Academic journal article Genetics

The Saccharomyces Cerevisiae 14-3-3 Proteins Are Required for the G^sub 1^/S Transition, Actin Cytoskeleton Organization and Cell Wall Integrity

Academic journal article Genetics

The Saccharomyces Cerevisiae 14-3-3 Proteins Are Required for the G^sub 1^/S Transition, Actin Cytoskeleton Organization and Cell Wall Integrity

Article excerpt

ABSTRACT

14-3-3 proteins are highly conserved polypeptides that participate in many biological processes by binding phosphorylated target proteins. The Saccharomyces cerevisiae BMH1 and BMH2 genes, whose concomitant deletion is lethal, encode two functionally redundant 14-3-3 isoforms. To gain insights into the essential function(s) shared by these proteins, we searched for high-dosage suppressors of the growth defects of temperature-sensitive bmh mutants. Both the protein kinase C1 (Pkc1) and its upstream regulators Wsc2 and Mid2 were found to act as high dosage suppressors of bmh mutants' temperature sensitivity, indicating a functional interaction between 14-3-3 and Pkc1. Consistent with a role of 14-3-3 proteins in Pkc1-dependent cellular processes, shift to the restrictive temperature of bmh mutants severely impaired initiation of DNA replication, polarization of the actin cytoskeleton, and budding, as well as cell wall integrity. Because Pkc1 acts in concert with the Swi4-Swi6 (SBF) transcriptional activator to control all these processes, the defective G^sub 1^/S transition of bmh mutants might be linked to impaired SBF activity. Indeed, the levels of the G^sub 1^ cyclin CLN2 transcripts, which are positively regulated by SBF, were dramatically reduced in bmh mutants. Remarkably, budding and DNA replication defects of bmh mutants were suppressed by CLN2 expression from an SBF-independent promoter, suggesting that 14-3-3 proteins might contribute to regulating the late G^sub 1^ transcriptional program.

THE 14-3-3 proteins are a large family of highly conserved, ubiquitously expressed acidic polypeptides of 28-33 kDa found in all eukaryotes. At least 7 isoforms are present in mammals and up to 15 isoforms are present in plants, while 2 isoforms have been identified in yeast, Drosophila melanogaster, and Caenorhabdiiis ekgans (reviewed in HERMEKING 2003; DOUGHERTY and MORRISON 2004). They form homo- and heterodimers able to bind protein ligands that are usually phosphorylated on serine or threonine residues of consensus binding motifs QONES et al. 1995; MUSLIN et al. 1996; YAFFE et al. 1997; CHAUDHRI et al. 2003). By inducing conformational changes or steric hindrance in protein ligands, 14-3-3 proteins can activate/repress their enzymatic activity, prevent their degradation, modulate their localization, and/or facilitate/inhibit their modifications and interactions (reviewed in HERMEKING 2003; DOUGHERTY and MORRISON 2004). Targets of 14-3-3 family members are found in all subcellular compartments and include transcription factors, biosynthetic enzymes, cytoskeletal proteins, signaling molecules, checkpoint and apoptosis factors, and tumor suppressors. This plethora of interacting proteins allows 14-3-3 to play important roles in a wide range of regulatory processes such as cell cycle control, mitogenic signal transduction, and apoptotic cell death and to be implicated in carcinogenesis and some human diseases (reviewed in DOUGHERTY and MORRISON 2004). However, because multiple 14-3-3 isoforms are present in mammals and 14-3-3 proteins have several binding targets, the mechanisms underlying 14-3-3 functions are not fully understood.

The two Saccharomyces cerevisiae members of the 14-3-3 family, sharing 93% amino acid identity, are encoded by the BMH1 and BMH2 genes. While single bmh1Δ and bmh2Δ mutants do not show detectable growth defects compared to wild type, the bmh1 bmh2 double disruption is lethal in most laboratory strains (VAN HEUSDEN et al. 1992, 1995; GELPERIN et al. 1995; ROBERTS et al. 1997).

Although their essential functions are not well understood, budding yeast Bmh proteins appear to be involved in many cellular processes. For example, they modulate the activity of several transcription factors. In fact, loss-of-function mutations impairing the SIN4 or the RTG3 genes, encoding a global transcriptional regulator and a basic helix-loop-helix transcription factor, respectively, suppress the temperature-sensitive phenotype of a bmh1 bmh2 mutant (VAN HEUSDEN and STEENSMA 2001). …

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