The 14-3-3 proteins constitute a family of conserved proteins present in all eukaryotic organisms so far investigated. These proteins have attracted interest because they are involved in important cellular processes such as signal transduction, cell-cycle control, apoptosis, stress response and malignant transformation and because at least 100 different binding partners for the 14-3-3 proteins have been reported. Although the exact function of 14-3-3 proteins is still unknown, they are known to (1) act as adaptor molecules stimulating protein-protein interactions, (2) regulate the subcellular localisation of proteins and (3) activate or inhibit enzymes. In this review, we discuss the role of the 14-3-3 proteins in three cellular processes: cell cycle control, signal transduction and apoptosis. These processes are regulated by the 14-3-3 proteins at multiple steps. The 14-3-3 proteins have an overall inhibitory effect on cell cycle progression and apoptosis, whereas in signal transduction they may act as stimulatory or inhibitory factors. This article contains supplementary material which may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/Suppmat/23/v23_10.936.

The 14‐3‐3 proteins comprise a family of highly conserved acidic proteins. Several activities have been ascribed to these proteins, including activation of tyrosine and tryptophan hydroxylases in the presence of calcium/calmodulin‐dependent protein kinase II, regulation of protein kinase C, phospholipase A2 activity, stimulation of exocytosis and activation of bacterial exoenzyme S (ExoS) during ADP‐ribosy‐lation of host proteins. In addition, a plant 14‐3‐3 protein is present in a G‐box DNA/protein‐binding complex. Previously, we isolated the BMH1 gene from Saccharomyces cerevisiae encoding a putative 14‐3‐3 protein. Using the polymerase chain reaction method, we have isolated a second yeast gene encoding a 14‐3‐3 protein (BMH2). While disruption of either BMH1 or BMH2 alone had little effect, it was impossible to obtain viable cells with both genes disrupted. The cDNA encoding a plant 14‐3‐3 protein under the control of the inducible GAL1 promoter complemented the double disruption. Transfer of the complemented double disruptant to a medium with glucose resulted in the appearance of a high percentage of large budded cells. After prolonged incubation, these cells became enlarged with irregular buds and chains of cells defective in cell‐cell separation became visible. These results suggest an essential role of the 14‐3‐3 proteins, possibly at a later stage of the yeast cell cycle.

Abstract14-3-3 proteins form a family of highly conserved proteins which are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. They interact with more than 200 different, mostly phosphorylated proteins. The molecular consequences of 14-3-3 binding are diverse: this binding may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization, to the interaction with other proteins or to shielding of binding sites. The binding partners, and hence the 14-3-3 proteins, are involved in almost every cellular process and 14-3-3 proteins have been linked to several diseases, such as cancer, Alzheimer's disease, the neurological Miller-Dieker and spinocerebellar ataxia type 1 diseases and bovine spongiform encephalopathy (BSE). The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both have two genes encoding 14-3-3 proteins, BMH1 and BMH2 and rad24 and rad25, respectively. In these yeasts, 14-3-3 proteins are essential in most laboratory strains. As in higher eukaryotes, yeast 14-3-3 proteins bind to numerous proteins involved in a variety of cellular processes. Recent genome-wide studies on yeast strains with impaired 14-3-3 function support the participation of 14-3-3 proteins in numerous yeast cellular processes. Given the high evolutionary conservation of the 14-3-3 proteins, the experimental accessibility and relative simplicity of yeasts make them excellent model organisms for elucidating the function of the 14-3-3 protein family.

We describe the identification and characterization of the BMH1 gene from the yeast Saccharomyces cerevisiae. The gene encodes a putative protein of 292 amino acids which is more than 50% identical with the bovine brain 14‐3‐3 protein and proteins isolated from sheep brain which are strong inhibitors of protein kinase C. Disruption mutants and strains with the BMH1 gene on multicopy plasmids have impaired growth on minimal medium with glucose as carbon source, i.e. a 30–50% increase in generation time. These observations suggest a regulatory function of the bmh1 protein. In contrast to strains with an intact or a disrupted BMH1 gene, strains with the BMH1 gene on multicopy plasmids hardly grew on media with acetate or glycerol as carbon source.

Cell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport proteins from the membrane. Arrestin-related trafficking proteins are important regulators of the endocytic pathway in yeast, facilitating selective ubiquitylation of target proteins by the E3 ubiquitin ligase, Rsp5. Specifically, Rod1 (Art4) has been reported to regulate the endocytosis of both the Hxt1, Hxt3, and Hxt6 glucose transporters and the Jen1 lactate transporter. Also, the AMP kinase homologue, Snf1, and 14-3-3 proteins have been shown to regulate Jen1 via Rod1. Here, we further characterized the role of Rod1, Snf1, and 14-3-3 in the signal transduction route involved in the endocytic regulation of the Hxt6 high affinity glucose transporter by showing that Snf1 interacts specifically with Rod1 and Rog3 (Art7), that the interaction between the Bmh2 and several arrestin-related trafficking proteins may be modulated by carbon source, and that both the 14-3-3 protein Bmh2 and the Snf1 regulatory domain interact with the arrestin-like domain containing the N-terminal half of Rod1 (amino acids 1-395). Finally, using both co-immunoprecipitation and bimolecular fluorescence complementation, we demonstrated the interaction of Rod1 with Hxt6 and showed that the localization of the Rod1-Hxt6 complex at the plasma membrane is affected by carbon source and is reduced upon overexpression of SNF1 and BMH2.An important component of the maintenance of cellular homeostasis and stress responses is the regulation of the composition of plasma membrane proteins responsible for the uptake and extrusion of nutrients and other nonpermeable small molecules. The general mechanisms controlling this regulatory process are highly conserved among eukaryotic organisms and involve both transcriptional regulation and modulation of the balance of secretion, recycling, and degradation of individual transporter proteins in response to changes in the extracellular environment (reviewed in Ref. 1). Regulated endocytosis, one important step in this process, has been well studied in both mammals and yeast, unveiling many mechanistic similarities that establish yeast as a relevant model system.In Saccharomyces cerevisiae, plasma membrane transporters that need to be down-regulated are ubiquitylated by the Rsp5 E3 ubiquitin ligase, endocytosed, sorted into multivesicular bodies in a process requiring the ESCRT machinery, and finally are delivered to the vacuole for degradation (2-5). Transporter ubiquitylation by the HECT family Rsp5 E3 ubiquitin ligase is mediated by one or more of several adaptor proteins that confer specificity and ensure the correct regulation of targeted proteins in response to changes in the extracellular environment (reviewed in Refs. 6). At least 18 Rsp5 adaptor proteins have been described, each having specificity for a subset of transport proteins and acting in response to specific stimuli.The molecular mechanisms governing th...