Cpc2, a fission yeast homologue of mammalian RACK1 protein, interacts with Ran1 (Pat1) kinase To regulate cell cycle progression and meiotic development

The Schizosaccharomyces pombe ran1/pat1 gene regulates the transition between mitosis and meiosis. Inactivation of Ran1 (Pat1) kinase is necessary and sufficient for cells to exit the cell cycle and undergo meiosis. The yeast two-hybrid interaction trap was used to identify protein partners for Ran1/Pat1. Here we report the identification of one of these, Cpc2. Cpc2 encodes a homologue of RACK1, a WD protein with homology to the beta subunit of heterotrimeric G proteins. RACK1 is a highly conserved protein, although its function remains undefined. In mammalian cells, RACK1 physically associates with some signal transduction proteins, including Src and protein kinase C. Fission yeast cells containing a cpc2 null allele are viable but cell cycle delayed. cpc2Delta cells fail to accumulate in G(1) when starved of nitrogen. This leads to defects in conjugation and meiosis. Copurification studies show that although Cpc2 and Ran1 (Pat1) physically associate, Cpc2 does not alter Ran1 (Pat1) kinase activity in vitro. Using a Ran1 (Pat1) fusion to green fluorescent protein, we show that localization of the kinase is impaired in cpc2Delta cells. Thus, in parallel with the proposed role of RACK1 in mammalian cells, fission yeast cpc2 may function as an anchoring protein for Ran1 (Pat1) kinase. All defects associated with loss of cpc2 are reversed in cells expressing mammalian RACK1, demonstrating that the fission yeast and mammalian gene products are indeed functional homologues.     

Receptor for Activated C-Kinase (RACK1) Homolog Cpc2 Facilitates the General Amino Acid Control Response through Gcn2 Kinase in Fission Yeast

General amino acid control (GAAC) is crucial for sensing and adaptation to nutrient availability. Amino acid starvation activates protein kinase Gcn2, which plays a central role in the GAAC response by phosphorylating the α-subunit of eukaryotic initiation factor 2 (eIF2α), leading to the translational switch to stimulate selective expression of stress-responsive genes. We report here that in fission yeast Schizosaccharomyces pombe, Cpc2, a homolog of mammalian receptor for activated C-kinase (RACK1), is important for the GAAC response. Deletion of S. pombe cpc2 impairs the amino acid starvation-induced phosphorylation of eIF2α and the expression of amino acid biosynthesis genes, thereby rendering cells severely sensitive to amino acid limitation. Unlike the Saccharomyces cerevisiae Cpc2 ortholog, which normally suppresses the GAAC response, our findings suggest that S. pombe Cpc2 promotes the GAAC response. We also found that S. pombe Cpc2 is required for starvation-induced Gcn2 autophosphorylation, which is essential for Gcn2 function. These results indicate that S. pombe Cpc2 facilitates the GAAC response through the regulation of Gcn2 activation and provide a novel insight for the regulatory function of RACK1 on Gcn2-mediated GAAC response.     

Fission yeast receptor of activated C kinase (RACK1) ortholog Cpc2 regulates mitotic commitment through Wee1 kinase

In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G(1)/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G(2)/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G(1)/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.     

Ste50p is involved in regulating filamentous growth in the yeast Saccharomyces cerevisiae and associates with Ste11p

STE50 is required to sustain pheromone-induced signal transduction in?S. cerevisiae. Here we report that Ste50p is involved in regulating pseudohyphal development. Both of these processes are also dependent on Ste11p. Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p. Overexpression of STE11 also suppresses the mating defects of ste50 mutants. We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various conditions. A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated, as well as from normally growing cells. Formation of the Ste50p-Ste11p complex does not require G_α, G_β, Ste20p or Ste5p. Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and occurs in the absence of Ste50p. We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation programmes: mating and filamentous growth.     

Rkp1/Cpc2, a fission yeast RACK1 homolog, is involved in actin cytoskeleton organization through protein kinase C, Pck2, signaling

The Rkp1/Cpc2, a fission yeast RACK1 homolog, interacted with Pck2, one of the known PKC homologs, in vivo and in vitro. The rkp1-deletion mutants (Deltarkp1) are elongated and the pck2-deletion mutant (Deltapck2) showed abnormal morphology. The double-deletion mutant (Deltarkp1Deltapck2) showed more aberrant cell shapes and was sensitive to high salt concentration. Both Deltarkp1 and Deltapck2 cells were sensitive to latrunculin B (Lat B) which inhibits actin polymerization. The cells expressing the human RACK1 homolog complemented the latrunculin B sensitivity of Deltarkp1 indicating that human RACK1 is a functional homolog of Rkp1/Cpc2. We propose that Rkp1/Cpc2 may function as a receptor for Pck2 in the regulation of actin cytoskeleton organization during cell wall synthesis and morphogenesis of Schizosaccharomyces pombe.     

A Role for a Protease in Morphogenic Responses during Yeast Cell Fusion

Cell fusion during yeast mating provides a model for signaling-controlled changes at the cell surface. We identified the AXL1 gene in a screen for genes required for cell fusion in both mating types during mating. AXL1 is a pheromone-inducible gene required for axial bud site selection in haploid yeast and for proteolytic maturation of a-factor. Two other bud site selection genes, RSR1, encoding a small GTPase, and BUD3, were also required for efficient cell fusion. Based on double mutant analysis, AXL1 in a MATα strain acted genetically in the same pathway with FUS2, a fusion-dedicated gene. Electron microscopy of axl1, rsr1, and fus2 prezygotes revealed similar defects in nuclear migration, vesicle accumulation, cell wall degradation, and membrane fusion during cell fusion. The axl1 and rsr1 mutants exhibited defects in pheromone-induced morphogenesis. AXL1 protease function was required in MATα strains for fusion during mating. The ability of the Rsr1p GTPase to cycle was required for efficient cell fusion, as it is for bud site selection. During conjugation, vegetative functions may be redeployed under the control of pheromone signaling for mating purposes. Since Rsr1p has been reported to physically associate with Cdc24p and Bem1p components of the pheromone response pathway, we suggest that the bud site selection genes Rsr1p and Axl1p may act to mediate pheromone control of Fus2p-based fusion events during mating.     

Ste18p Is a Positive Control Element in the Mating Process of Candida albicans

Heterotrimeric G proteins are an important class of eukaryotic signaling molecules that have been identified as central elements in the pheromone response pathways of many fungi. In the fungal pathogen Candida albicans, the STE18 gene (ORF19.6551.1) encodes a potential γ subunit of a heterotrimeric G protein; this protein contains the C-terminal CAAX box characteristic of γ subunits and has sequence similarity to γ subunits implicated in the mating pathways of a variety of fungi. Disruption of this gene was shown to cause sterility of MTLa mating cells and to block pheromone-induced gene expression and shmoo formation; deletion of just the CAAX box residues is sufficient to inactivate Ste18 function in the mating process. Intriguingly, ectopic expression behind the strong ACT1 promoter of either the Gα or the Gβ subunit of the heterotrimeric G protein is able to suppress the mating defect caused by deletion of the Gγ subunit and restore both pheromone-induced gene expression and morphology changes.     

Interaction between a negative regulator (Msa2/Nrd1) and a positive regulator (Cpc2) of sexual differentiation in Schizosaccharomyces pombe

The sexual differentiation of Schizosaccharomyces pombe is controlled by many cellular components which have not been fully characterized. We isolated a gene called msa2 as a multi-copy suppressor of a sporulation abnormal mutant (sam1). Msa2p is identical with Nrd1p which has been characterized as a factor that blocks the onset of sexual differentiation. The yeast two-hybrid system was used to identify Cpc2p, a fission yeast homolog of the RACK1 protein, that interacted with Msa2p/Nrd1p. We confirmed that Msa2p/Nrd1p interacted with Cpc2p in S. pombe cells. An epistatic analysis of msa2/nrd1 and cpc2 suggests that Msa2p/Nrd1p was an upstream regulator for Cpc2p. A localization analysis of Cpc2p and Msa2p/Nrd1p indicates that both proteins were predominantly localized in the cytoplasm. The interaction of negative regulator Msa2p/Nrd1p with positive regulator Cpc2p suggests a new regulatory circuit in the sexual differentiation of S. pombe.     

Phosphorylation of the pheromone-responsive Gβ protein of Saccharomyces cerevisiae does not affect its mating-specific signaling function

The pheromone-responsive G_β subunit of Saccharomyces cerevisiae (encoded by STE4) is rapidly phosphorylated at multiple sites when yeast cells are exposed to mating pheromone. It has been shown that a mutant form of Ste4 lacking residues 310–346, ste4Δ310–346, cannot be phosphorylated, and that its expression leads to defects in recovery from pheromone stimulation. Based on these observations, it was proposed that phosphorylation of Ste4 is associated with an adaptive response to mating pheromone. In this study we used site-directed mutagenesis to create two phosphorylation null (Pho^?) alleles of STE4: ste4-T320?A/S335A and ste4-T322 A/S335A and ste4-T322A/S335A. When expressed in yeast, these mutant forms of Ste4 remained unphosphorylated upon pheromone stimulation. The elimination of Ste4 phosphorylation has no discernible effect on either signaling or adaptation. In addition, disruption of the FUS3 gene, which encodes a pheromone-specific MAP kinase, leads to partial loss of pheromone-induced Ste4 phosphorylation. Two-hybrid analysis suggests that the ste4Δ310–346 deletion mutant is impaired in its interaction with Gpa1, the pheromone-responsive G_α of yeast, whereas the Ste4-T320A/S335A mutant has normal affinity for Gpa1. Taken together, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhibited by the 310–346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1.     

Ste50p is involved in regulating filamentous growth in the yeast Saccharomyces cerevisiae and associates with Ste11p

STE50 is required to sustain pheromone-induced signal transduction in S. cerevisiae. Here we report that Ste50p is involved in regulating pseudohyphal development. Both of these processes are also dependent on Ste11p. Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p. Overexpression of STE11 also suppresses the mating defects of ste50 mutants. We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various conditions. A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated, as well as from normally growing cells. Formation of the Ste50p-Ste11p complex does not require G_α, G_β, Ste20p or Ste5p. Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and occurs in the absence of Ste50p. We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation programmes: mating and filamentous growth. Received: 20 February 1998 / Accepted: 17 March 1998     

Ste50p sustains mating pheromone-induced signal transduction in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the hetero-trimeric G protein transduces the mating pheromone signal from a cell-surface receptor. Free Gβγ then activates a mitogen-activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal-transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone-induced signal which leads cells to hormone-induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone-induced signal transduction, and overexpression of STE50 intensifies the pheromone-induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the α-pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two-hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a new component, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP kinase complex in S. cerevisiae.     

Saccharomyces cerevisiae Mpt5p interacts with Sst2p and plays roles in pheromone sensitivity and recovery from pheromone arrest.

SST2 plays an important role in the sensitivity of yeast cells to pheromone and in recovery from pheromone-induced G1 arrest. Recently, a family of Sst2p homologs that act as GTPase-activating proteins (GAPs) for G alpha subunits has been identified. We have identified an interaction between Sst2p and the previously identified Mpt5p by using the two-hybrid system. Loss of Mpt5p function resulted in a temperature-sensitive growth phenotype, an increase in pheromone sensitivity, and a defect in recovery from pheromone-induced G1 arrest, although the effects on pheromone response and recovery were mild in comparison to those of sst2 mutants. Overexpression of either Sst2p or Mpt5p promoted recovery from G1 arrest. Promotion of recovery by overexpression of Mpt5p required Sst2p, but the effect of overexpression of Sst2p was only partially dependent on Mpt5p. Mpt5p was also found to interact with the mitogen-activated protein kinase homologs Fus3p and Kss1p, and an mpt5 mutation was able to suppress the pheromone arrest and mating defects of a fus3 mutant. Because either mpt5 or cln3 mutations suppressed the fus3 phenotypes, interactions of Mpt5p with the G1 cyclins and Cdc28p were tested. An interaction between Mpt5p and Cdc28p was detected. We discuss these results with respect to a model in which Sst2p plays a role in pheromone sensitivity and recovery that acts through Mpt5p in addition to a role as a G alpha GAP suggested by the analysis of the Sst2p homologs.     

The sixth transmembrane region of a pheromone G-protein coupled receptor,Map3, is implicated in discrimination of closely related pheromones in Schizosaccharomyces pombe

Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycete fungi including yeasts, mating between cells of opposite mating type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focused on the stringency of pheromone receptors. The fission yeast Schizosaccharomyces pombe has two mating types, Plus (P) and Minus (M). Here, we investigated the stringency of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs between S. pombe and the closely related species Schizosaccharomyces octosporus, which showed that SoMam2 (Mam2 of S. octosporus) is partially functional in S. pombe, whereas SoMap3 (Map3 of S. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the activation by the pheromone. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are key residues important for discrimination of closely related M-factors. Thus, the differences in these two GPCRs might reflect the significantly distinct stringency/flexibility of their respective pheromone/receptor systems; nevertheless, species-specific pheromone recognition remains incomplete.