Components required for cytokinesis are important for bud site selection in yeast

Polarized cell division is a fundamental process that occurs in a variety of organisms; it is responsible for the proper positioning of daughter cells and the correct segregation of cytoplasmic components. The SPA2 gene of yeast encodes a nonessential protein that localizes to sites of cell growth and to the site of cytokinesis. spa2 mutants exhibit slightly altered budding patterns. In this report, a genetic screen was used to isolate a novel ochre allele of CDC10, cdc10-10; strains containing this mutation require the SPA2 gene for growth. CDC10 encodes a conserved potential GTP-binding protein that previously has been shown to localize to the bud neck and to be important for cytokinesis. The genetic interaction of cdc10-10 and spa2 suggests a role for SPA2 in cytokinesis. Most importantly, strains that contain a cdc10-10 mutation and those containing mutations affecting other putative neck filament proteins do not form buds at their normal proximal location. The finding that a component involved in cytokinesis is also important in bud site selection provides strong evidence for the cytokinesis tag model; i.e., critical components at the site of cytokinesis are involved in determining the next site of polarized growth and division.     

Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae

The establishment of cell polarity was examined in the budding yeast, S. cerevisiae. The distribution of a polarized protein, the SPA2 protein, was followed throughout the yeast cell cycle using synchronized cells and cdc mutants. The SPA2 protein localizes to a patch at the presumptive bud site of G1 cells. Later it concentrates at the bud tip in budded cells. At cytokinesis, the SPA2 protein is at the neck between the mother and daughter cells. Analysis of unbudded haploid cells has suggested a series of events that occurs during G1. The SPA2 patch is established very early in G1, while the spindle pole body residues on the distal side of the nucleus. Later, microtubules emanating from the spindle pole body intersect the SPA2 crescent, and the nucleus probably rotates towards the SPA2 patch. By middle G1, most cells contain the SPB on the side of the nucleus proximal to the SPA2 patch, and a long extranuclear microtubule bundle intersects this patch. We suggest that a microtubule capture site exists in the SPA2 staining region that stabilizes the long microtubule bundle; this capture site may be responsible for rotation of the nucleus. Cells containing a polarized distribution of the SPA2 protein also possess a polarized distribution of actin spots in the same region, although the actin staining is much more diffuse. Moreover, cdc4 mutants, which form multiple buds at the restrictive temperature, exhibit simultaneous staining of the SPA2 protein and actin spots in a subset of the bud tips. spa2 mutants contain a polarized distribution of actin spots, and act1-1 and act1-2 mutants often contain a polarized distribution of the SPA2 protein suggesting that the SPA2 protein is not required for localization of the actin spots and the actin spots are not required for localization of the SPA2 protein. cdc24 mutants, which fail to form buds at the restrictive temperature, fail to exhibit polarized localization of the SPA2 protein and actin spots, indicating that the CDC24 protein is directly or indirectly responsible for controlling the polarity of these proteins. Based on the cell cycle distribution of the SPA2 protein, a "cytokinesis tag" model is proposed to explain the mechanism of the non-random positioning of bud sites in haploid yeast cells.     

The SPA2 gene of Saccharomyces cerevisiae is important for pheromone- induced morphogenesis and efficient mating

Upon exposure to mating pheromone, Saccharomyces cerevisiae undergoes cellular differentiation to form a morphologically distinct cell called a "shmoo". Double staining experiments revealed that both the SPA2 protein and actin localize to the shmoo tip which is the site of polarized cell growth. Actin concentrates as spots throughout the shmoo projection, while SPA2 localizes as a sharp patch at the shmoo tip. DNA sequence analysis of the SPA2 gene revealed an open reading frame 1,466 codons in length; the predicted protein sequence contains many internal repeats including a nine amino acid sequence that is imperfectly repeated 25 times. Portions of the SPA2 sequence exhibit a low-level similarity to proteins containing coiled-coil structures. Yeast cells containing a large deletion of the SPA2 gene are similar in growth rate to wild-type cells. However, spa2 mutant cells are impaired in their ability to form shmoos upon exposure to mating pheromone, and they do not mate efficiently with other spa2 mutant cells. Thus, we suggest that the SPA2 protein plays a critical role in cellular morphogenesis during mating, perhaps as a cytoskeletal protein.     

Polarized growth controls cell shape and bipolar bud site selection in Saccharomyces cerevisiae

We examined the relationship between polarized growth and division site selection, two fundamental processes important for proper development of eukaryotes. Diploid Saccharomyces cerevisiae cells exhibit an ellipsoidal shape and a specific division pattern (a bipolar budding pattern). We found that the polarity genes SPA2, PEA2, BUD6, and BNI1 participate in a crucial step of bud morphogenesis, apical growth. Deleting these genes results in round cells and diminishes bud elongation in mutants that exhibit pronounced apical growth. Examination of distribution of the polarized secretion marker Sec4 demonstrates that spa2Delta, pea2Delta, bud6Delta, and bni1Delta mutants fail to concentrate Sec4 at the bud tip during apical growth and at the division site during repolarization just prior to cytokinesis. Moreover, cell surface expansion is not confined to the distal tip of the bud in these mutants. In addition, we found that the p21-activated kinase homologue Ste20 is also important for both apical growth and bipolar bud site selection. We further examined how the duration of polarized growth affects bipolar bud site selection by using mutations in cell cycle regulators that control the timing of growth phases. The grr1Delta mutation enhances apical growth by stabilizing G(1) cyclins and increases the distal-pole budding in diploids. Prolonging polarized growth phases by disrupting the G(2)/M cyclin gene CLB2 enhances the accuracy of bud site selection in wild-type, spa2Delta, and ste20Delta cells, whereas shortening the polarized growth phases by deleting SWE1 decreases the fidelity of bipolar budding. This study reports the identification of components required for apical growth and demonstrates the critical role of polarized growth in bipolar bud site selection. We propose that apical growth and repolarization at the site of cytokinesis are crucial for establishing spatial cues used by diploid yeast cells to position division planes.     

Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle

Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i.e., increase in cell mass and cell volume) at restrictive temperature (36 degrees C) but were unable to form buds. Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36 degrees C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanate-conjugated concanavalin A suggested that mannan incorporation was also delocalized in mutant cells grown at 36 degrees C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded as well as in unbudded cells. Growth of the mutant strains at temperatures less than 36 degrees C revealed allele-specific differences in behavior. Two strains produced buds of abnormal shape during growth at 33 degrees C. Moreover, these same strains displayed abnormal localization of budding sites when growth at 24 degrees C (the normal permissive temperature for the mutants); in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses. Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape.     

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.     

SPA1: a gene important for chromosome segregation and other mitotic functions in S. cerevisiae

Human autoantibodies that recognize the spindle poles of mammals, plants, and insects were found to recognize two antigens in yeast. One of these proteins, called SPA1 (for Spindle Pole Antigen), is antigenically related to the spindle poles of a diverse set of organisms. The gene encoding SPA1 was cloned by immunoscreening a lambda gt11 yeast genomic DNA expression library with autoantibody probes. Mutational analysis of the SPA1 gene demonstrates that it is important for cell growth, chromosome segregation, and other cellular processes; spa1 mutants are viable but grow poorly at 30 degrees C, missegregate chromosomes at an increased frequency, and often contain deformed spindles. A significant fraction of spa1 mutant cells contain two or more nuclei, and others contain none; these abnormal cells may arise through a nuclear migration defect. Thus SPA1 represents a new fidelity gene that is important for chromosome segregation and other mitotic functions.     

Polarized morphogenesis regulator Spa2 is required for the function of putative stretch-activated Ca2+-permeable channel component Mid1 in Saccharomyces cerevisiae

Mid1 is a putative stretch-activated Ca2+ channel component and is required for the maintenance of viability in the mating process. In response to mating pheromone, the mid1 mutant normally forms a pointed mating projection but eventually dies. This phenotype is called the mid phenotype. To identify a protein regulating Mid1 or regulated by Mid1, we isolated a multicopy suppressor that rescues the mid1-1 mutant from mating pheromone-induced death and found that it encodes a truncated Spa2 protein lacking an amino-terminal region responsible for interaction with components of the mitogen-activated protein kinase cascades. One of these SPA2 alleles was SPA2DeltaN, whose product lacked the region from Ser5 to Leu230. SPA2DeltaN on a multicopy plasmid (YEpSPA2DeltaN) complemented the mid phenotype but not another phenotype, low Ca2+ accumulation, of the mid1-1 mutant. Neither SPA2DeltaN on a low-copy plasmid nor wild-type SPA2 on a multicopy plasmid had suppressive activity. The SPA2 gene is involved in the formation of a pointed mating projection, and cells of the spa2Delta mutant lacking Spa2 are viable and develop a peanut shell-like structure when exposed to mating pheromone. Like the spa2Delta mutant, the mid1-1 spa2Delta double mutant and the mid1-1/YEpSPA2DeltaN strain developed the peanut shell-like structure. The mid1-1 spa2Delta double mutant did not have the mid phenotype, indicating that SPA2 is epistatic to MID1. Overexpression of Spa2DeltaN abolished the localization of Spa2-green fluorescent protein to the tip of the mating projection. These results suggest that the Spa2DeltaN protein interferes with the localization of the normal Spa2 protein and thereby prevents cells from entering the mating process. Therefore, we suggest that Mid1 function is influenced by Spa2 function through polarized morphogenesis.     

A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth.

The Saccharomyces cerevisiae SPA2 protein localizes at sites involved in polarized cell growth in budding cells and mating cells. spa2 mutants have defects in projection formation during mating but are healthy during vegetative growth. A synthetic lethal screen was devised to identify mutants that require the SPA2 gene for vegetative growth. One mutant, called slk-1 (for synthetic lethal kinase), has been characterized extensively. The SLK1 gene has been cloned, and sequence analysis predicts that the SLK1 protein is 1,478 amino acid residues in length. Approximately 300 amino acids at the carboxy terminus exhibit sequence similarity with the catalytic domains of protein kinases. Disruption mutations have been constructed in the SLK1 gene. slk1 null mutants cannot grow at 37 degrees C, but many cells can grow at 30, 24, and 17 degrees C. Dead slk1 mutant cells usually have aberrant cell morphologies, and many cells are very small, approximately one-half the diameter of wild-type cells. Surviving slk1 cells also exhibit morphogenic defects; these cells are impaired in their ability to form projections upon exposure to mating pheromones. During vegetative growth, a higher fraction of slk1 cells are unbudded compared with wild-type cells, and under nutrient limiting conditions, slk1 cells exhibit defects in cell cycle arrest. The different slk1 mutant defects are partially rescued by an extra copy of the SSD1/SRK1 gene. SSD1/SRK1 has been independently isolated as a suppressor of mutations in genes involved in growth control, sit4, pde2, bcy1, and ins1 (A. Sutton, D. Immanuel, and K.T. Arnat, Mol. Cell. Biol. 11:2133-2148, 1991; R.B. Wilson, A.A. Brenner, T.B. White, M.J. Engler, J.P. Gaughran, and K. Tatchell, Mol. Cell. Biol. 11:3369-3373, 1991). These data suggest that SLK1 plays a role in both cell morphogenesis and the control of cell growth. We speculate that SLK1 may be a regulatory link for these two cellular processes.     

Saccharomyces cerevisiae cells execute a default pathway to select a mate in the absence of pheromone gradients

During conjugation, haploid S. cerevisiae cells find one another by polarizing their growth toward each other along gradients of pheromone (chemotropism). We demonstrate that yeast cells exhibit a second mating behavior: when their receptors are saturated with pheromone, wild-type a cells execute a default pathway and select a mate at random. These matings are less efficient than chemotropic matings, are induced by the same dose of pheromone that induces shmoo formation, and appear to use a site near the incipient bud site for polarization. We show that the SPA2 gene is specifically required for the default pathway: spa2 delta mutants cannot mate if pheromone concentrations are high and gradients are absent, but can mate if gradients are present. ste2 delta, sst2 delta, and far1 delta mutants are chemotropism-defective and therefore must choose a mate by using a default pathway; consistent with this deduction, these strains require SPA2 to mate. In addition, our results suggest that far1 mutants are chemotropism-defective because their mating polarity is fixed at the incipient bud site, suggesting that the FAR1 gene is required for inhibiting the use of the incipient bud site during chemotropic mating. These observations reveal a molecular relationship between the mating and budding polarity pathways.     

The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in arabidopsis

The Arabidopsis thaliana proteins suppressor of phytochrome A-105 1 (SPA1), SPA3, and SPA4 of the four-member SPA1 protein family have been shown to repress photomorphogenesis in light-grown seedlings. Here, we demonstrate that spa quadruple mutant seedlings with defects in SPA1, SPA2, SPA3, and SPA4 undergo strong constitutive photomorphogenesis in the dark. Consistent with this finding, adult spa quadruple mutants are extremely small and dwarfed. These extreme phenotypes are only observed when all SPA genes are mutated, indicating functional redundancy among SPA genes. Differential contributions of individual SPA genes were revealed by analysis of spa double and triple mutant genotypes. SPA1 and SPA2 predominate in dark-grown seedlings, whereas SPA3 and SPA4 prevalently regulate the elongation growth in adult plants. Further analysis of SPA2 function indicated that SPA2 is a potent repressor of photomorphogenesis only in the dark but not in the light. The SPA2 protein is constitutively nuclear localized in planta and can physically interact with the repressor COP1. Epistasis analysis between spa2 and cop1 mutations provides strong genetic support for a biological significance of a COP1-SPA2 interaction in the plant. Taken together, our results have identified a new family of proteins that is essential for suppression of photomorphogenesis in darkness.     

Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae

Calmodulin was localized in Saccharomyces cerevisiae by indirect immunofluorescence using affinity-purified polyclonal antibodies. Calmodulin displays an asymmetric distribution that changes during the cell cycle. In unbudded cells, calmodulin concentrates at the presumptive site of bud formation approximately 10 min before bud emergence. In small budded cells, calmodulin accumulates throughout the bud. As the bud grows, calmodulin concentrates at the tip, then disperses, and finally concentrates in the neck region before cytokinesis. An identical staining pattern is observed when wild-type calmodulin is replaced with mutant forms of calmodulin impaired in binding Ca2+. Thus, the localization of calmodulin does not depend on its ability to bind Ca2+ with a high affinity. Double labeling of yeast cells with affinity-purified anti-calmodulin antibody and rhodamine-conjugated phalloidin indicates that calmodulin and actin concentrate in overlapping regions during the cell cycle. Furthermore, disrupting calmodulin function using a temperature-sensitive calmodulin mutant delocalizes actin, and act1-4 mutants contain a random calmodulin distribution. Thus, calmodulin and actin distributions are interdependent. Finally, calmodulin localizes to the shmoo tip in cells treated with alpha-factor. This distribution, at sites of cell growth, implicates calmodulin in polarized cell growth in yeast.     

Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth sites, including the bud tip and the cytokinesis site, and is required for bud formation. We have recently shown that Pkc1p, a yeast homolog of mammalian protein kinase C, and glucan synthase are targets of Rho1p. Using the two-hybrid screening system, we cloned a gene encoding a protein which interacted with the GTP-bound form of Rho1p. This gene was identified as BNI1, known to be implicated in cytokinesis or establishment of cell polarity in S.cerevisiae. Bni1p shares homologous domains (FH1 and FH2 domains) with proteins involved in cytokinesis or establishment of cell polarity, including formin of mouse, capu and dia of Drosophila and FigA of Aspergillus. A temperature-sensitive mutation in which the RHO1 gene was replaced by the mammalian RhoA gene showed a synthetically lethal interaction with the bni1 mutation and the RhoA bni1 mutant accumulated cells with a deficiency in cytokinesis. Furthermore, this synthetic lethality was caused by the incapability of RhoA to activate Pkc1p, but not glucan synthase. These results suggest that Rho1p regulates cytoskeletal reorganization at least through Bni1p and Pkc1p.     

Localized secretion of acid phosphatase reflects the pattern of cell surface growth in saccharomyces cerevisiae

Secretion of cell wall-bound acid phosphatase by Saccharomyces cerevisiae occurs along a restricted portion of the cell surface. Acid phosphatase activity produced during derepressed synthesis on a phosphate-limited growth medium is detected with an enzyme-specific stain and is localized initially to the bud portion of a dividing cell. After two to three generations of phosphate-limited growth, most of the cells can be stained; if further phosphatase synthesis is repressed by growth in excess phosphate, dividing cells are produced in which the parent but not the bud can be stained. Budding growth is interrupted in α-mating-type cells by a pheromone (α-factor) secreted by the opposite mating type; cell surface growth continues in the presence of α-factor and produces a characteristic cell tip. When acid phosphatase synthesis is initiated during α-factor treatment, only the cell tip can br stained; when phosphate synthesis is repressed during α-factor treatment, the cell body but not the tip can be stained. A mixture of derepressed α cells and phosphatase-negative α cells form zygotes in which mainly one parent cell surface can be stained. The cell cycle mutant, cdc 24 (Hartwell, L.H. 1971. Exp. Cell Res. 69:265-276), fails to bud and, instead, expands symmetrically as a sphere at a nonpermissive temperature (37 degrees C). This mutant does not form a cell tip during α-factor treatment at 37 degrees C, and although acid phosphatade secretion occurs at this temperature, it is not localized. These results suggest that secretion reflects a polar mode of yeast cell- surface growth, and that this organization requires the cdc 24 gene product.     

Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A

The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.     

Identification of a motor protein required for filamentous growth in Ustilago maydis.

The phytopathogenic fungus Ustilago maydis exists in two stages, the yeast-like haploid form and the filamentous dikaryon. Both pathogenicity and dimorphism are genetically controlled by two mating-type loci, with only the filamentous stage being pathogenic on corn. We have identified two genes (kin1 and kin2) encoding motor proteins of the kinesin family. Kin1 is most similar to the human CENP-E gene product, while Kin2 is most closely related to the conventional kinesin Nkin of Neurospora crassa. Deletion mutants of kin1 had no discernible phenotype; delta kin2 mutants, however, were severely affected in hyphal extension and pathogenicity. The wild-type dikaryon showed rapid tip growth, with all the cytoplasm being moved to the tip compartment. Left behind are septate cell wall tubes devoid of cytoplasm. In delta kin2 mutants, dikaryotic cells were formed after cell fusion, but these hyphal structures remained short and filled with cytoplasm. A functional green fluorescent protein (GFP)-Kin2 fusion was generated and used to determine the localization of the motor protein by fluorescence microscopy. Inspection of the hyphal tips by electron microscopy revealed a characteristic accumulation of darkly stained vesicles which was absent in mutant cells. We suggest that the motor protein Kin2 is involved in organizing this specialized growth zone at the hyphal tip, probably by affecting the vectorial transport of vesicles.     

The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light

Suppressor of phyA-105 (SPA1) is a phytochrome A-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. SPA1 is part of a small gene family comprising three genes: SPA1-related 2 (SPA2), SPA1-related 3 (SPA3), and SPA1-related 4 (SPA4). Here, we investigate the functions of SPA3 and SPA4, two very closely related genes coding for proteins with 74% identical amino acids. Seedlings with mutations in SPA3 or SPA4 exhibit enhanced photomorphogenesis in the light, but show no phenotype in darkness. While there are small differences between the effects of spa3 and spa4 mutations, it is apparent that SPA3 and SPA4 function to inhibit light responses in continuous far-red, red, and blue light. Phytochrome A is necessary for all aspects of the spa4 mutant phenotype, suggesting that SPA4, like SPA1, acts specifically in phytochrome A signaling. Enhanced photoresponsiveness of spa3 mutants is also fully dependent on phytochrome A in far-red and blue light, but not in red light. Hence, SPA3 function in red light may be dependent on other phytochromes in addition to phytochrome A. Using yeast two-hybrid and in vitro interaction assays, we further show that SPA3 as well as SPA4 can physically interact with the constitutive repressor of light signaling COP1. Deletion analyses suggest that SPA3 and SPA4, like SPA1, bind to the coiled-coil domain of COP1. Taken together, our results have identified two new loci coding for negative regulators that may be involved in fine tuning of light responses by interacting with COP1.     

Actin recovery and bud emergence in osmotically stressed cells requires the conserved actin interacting mitogen-activated protein kinase kinase kinase Ssk2p/MTK1 and the scaffold protein Spa2p

Osmotic stress causes actin cytoskeleton disassembly, a cell cycle arrest, and activation of the high osmolarity growth mitogen-activated protein kinase pathway. A previous study showed that Ssk2p, a mitogen-activated protein kinase kinase kinase of the high osmolarity growth pathway, promotes actin cytoskeleton recovery to the neck of late cell cycle, osmotically stressed yeast cells. Data presented herein examined the role of Ssk2p in actin recovery early in the cell cycle. We found that actin recovery at all stages of the cell cycle is not controlled by Ssk1p, the known activator of Ssk2p, but required a polarized distribution of Ssk2p as well as its actin-interacting and kinase activity. Stress-induced localization of Ssk2p to the neck required the septin Shs1p, whereas localization to the bud cortex depended on the polarity scaffold protein Spa2p. spa2delta cells, like ssk2delta cells, were defective for actin recovery from osmotic stress. These spa2delta defects could be suppressed by overexpression of catalytically active Ssk2p. Furthermore, Spa2p could be precipitated by GST-Ssk2p from extracts of osmotically stressed cells. The Ssk2p mediated actin recovery pathway seems to be conserved; MTK1, a human mitogen-activated protein kinase kinase kinase of the p38 stress response pathway and Ssk2p homolog, was also able to localize at polarized growth sites, form a complex with actin and Spa2p, and complement actin recovery defects in osmotically stressed ssk2delta and spa2delta yeast cells. We hypothesize that osmotic stress-induced actin disassembly leads to the formation of an Ssk2p-actin complex and the polarized localization of Ssk2p. Polarized Ssk2p associates with the scaffold protein Spa2p in the bud and Shs1p in the neck, allowing Ssk2p to regulate substrates involved in polarized actin assembly.