Cdc42p and Fus2p act together late in yeast cell fusion

Cell fusion is the key event of fertilization that gives rise to the diploid zygote and is a nearly universal aspect of eukaryotic biology. In the yeast Saccharomyces cerevisiae, several mutants have been identified that are defective for cell fusion, and yet the molecular mechanism of this process remains obscure. One obstacle has been that genetic screens have mainly focused on mating-specific factors, whereas the process likely involves housekeeping proteins as well. Here we implicate Cdc42p, an essential protein with roles in multiple aspects of morphogenesis, as a core component of the yeast cell fusion pathway. We identify a point mutant in the Rho-insert domain of CDC42, called cdc42-138, which is specifically defective in cell fusion. The cell fusion defect is not a secondary consequence of ineffective signaling or polarization. Genetic and morphological data show that Cdc42p acts at a late stage in cell fusion in concert with a key cell fusion regulator, Fus2p, which contains a Dbl-homology domain. We find that Fus2p binds specifically with activated Cdc42p, and binding is blocked by the cdc42-138 mutation. Thus, in addition to signaling and morphogenetic roles in mating, Cdc42p plays a role late in cell fusion via activation of Fus2p.     

Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway

The adaptive response to hyperosmotic stress in yeast, termed the high osmolarity glycerol (HOG) response, is mediated by two independent upstream pathways that converge on the Pbs2 MAP kinase kinase (MAPKK), leading to the activation of the Hog1 MAP kinase. One branch is dependent on the Sho1 transmembrane protein, whose primary role was found to be the binding and translocation of the Pbs2 MAPKK to the plasma membrane, and specifically to sites of polarized growth. The yeast PAK homolog Ste20 is essential for the Sho1-dependent activation of the Hog1 MAP kinase in response to severe osmotic stress. This function of Ste20 in the HOG pathway requires binding of the small GTPase Cdc42. Overexpression of Cdc42 partially complements the osmosensitivity of ste20Delta mutants, perhaps by activating another PAK-like kinase, while a dominant-negative Cdc42 mutant inhibited signaling through the SHO1 branch of the HOG pathway. Since activated Cdc42 translocates Ste20 to sites of polarized growth, the upstream and downstream elements of the HOG pathway are brought together through the membrane targeting function of Sho1 and Cdc42.     

Specific protein targeting during cell differentiation: polarized localization of Fus1p during mating depends on Chs5p in Saccharomyces cerevisiae

In budding yeast, chs5 mutants are defective in chitin synthesis and cell fusion during mating. Chs5p is a late-Golgi protein required for the polarized transport of the chitin synthase Chs3p to the membrane. Here we show that Chs5p is also essential for the polarized targeting of Fus1p, but not of other cell fusion proteins, to the membrane during mating.     

The functional relationship between the Cdc50p-Drs2p putative aminophospholipid translocase and the Arf GAP Gcs1p in vesicle formation in the retrieval pathway from yeast early endosomes to the TGN

Drs2p, the catalytic subunit of the Cdc50p-Drs2p putative aminophospholipid translocase, has been implicated in conjunction with the Arf1 signaling pathway in the formation of clathrin-coated vesicles (CCVs) from the TGN. Herein, we searched for Arf regulator genes whose mutations were synthetically lethal with cdc50Delta, and identified the Arf GAP gene GCS1. Most of the examined transport pathways in the Cdc50p-depleted gcs1Delta mutant were nearly normal, including endocytic transport to vacuoles, carboxypeptidase Y sorting, and the processing and secretion of invertase. In contrast, this mutant exhibited severe defects in the early endosome-to-TGN transport pathway; proteins that are transported via this pathway, such as the v-SNARE Snc1p, the t-SNARE Tlg1p, and the chitin synthase III subunit Chs3p, accumulated in TGN-independent aberrant membrane structures. We extended our analyses to clathrin adaptors, and found that Gga1p/Gga2p and AP-1 were also involved in this pathway. The Cdc50p-depleted gga1Delta gga2Delta mutant and the gcs1Delta apl2Delta (the beta1 subunit of AP-1) mutant exhibited growth defects and intracellular Snc1p-containing membranes accumulated in these cells. These results suggest that Cdc50p-Drs2p plays an important role in the Arf1p-mediated formation of CCVs for the retrieval pathway from early endosomes to the TGN.     

Cdc37p is involved in osmoadaptation and controls high osmolarity-induced cross-talk via the MAP kinase Kss1p

Cdc37p, the p50 homolog of Saccharomyces cerevisiae, is an Hsp90 cochaperone involved in the targeting of protein kinases to Hsp90. Here we report a role for Cdc37p in osmoadaptive signalling in this yeast. The osmosensitive phenotype that is displayed by the cdc37-34 mutant strain appears not to be the consequence of deficient signalling through the high osmolarity glycerol (HOG) MAP kinase pathway. Rather, Cdc37p appears to play a role in the filamentous growth (FG) pathway, which mediates adaptation to high osmolarity parallel to the HOG pathway. The osmosensitive phenotype of the cdc37-34 mutant strain is aggravated upon the deletion of the HOG gene. We report that the hyper-osmosensitive phenotype of the cdc37-34, hog1 mutant correlates to a reduced of activity of the FG pathway. We utilized this phenotype to isolate suppressor genes such as KSS1 that encodes a MAP kinase that functions in the FG pathway. We report that Kss1p interacts physically with Cdc37p. Like Kss1p, the second suppressor that we isolated, Dse1p, is involved in cell wall biogenesis or maintenance, suggesting that Cdc37p controls osmoadapation by regulating mitogen-activated protein kinase signalling aimed at adaptive changes in cell wall organization.     

Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway

The yeast high osmolarity glycerol (HOG) signaling pathway can be activated by either of the two upstream pathways, termed the SHO1 and SLN1 branches. When stimulated by high osmolarity, the SHO1 branch activates an MAP kinase module composed of the Ste11 MAPKKK, the Pbs2 MAPKK, and the Hog1 MAPK. To investigate how osmostress activates this MAPK module, we isolated both gain-of-function and loss-of-function alleles in four key genes involved in the SHO1 branch, namely SHO1, CDC42, STE50, and STE11. These mutants were characterized using an HOG-dependent reporter gene, 8xCRE-lacZ. We found that Cdc42, in addition to binding and activating the PAK-like kinases Ste20 and Cla4, binds to the Ste11-Ste50 complex to bring activated Ste20/Cla4 to their substrate Ste11. Activated Ste11 and its HOG pathway-specific substrate, Pbs2, are brought together by Sho1; the Ste11-Ste50 complex binds to the cytoplasmic domain of Sho1, to which Pbs2 also binds. Thus, Cdc42, Ste50, and Sho1 act as adaptor proteins that control the flow of the osmostress signal from Ste20/Cla4 to Ste11, then to Pbs2.     

The Exomer Coat Complex Transports Fus1p to the Plasma Membrane via a Novel Plasma Membrane Sorting Signal in Yeast

Sorting of transmembrane cargo proteins to different cellular compartments is mediated by sorting signals that are recognized by coat proteins involved in vesicle biogenesis. We have identified a sorting signal in the yeast cell fusion protein Fus1p that is required for its transport from the trans-Golgi compartment to the plasma membrane. Transport of Fus1p from the trans-Golgi to the cell surface is dependent on Chs5p, a component of the multisubunit exomer complex. We show that Fus1p transport is also dependent on the exomer components Bch1p and Bud7p. Disruption of the clathrin adaptor protein complex 1 (AP-1) restores Fus1p localization to the cell surface in the absence of exomer, possibly by promoting an alternate, exomer-independent route of transport. Mutation of an IXTPK sequence in the cytosolic tail of Fus1p abolishes its physical interaction with Chs5p, results in mislocalization of Fus1p, and therefore causes a cell fusion defect. These defects are suppressed by disruption of AP-1. We suggest that IXTPK comprises a novel sorting signal that is recognized and bound by exomer leading to the capture of Fus1p into coated vesicles en route to the cell surface.     

Requirement for Bbp1p in the proper mitotic functions of Cdc5p in Saccharomyces cerevisiae

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pDeltaC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p(243-385)), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pDeltaC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5DeltaC-BBP1(243-385) under CDC5 promoter control partially complemented the cdc5Delta defect. These data suggest that Bbp1pDeltaC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.     

Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Galpha subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.     

Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe

Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.     

Candidate osmosensors from Candida utilis and Kluyveromyces lactis: structural and functional homology to the Sho1p putative osmosensor from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, increases in external osmolarity evoke osmostress-induced signalling via the HOG MAP kinase pathway. One of the upstream components of this signal transduction route is the putative osmosensor, Sho1p. With the aim to elucidate the molecular basis of osmosensing in budding yeast, we have cloned SHO1 homologues from Candida utilis and Kluyveromyces lactis which allowed determination of conserved domains of Sho1p. Results obtained from sequence comparisons, confirmed the importance of the transmembrane domains and the SH3 domain for Sho1p function. The K. lactis and S. cerevisiae Sho1p show the highest degree of homology, the isoform from C. utilis is a shorter protein. SHO1 from C. utilis, however, did complement the osmosensitivity of the sho1ssk2ssk22 strain by restoring HOG pathway function, since Hog1p dual phosphorylation after high osmotic challenge was restored in this strain after transformation with a plasmid bearing this SHO1 homologue.     

The stress-activated mitogen-activated protein kinase signaling cascade promotes exit from mitosis

In budding yeast, a signaling network known as the mitotic exit network (MEN) triggers exit from mitosis. We find that hypertonic stress allows MEN mutants to exit from mitosis in a manner dependent on the high osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase cascade. The HOG pathway drives exit from mitosis in MEN mutants by promoting the activation of the MEN effector, the protein phosphatase Cdc14. Activation of Cdc14 depends on the Cdc14 early anaphase release network, a group of proteins that functions in parallel to the MEN to promote Cdc14 function. Notably, exit from mitosis is promoted by the signaling branch defined by the Sho1 osmosensing system, but not by the Sln1 osmosensor of the HOG pathway. Our results suggest that the stress MAP kinase pathway mobilizes programs to promote completion of the cell cycle and entry into G1 under unfavorable conditions.     

Transmembrane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG pathway

To cope with life-threatening high osmolarity, yeast activates the high-osmolarity glycerol (HOG) signaling pathway, whose core element is the Hog1 MAP kinase cascade. Activated Hog1 regulates the cell cycle, protein translation, and gene expression. Upstream of the HOG pathway are functionally redundant SLN1 and SHO1 signaling branches. However, neither the osmosensor nor the signal generator of the SHO1 branch has been clearly defined. Here, we show that the mucin-like transmembrane proteins Hkr1 and Msb2 are the potential osmosensors for the SHO1 branch. Hyperactive forms of Hkr1 and Msb2 can activate the HOG pathway only in the presence of Sho1, whereas a hyperactive Sho1 mutant activates the HOG pathway in the absence of both Hkr1 and Msb2, indicating that Hkr1 and Msb2 are the most upstream elements known so far in the SHO1 branch. Hkr1 and Msb2 individually form a complex with Sho1, and, upon high external osmolarity stress, appear to induce Sho1 to generate an intracellular signal. Furthermore, Msb2, but not Hkr1, can also generate an intracellular signal in a Sho1-independent manner.     

Functional analysis of the interaction between Afr1p and the Cdc12p septin, two proteins involved in pheromone-induced morphogenesis.

Saccharomyces cerevisiae mating pheromones induce production of Afr1p, a protein that negatively regulates pheromone receptor signaling and is required for normal formation of the projection of cell growth that becomes the site of cell fusion during conjugation. Afr1p interacts with Cdc12p, which belongs to a family of filament-forming proteins termed septins that have been studied primarily for their role in bud morphogenesis and cytokinesis. The significance of the interaction between Afr1p and Cdc12p was tested in this study by examining the effects of AFR1 mutations that destroy the Cdc12p-binding domain. The results demonstrate that sequences in the C-terminal half of Afr1p are required for interaction with Cdc12p and for proper localization of Afr1p to the base of the mating projection. However, the Cdc12p-binding domain was not required for regulation of receptor signaling or for mating projection formation. This result was surprising because cells carrying a temperature-sensitive cdc12-6 mutation were defective in projection formation, indicating a role for Cdc12p in this process. Although the Cdc12p-binding domain was no essential for Afr1p function, this domain did improve the ability of Afr1p to promote morphogenesis, suggesting that the proper localization of Afr1p is important for its function.     

Aspergillus nidulans HOG pathway is activated only by two-component signalling pathway in response to osmotic stress

Genome sequencing analyses revealed that Aspergillus nidulans has orthologous genes to all those of the high-osmolarity glycerol (HOG) response mitogen-activated protein kinase (MAPK) pathway of Saccharomyces cerevisiae. A. nidulans mutant strains lacking sskA, sskB, pbsB, or hogA, encoding proteins orthologous to the yeast Ssk1p response regulator, Ssk2p/Ssk22p MAPKKKs, Pbs2p MAPKK and Hog1p MAPK, respectively, showed growth inhibition under high osmolarity, and HogA MAPK in these mutants was not phosphorylated under osmotic or oxidative stress. Thus, activation of the A. nidulans HOG (AnHOG) pathway depends solely on the two-component signalling system, and MAPKK activation mechanisms in the AnHOG pathway differ from those in the yeast HOG pathway, where Pbs2p is activated by two branches, Sln1p and Sho1p. Expression of pbsB complemented the high-osmolarity sensitivity of yeast pbs2Delta, and the complementation depended on Ssk2p/Ssk22p, but not on Sho1p. Pbs2p requires its Pro-rich motif for binding to the Src-homology3 (SH3) domain of Sho1p, but PbsB lacks a typical Pro-rich motif. However, a PbsB mutant (PbsB(Pro)) with the yeast Pro-rich motif was activated by the Sho1p branch in yeast. In contrast, HogA in sskADelta expressing PbsB(Pro) was not phosphorylated under osmotic stress, suggesting that A. nidulans ShoA, orthologous to yeast Sho1p, is not involved in osmoresponsive activation of the AnHOG pathway. We also found that besides HogA, PbsB can activate another Hog1p MAPK orthologue, MpkC, in A. nidulans, although mpkC is dispensable in osmoadaptation. In this study, we discuss the differences between the AnHOG and the yeast HOG pathways.     

Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating

Fus2p is a pheromone-induced protein associated with the amphiphysin homologue Rvs161p, which is required for cell fusion during mating in Saccharomyces cerevisiae. We constructed a functional Fus2p-green fluorescent protein (GFP), which exhibits highly dynamic localization patterns in pheromone-responding cells (shmoos): diffuse nuclear, mobile cytoplasmic dots and stable cortical patches concentrated at the shmoo tip. In mitotic cells, Fus2p-GFP is nuclear but becomes cytoplasmic as cells form shmoos, dependent on the Fus3p protein kinase and high levels of pheromone signaling. The rapid cytoplasmic movement of Fus2p-GFP dots requires Rvs161p and polymerized actin and is aberrant in mutants with compromised actin organization, which suggests that the Fus2p dots are transported along actin cables, possibly in association with vesicles. Maintenance of Fus2p-GFP patches at the shmoo tip cortex is jointly dependent on actin and a membrane protein, Fus1p, which suggests that Fus1p is an anchor for Fus2p. In zygotes, Fus2p-GFP forms a dilating ring at the cell junction, returning to the nucleus at the completion of cell fusion.     

The Signaling Mucins Msb2 and Hkr1 Differentially Regulate the Filamentation Mitogen-activated Protein Kinase Pathway and Contribute to a Multimodal Response

A central question in the area of signal transduction is why pathways utilize common components. In the budding yeast Saccharomyces cerevisiae, the HOG and filamentous growth (FG) MAPK pathways require overlapping components but are thought to be induced by different stimuli and specify distinct outputs. To better understand the regulation of the FG pathway, we examined FG in one of yeast''s native environments, the grape-producing plant Vitis vinifera. In this setting, different aspects of FG were induced in a temporal manner coupled to the nutrient cycle, which uncovered a multimodal feature of FG pathway signaling. FG pathway activity was modulated by the HOG pathway, which led to the finding that the signaling mucins Msb2p and Hkr1p, which operate at the head of the HOG pathway, differentially regulate the FG pathway. The two mucins exhibited different expression and secretion patterns, and their overproduction induced nonoverlapping sets of target genes. Moreover, Msb2p had a function in cell polarization through the adaptor protein Sho1p that Hkr1p did not. Differential MAPK activation by signaling mucins brings to light a new point of discrimination between MAPK pathways.     

Analysis of mitogen-activated protein kinase signaling specificity in response to hyperosmotic stress: use of an analog-sensitive HOG1 allele

When confronted with a marked increase in external osmolarity, budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high-osmolarity glycerol or HOG pathway) to elicit cellular responses necessary to permit continued growth. One input that stimulates the HOG pathway requires the integral membrane protein and putative osmosensor Sho1, which recruits and enables activation of the MAPK kinase kinase Ste11. In mutants that lack the downstream MAPK kinase (pbs2Delta) or the MAPK (hog1Delta) of the HOG pathway, Ste11 activated by hyperosmotic stress is able to inappropriately stimulate the pheromone response pathway. This loss of signaling specificity is known as cross talk. To determine whether it is the Hog1 polypeptide per se or its kinase activity that is necessary to prevent cross talk, we constructed a fully functional analog-sensitive allele of HOG1 to permit acute inhibition of this enzyme without other detectable perturbations of the cell. We found that the catalytic activity of Hog1 is required continuously to prevent cross talk between the HOG pathway and both the pheromone response and invasive growth pathways. Moreover, contrary to previous reports, we found that the kinase activity of Hog1 is necessary for its stress-induced nuclear import. Finally, our results demonstrate a role for active Hog1 in maintaining signaling specificity under conditions of persistently high external osmolarity.