Alteration of a yeast SH3 protein leads to conditional viability with defects in cytoskeletal and budding patterns.

Abstract

Mutations in genes necessary for survival in stationary phase were isolated to understand the ability of wild-type Saccharomyces cerevisiae to remain viable during prolonged periods of nutritional deprivation. Here we report results concerning one of these mutants, rvsl67, which shows reduced viability and abnormal cell morphology upon carbon and nitrogen starvation. The mutant exhibits the same response when cells are grown in high salt concentrations and other unfavorable growth conditions. The RVS167 gene product displays significant homology with the Rvs161 protein and contains a SH3 domain at the C-terminal end. Abnormal actin distribution is associated with the mutant phenotype. In addition, while the budding pattern of haploid strains remains axial in standard growth conditions, the budding pattern of diploid mutant strains is random. The gene RVS167 therefore could be implicated in cytoskeletal reorganization in response to environmental stresses and could act in the budding site selection mechanism.In Saccharomyces cerevisiae, nutrient availability coordinates cell growth and proliferation. In medium containing all essential growth elements, yeast cells proliferate and cell growth and division are held in balance by the necessity of a minimum cell size before beginning a new division cycle (36). If one of the essential elements, for example, carbon or nitrogen, becomes exhausted, yeast cells stop division in the nonbudding G1 phase of the cell cycle (81). The culture then is in stationary phase, and cells can remain in the living state for prolonged periods under conditions which are not propitious for growth. Indeed, in their natural environment, yeast cells spend only a small fraction of their existence in exponential growth, because of the limited availability of nutriments.The transition from exponentially growing cells to arrested stationary-phase cells following nutrient starvation is accompanied by a number of molecular and physiological changes. At the molecular level, accumulation of glycogen and trehalose (48) is observed. The global analysis of proteins synthesized under starvation conditions reveals a subset of proteins whose synthesis increases in nutritional deprivation (9, 32). In addition, synthesis of most of the proteins expressed during exponential phase ceases (9). Physiological changes concern a higher resistance to heat shock (60), to lytic enzymes (20), and to a large number of other environmental stresses.There are mainly two questions related to stationaryphase entry. The first question concerns the mechanisms implied in cell proliferation control in response to the nutrient starvation. Some of the molecular regulatory elements of cell proliferation control are now relatively well-known in S. cerevisiae. Thus a multitude of observations has been taken as evidence that the cyclic AMP (cAMP) pathway could be a signal-transmitting pathway for growth arrest following nutrient exhaustion (35,50,77 sponding to the nutritional environment. Genetic evidence for the existence of at least...

Mechanisms of apoptosis by c-Myc

“…The N and C-terminal regions of Bin1 are related to amphiphysin, a neuronal protein that is a paraneoplastic autoimmune antigen in breast and lung cancer (David et al, 1994;Dropcho, 1996). The same regions are also related to Rvs167 and Rvs161, two negative regulators of the cell cycle in yeast (Crouzet et al, 1991;Bauer et al, 1993) [Bin1 is related but not homologous since it cannot complement Rvs functions (Sakamuro et al, 1996)]. Amphiphysin and brainspeci®c splice forms of Bin1, also termed amphiphysin II or amphiphysin isoform, have been implicated in receptor-mediated endocytosis (David et al, 1996;Wigge et al, 1997;Owen et al, 1998).…”

Bin1 functionally interacts with Myc and inhibits cell proliferation via multiple mechanisms

“…First, Bin1 inhibits cell transformation by Myc or adenovirus E1A . Second, Bin1 is related to RVS167, a negative regulator of the cell cycle in yeast (Bauer et al, 1993). Third, although widely expressed in normal cells, Bin1 is poorly expressed or undetectable in *50% of carcinoma cell lines and primary breast carcinomas examined .…”

Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

“…Vertebrate actin-associated proteins containing SH3 domains include proteins with structural roles such as fodrin and nebulin (Merilainen et al, 1993;Wang et al, 1996), unconventional myosins (Bement et al, 1994;Stoffler et al, 1995;Goodson et al, 1996), kinases and associated molecules (Flynn et al, 1993;Turner and Miller, 1994;Hildebrand et al, 1996), and other actin-binding proteins such as cortactin (Wu and Parsons, 1993). In S. cerevisiae, SH3-containing proteins include the actin-associated proteins Abplp (Drubin et al, 1990), Slalp (Holtzman et al, 1993), and Rvs167p (Bauer et al, 1993), as well as Bemlp and the recently identified Boilp and Boi2p, which have roles in the maintenance of normal cellular and cytoskeletal morphology (Chenevert et al, 1992;Bender et al, 1996;Matsui et al, 1996). A number of yeast proteins implicated in cytoskeletal function, including Abplp, Slalp, Boilp, Boi2p, and Srv2p (Field et al, 1990), contain proline-rich regions which include amino acid sequences similar to known binding sites for SH3 domains.…”

“…Like Abplp and Srv2p, Slalp (synthetic lethal-ABP1), Sla2p, and Sac6p (yeast fimbrin) coimmunolocalize in the cortical actin cytoskeleton (Drubin et al, 1988;Adams et al, 1989; Lila, Yang, and Drubin, unpublished observations) and were identified in a screen for strong genetic enhancers of an ABP1 null mutation (synthetic lethals; Holtzman et al, 1993). Rvsl67p interacts with actin in the yeast two-hybrid system (Amberg et al, 1995), and has functions important for sporulation and viability under adverse growth conditions (Bauer et al, 1993). Rvs167p and Slalp also contain one and three SH3 domains, respectively, suggesting that they too may interact physically with other cytoskeletally associated proteins.…”