunit of the pheromone-responsive G protein. We show that Gpal is myristoylated by Nmtl, and without this normally stable modification, Gpal is unable to inhibit pheromone signaling. This loss of Gpal function is probably not the result of improper subcellular localization. Unlike the mammalian God proteins cq and ~o, nonmyristoylated Gpal is able to associate with membranes. In addition to Gpal, our data indicate that Nmtl myristoylates other proteins essential to vegetative growth.

The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli I-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed ,I-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3-to 12-fold, in good agreeement with previous mRNA analyses. When MATa cells were arrested in Gl with a-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL) promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.The regulation of DNA repair and recombination in the procaryote Escherichia coli has been extensively studied. One result of these studies has been the elucidation of the inducible SOS repair pathway, a component of which, the recA gene produpt, appears to control a number of genes involved in both the repair of DNA lesions and general DNA recombination (for a review, see reference 38). The regulation of such activities in eucaryotic systems has not yet been thoroughly elucidated. In Saccharomyces cerevisiae, mutations in three separate epistasis groups have been demonstrated to cause sensitivity to DNA-damaging agents such as UV light, X-rays, and chemical mutagens (12,14). However, each of these groups has a different phenotypic response to different kinds of damage, leading to the hypothesis that each group is involved in a separate repair pathway (13,25). Some mutations in one of these epistasis groups, the RAD50-57 group of genes, have been shown to be not only sensitive to the effects of ionizing radiation, but also to block meiotic recombination and to reduce some types of mitotic recombination (16,27). For this reason, the repair mediated by the RAD50-57 pathway has been postulated to be recombinational repair. Mutations in RAD54 and RAD52, the genes whose regulation we describe here, have been shown to be blocked in double-strand break repair (6,20,28). Additionally, rad52 mutations block significant levels of recombination in meiosis and yield inviable spores (15, 16). Although rad54 mutations previously had been reported to have little if any effect on meiosis, deletion mutations of the RAD54 gene have recently been isolated and shown to decrease bot...

The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNAdamaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not effect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damageinducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae.The yeast Saccharomyces cerevisiae offers an attractive model for the study of DNA repair in eucaryotic organisms. Extensive genetic analyses have established that mutations in many genes which lead to increased sensitivity to DNAdamaging agents can be ordered into three epistasis groups, the RAD3, RAD6, and RAD52 groups (3,5,9,21,24). The three groups are thought to represent separate repair systems, with the RAD3 group responsible for excision repair, the RAD6 group for an error-prone pathway, and the RAD52 group for a repair activity involving recombination (reviewed in references 7, 8, and 12). Phenotypes of representative mutants from each group support such a conclusion. In particular, several RADS2 group mutants, including rad54 mutants, show reduced sporulation and spore viability (10; E. Dowling, Ph.D. Thesis, University of California, Berkeley, 1982) and are unable to carry out homothallic mating type switching, an event which is presumed to be lethal in such mutants since rad52 HO and rad54 HO spores die (27; J. Game, personal communication). Additionally, RAD52 group mutants are very sensitive to ionizing radiation, which results in double-strand breaks that are thought to require genetic recombination for repair (10,22).In Escherichia coli, many DNA repair activities are coordinately regulated by the lexAlrecA regulatory network, in which the lexA protein represses transcription of many DNA repair genes by binding to their operators and recA, activated by DNA damage (or other stress), causes proteolysis of lexA and enhanced transcription of these genes, a process known as the SOS response (reviewed in reference 32 have been shown to be damage inducible, including members of two epistasis groups (4, 25). At least two of these genes, RAD2 and RAD54, are induced by several different agents, despite the fact that rad2 and radS4 mutants are only slightly sensitive to some of these agent...