General construction pattern of histamine H3-receptor antagonists: change of a paradigm

The RHO1 gene encodes a homolog of mammalian RhoA small G protein in the yeast Saccharomyces cerevisiae. We have shown that Bni1p is one of the downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction with profilin, an actin monomer-binding protein. A Bni1p-binding protein was affinity purified from the yeast cytosol fraction and was identified to be Tef1p/Tef2p, translation elongation factor 1alpha (EF1alpha). EF1alpha is an essential component of the protein synthetic machinery and also possesses the actin filament (F-actin)-binding and -bundling activities. EF1alpha bound to the 186 amino acids region of Bni1p, located between the FH1 domain, the proline-rich profilin-binding domain, and the FH2 domain, of which function is not known. The binding of Bni1p to EF1alpha inhibited its F-actin-binding and -bundling activities. The BNI1 gene deleted in the EF1alpha-binding region did not suppress the bni1 bnr1 mutation in which the actin organization was impaired. These results suggest that the Rho1p-Bni1p system regulates reorganization of the actin cytoskeleton through the interaction with both EF1alpha and profilin.     

Acetabular fractures: easier classification with a systematic approach

Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. We describe a new formin homology region, FH3. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. We replaced genomic fus1+ with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolished mating. FH3, but neither FH1 nor FH2, was required for Fus1 localization. An FH3 domain-GFP fusion protein localized to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affected cytokinesis. Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains.     

Rho1p-Bni1p-Spa2p interactions: implication in localization of Bni1p at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213-1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826-987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.     

Two functional soybean genes encoding p34cdc2 protein kinases are regulated by different plant developmental pathways

We have isolated two cDNA clones (cdc2-S5 and cdc2-S6) encoding p34cdc2 protein kinases, homologs of yeast cdc2/CDC28 genes, from a soybean nodule cDNA library. The two sequences share 90% sequence homology in the coding regions. The 5' and 3' noncoding regions are distinct from each other, however, indicating that at least two genes encode p34cdc2 protein kinases in soybean. Both sequences can rescue the cdc28 mutation in Saccharomyces cerevisiae but rescue it with different efficiency. Genomic Southern analysis showed the existence of two copies for each of these genes, which are not closely linked and are nonallelic. The relative expression level of the two soybean p34cdc2 genes varies in different tissues. Expression of cdc2-S5 is higher in roots and root nodules, whereas cdc2-S6 is more actively expressed in aerial tissues, indicating that regulation of these two p34cdc2 genes is coupled with plant developmental pathways. Expression of cdc2-S5 is, furthermore, enhanced after Rhizobium infection, whereas cdc2-S6 fails to respond, suggesting that cdc2-S5 plays a role in nodule initiation and organogenesis. This latter gene preferentially responds to auxin (alpha-naphthaleneacetic acid) treatment, indicating that phytohormones may be involved in the control of cell division mediated by Rhizobium infection. Thus, different p34cdc2 protein kinases may control cell division in different tissues in a multicellular organism and respond to different signals--e.g., phytohormones.     

Cellular and molecular mechanisms of breast and prostate cancer metastasis to bone

Coordination between karyokinesis and cytokinesis in the cell division cycle is fundamental to a precise transmission of duplicated genome into dividing daughter cells. byr4, a previously isolated essential gene, affects the mitotic cell cycle and cytokinesis in S. pombe. Phenotypic analyses of the null alleles and the overexpression of byr4 suggest that byr4 is a dosage-dependent coordinator of karyokinesis and cytokinesis (Song et al., 1996). In this study, the functional mechanisms of byr4 were investigated using a byr4 mutant that exhibits byr4 overexpression phenotypes in thiamine deficient media. Genetic suppression analyses of this byr4 mutant with other cytokinesis regulatory genes in S. pombe, cdc16, cdc7, cdc15, cdc14, and plo1, show that byr4 overexpression phenotypes are suppressed by the overexpression of cdc16 and cdc7, but not by plo1, cdc14, and cdc15. Also, the basal expression of byr4 and cdc7 suppresses the temperature-sensitive cdc16 mutation. However, the basal expression of either byr4 or cdc16 does not suppress the temperature-sensitive cdc7 mutation. The results of these suppression tests suggest that byr4 genetically interacts with cdc16 and cdc7: byr4 functions at the same level with or downstream of cdc16 and upstream of cdc7. In the present study, we also show that Byr4 interacts with Cdc16 and Spg1 in the yeast two-hybrid assays. Recent reports suggest a possible small GTPase pathway to regulate the timing of cytokinesis where Cdc16 functions as a GAP (GTPase activating protein), Spg1 as a GTPase, and Cdc7 as a downstream effector. Combined genetic and two-hybrid analyses of this study strongly suggest that Byr4 directly interacts with this possible small GTPase pathway including Cdc16, Spg1, and Cdc7 to regulate cytokinesis in S. pombe.     

A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.     

Modification of yeast Cdc53p by the ubiquitin-related protein rub1p affects function of the SCFCdc4 complex

The RUB1/NEDD-8 family of ubiquitin-related genes is widely represented among eukaryotes. Here we report that Cdc53p in Saccharomyces cerevisiae, a member of the Cullin family of proteins, is stably modified by the covalent attachment of a single Rub1p molecule. Two genes have been identified that are required for Rub1p conjugation to Cdc53p. The first gene, designated ENR2, encodes a protein with sequence similarity to the amino-terminal half of the ubiquitin-activating enzyme. By analogy with Aos1p, we infer that Enr2p functions in a bipartite Rub1p-activating enzyme. The second gene is SKP1, shown previously to be required for some ubiquitin-conjugation events. A deletion allele of ENR2 is lethal with temperature-sensitive alleles of cdc34 and enhances the phenotypes of cdc4, cdc53, and skp1, strongly implying that Rub1p conjugation to Cdc53p is required for optimal assembly or function of the E3 complex SCFCdc4. Consistent with this model, both enr2delta and an allele of Cdc53p that is not Rub1p modified, render cells sensitive to alterations in the levels of Cdc4p, Cdc34p, and Cdc53p.     

Differences in binding of PI 3-kinase to the src-homology domains 2 and 3 of p56 lck and p59 fyn tyrosine kinases

Two T cell-specific src-family tyrosine kinases, p56 lck (lck) and p59 fyn (fyn), are implicated in regulating PI 3-kinase activity in response to interleukin-2 (IL-2), a cytokine that induces T cell proliferation. The src- homology domains 3 (SH3) of src-family kinases can directly interact with the PI 3-kinase regulatory subunit p85 and this may be a mechanism to regulate PI 3-kinase activity. In order to understand the mode of PI 3-kinase activation by the IL-2 receptor, we examined the association of PI 3-kinase to SH2 and SH3 domains of lck and fyn in IL-2-dependent kit 225 cells. The fyn SH3 domain bound more PI 3-kinase and its p85 subunit than the lck SH3 domain, while the lck SH2 domain bound more PI 3-kinase than the fyn SH2 domain. None of these interactions were regulated by IL-2. Low binding of PI 3-kinase to the lck SH3 domain was not observed in IL-2-independent Jurkat T cells. Thus, SH3 and SH2 domains of lck and fyn bound different amounts of PI 3-kinase, a feature that was dependent on a T cell type, but was not influenced by IL-2.     

Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function

To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother-bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Delta vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Delta cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605-616).     

The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised

Premature initiation of cytokinesis can lead to loss of chromosomes, and 'cutting' of the nucleus. Therefore, the proper spatial and temporal co-ordination of mitosis and cytokinesis is essential for maintaining the integrity of the genome. The fission yeast cdc16 gene is implicated both in the spindle assembly checkpoint and control of septum formation. To identify other proteins involved in these controls, we have isolated multicopy suppressors of the cdc16-116 mutation, and the characterization of one of these, dma1 (defective in mitotic arrest), is presented here. dma1 is not an essential gene, but in a dma1 null background (dma1-D1) the function of the spindle assembly checkpoint is compromised. If assembly of the spindle is prevented, dma1-D1 cells do not arrest, the activity of cdc2 kinase decays and cells form a division septum without completing a normal mitosis. dma1-D1 cells also show an increased rate of chromosome loss during exponential growth. Upon ectopic expression from an inducible promoter, dma1p delays progress through mitosis and inhibits septum formation, giving rise to elongated, multinucleate cells. We propose that dma1 is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is impaired.     

SHP-1 associates with both platelet-derived growth factor receptor and the p85 subunit of phosphatidylinositol 3-kinase

The Src homology 2 (SH2)-containing protein tyrosine phosphatase 1, SHP-1, is highly expressed in all hematopoietic cells as well as in many non-hematopoietic cells, particularly in some malignant epithelial cell lines. In hematopoietic cells, SHP-1 negatively regulates multiple cytokine receptor pathways. The precise function and the targets of SHP-1 in non-hematopoietic cells, however, are largely unknown. Here we demonstrate that SHP-1 associates with both the tyrosine-phosphorylated platelet-derived growth factor (PDGF) receptor and the p85 subunit of phosphatidylinositol 3-kinase in MCF-7 and TRMP cells. Through the use of mutant PDGF receptors and performing peptide competition for immunoprecipitation, it was determined that SHP-1 independently associates with the PDGF receptor and p85 and that its N-terminal SH2 domain is directly responsible for the interactions. Overexpression of SHP-1 in TRMP cells transfected with the PDGF receptor markedly inhibited PDGF-induced c-fos promoter activation, whereas the expression of three catalytically inactive SHP-1 mutants increased the c-fos promoter activation in response to PDGF stimulation. These results indicate that SHP-1 might negatively regulate PDGF receptor-mediated signaling in these cells. Identification of the association of SHP-1 with the PDGF receptor and p85 in MCF-7 and TRMP cells furthers our understanding of the function of SHP-1 in non-hematopoietic cells.     

Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body

BACKGROUND: An actomyosin-based contractile ring plays a pivotal role in cytokinesis. Despite the identification of many components of the ring, the steps involved in its assembly are unknown. The fission yeast Schizosaccharomyces pombe is an attractive organism in which to study cytokinesis because its cell cycle has been well characterized; it divides by medial fission using an actomyosin ring; and a number of S. pombe mutants defective in actomyosin ring assembly have been isolated. Here, we have characterized one such mutant, rng2. RESULTS: Temperature-sensitive rng2 mutants accumulated F-actin cables in the medial region of the cell but failed to organize the cables into a ring. In rng2-null mutants, only a spot-like structure containing F-actin was detected. The rng2+ gene encodes a protein related to human IQGAP1, a protein that binds actin and calmodulin and is a potential effector for the Rho family of GTPases. Rng2p localized to the actomyosin ring and to the spindle pole body (SPB) of interphase and mitotic cells. Localization of Rng2p to the actomyosin ring but not the SPB required F-actin. Rng2p interacted with calmodulin, a component of the SPB and the actomyosin ring. The rng2 gene showed genetic interactions with three other actomyosin ring assembly mutants, cdc4, cdc12, and rng5. CONCLUSIONS: The S. pombe IQGAP-related protein Rng2p is a component of the actomyosin ring and the SPB and is required for actomyosin ring construction following assembly of F-actin at the division site.     

The multiple roles of Cyk1p in the assembly and function of the actomyosin ring in budding yeast

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOH-terminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis.     

Dual function of Cyk2, a cdc15/PSTPIP family protein, in regulating actomyosin ring dynamics and septin distribution

We previously showed that the budding yeast Saccharomyces cerevisiae assembles an actomyosin-based ring that undergoes a contraction-like size change during cytokinesis. To learn more about the biochemical composition and activity of this ring, we have characterized the in vivo distribution and function of Cyk2p, a budding yeast protein that exhibits significant sequence similarity to the cdc15/PSTPIP family of cleavage furrow proteins. Video microscopy of cells expressing green fluorescent protein (GFP)-tagged Cyk2p revealed that Cyk2p forms a double ring that coincides with the septins through most of the cell cycle. During cytokinesis, however, the Cyk2 double ring merges with the actomyosin ring and exhibits a contraction-like size change that is dependent on Myo1p. The septin double ring, in contrast, does not undergo the contraction-like size change but the separation between the two rings increases during cytokinesis. These observations suggest that the septin-containing ring is dynamically distinct from the actomyosin ring and that Cyk2p transits between the two types of structures. Gene disruption of CYK2 does not affect the assembly of the actomyosin ring but results in rapid disassembly of the ring during the contraction phase, leading to incomplete cytokinesis, suggesting that Cyk2p has an important function in modulating the stability of the actomyosin ring during contraction. Overexpression of Cyk2p also blocks cytokinesis, most likely due to a loss of the septins from the bud neck, indicating that Cyk2p may also play a role in regulating the localization of the septins.     

Overexpression of MID2 suppresses the profilin-deficient phenotype of yeast cells

Profilin-deficient Saccharomyces cerevisiae cells show abnormal growth, actin localization, chitin deposition, bud formation and cytokinesis. Previous studies have also revealed a synthetic lethality between pfy1 and late secretory mutants, suggesting a role for profilin in intracellular transport. In this work, we document further the secretion defect associated with the pfy1delta mutant. Electron microscopic observations reveal an accumulation of glycoproteins in the bud and in the mother cell. The MATa, pfy1delta cells mate as well as wild-type cells, while the mating efficiency of MAT alpha, pfy1delta cells is reduced. Pulse-chase experiments demonstrate an accumulation of the 19 kDa alpha-factor precursor and delayed secretion of the mature alpha-factor. The TGN protein Kex2p is the principal enzyme responsible for the endoproteolytic cleavage of the alpha-factor precursor. An immunofluorescence detection of Kex2p shows an altered localization in pfy1delta cells. Instead of a discrete punctate distribution, the enzyme is dispersed throughout the cytoplasm. A high-copy-number plasmid containing MID2, which encodes a potential transmembrane protein involved in cell cycle control, suppresses the abnormal growth, actin distribution, alpha-factor maturation and the accumulation of intracellular membranous structures in pfy1delta cells.     

The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators, Ssk1p and Skn7p

The Saccharomyces cerevisiae Sln1 protein is a 'two-component' regulator involved in osmotolerance. Two-component regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.     

Bet1p activates the v-SNARE Bos1p

Bet1p is a type II membrane protein that is required for vesicular transport between the endoplasmic reticulum and Golgi complex in the yeast Saccharomyces cerevisiae. A domain of Bet1p, that shows potential to be involved in a coiled-coil interaction, is homologous to a region of the neuronal protein SNAP-25. Here, we used in vitro binding studies to demonstrate that Bet1p plays a role in potentiating soluble NSF attachment protein receptor (SNARE) interactions. Mutational analysis points to the coiled-coil region as necessary for Bet1p function, and circular dichroism experiments support this theory. In vitro binding studies were also used to demonstrate that a direct interaction between Bet1p and Bos1p is required for the efficient interaction of the vesicle SNARE with its SNARE target. Genetic studies suggest that the interactions of Bet1p with Bos1p are regulated by the small GTP-binding protein Ypt1p.     

Actin cytoskeleton organization regulated by the PAK family of protein kinases

Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton.     

Cse1p is required for export of Srp1p/importin-alpha from the nucleus in Saccharomyces cerevisiae

In metazoan cells, the CAS protein has been shown to function as a recycling factor for the importin-alpha subunit of the classical nuclear localization signal receptor, exporting importin-alpha from the nucleus to allow its participation in multiple rounds of nuclear import. CAS is a member of a family of proteins that bear homology to the larger subunit of the nuclear localization signal receptor, importin-beta, and that are found in all eukaryotes from yeast to humans. Sequence similarity identifies the product of the Saccharomyces cerevisiae CSE1 gene as a potential CAS homologue. Here we present evidence that Cse1p is the functional homologue of CAS: Cse1p is required to prevent accumulation of Srp1p/importin-alpha in the nucleus, it localizes to the nuclear envelope in a pattern typical of nuclear transport receptors, and it associates in vivo with Srp1p in a nucleotide-specific manner. We show further that mutations in CSE1 and SRP1 have specific effects on their association and on the intracellular localization of Cse1p.