Shmoo Formation Research Articles

Mdy2, a ubiquitin-like (UBL)-domain protein, is required for efficient mating in Saccharomyces cerevisiae

MDY2, a gene required for efficient mating of the yeast Saccharomyces cerevisiae, was characterized in this study. The gene encodes a protein of 212 amino acids, which contains a ubiquitin-like (UBL) domain (residues 74-149). Deletion of MDY2 is associated with a five- to sevenfold reduction in mating efficiency, mainly due to defects in nuclear migration and karyogamy at the prezygotic stage. However, prior to mating pair fusion, shmoo formation is reduced by 30%, with a concomitant failure to form mating pairs. Strikingly, migration of the nucleus into the shmoo tip is also delayed or fails to occur. In addition, we show that in mdy2 mutants, microtubule bundles, as well as the microtubule end-binding protein Kar9, fail to localize properly to the shmoo tip, suggesting that the nuclear migration defect could be due to aberrant localization of Kar9. Pheromone signal transduction (as measured by FUS1 induction by alpha-factor) is not affected in mdy2delta mutants and mitosis is also normal in these cells. MDY2 is not induced by mating pheromone. In vegetatively growing cells, GFP-Mdy2 is localized in the nucleus, and remains nuclear after exposure of cells to alpha-factor. His-tagged Mdy2 shows no evidence of the C-terminal processing typical of ubiquitin, and also localizes to the nucleus. Thus MDY2 is a novel gene, whose product plays a role in shmoo formation and in nuclear migration in the pre-zygote, possibly by interacting with other UBL-type proteins that possess ubiquitin association (UBA) domains.

Replicative aging of the yeast does not require DNA replication

Mating pheromone treatment resulting in shmoo formation is a physiologically relevant model for separation of cell growth and division processes in the yeast Saccharomyces cerevisiae. Using this attitude we demonstrate that yeast loses its capacity for division at a faster rate when engaged in intensive growth and metabolism without cell divisions (in the shmoo state) than during normal reproductive growth. These results suggest that limitation of the division potential in the yeast is not due to a counter of cell divisions but is of growth/metabolic nature, perhaps involving attaining a limitation of cell volume.

Yeast pheromone signaling pathway as a bioassay to assess the effect of chemicals on mammalian peptide hormones

The intercellular pheromone signal transduction pathways involved in sexual reproduction in the yeast Saccharomyces cerevisiae constitute an extracellular network system involving cell surface receptors. The system is analogous to the signaling pathway of mammalian peptide hormones. The yeast mating pheromone α factor is homologous to mammalian gonadoliberins such as luteinizing hormone-releasing hormone (LHRH). In this study, we used the yeast pheromone signaling pathway as a model system to evaluate the effect of industrial chemicals on mammalian peptide hormones. Haploid a- and α-cell types conjugate, using mating pheromones, to form diploid cells. However, in a cells treated with certain chemicals used in pesticides, fungicides, and industrial products (i.e., TPN (CAS No. 1897-45-6), thiuram (CAS No. 137-26-8), captan (CAS No. 133-06-2), oxine–copper (CAS No. 10380-28-6), zineb (CAS No. 12122-67-7), and ziram (CAS No. 137-30-4)) the induction of shmoo formation was suppressed even when commercial α-factor was added. The FUS1–lacZ gene, which is transcriptionally regulated by a pheromone, was transferred into yeast and the effects of TPN, captan, zineb, and ziram, under sublethal conditions, were investigated: β-Galactosidase levels declined to levels similar to that of untreated control cells when in the absence of the α-factor. Furthermore, these chemicals influenced conjugation to α-cells, and mating efficiency declined as chemical concentration increased. Analysis of the yeast pheromone signaling pathway helps to establish chemical toxicity assay models for mammalian peptide signal transduction pathways.

The vesicular transport protein Cgp1p/Vps54p/Tcs3p/Luv1p is required for the integrity of the actin cytoskeleton.

The CGP1 gene was identified in a screen for mutations that were synthetic lethal in combination with a deletion of the gene (CPF1) for centromere and promoter factor 1. Cells deleted for CGP1 showed reduced viability, were temperature sensitive for growth and exhibited altered sensitivity to microtubule-destabilizing drugs. Furthermore, Deltacgp1 cells showed increased rates of loss of a circular minichromosome and defects in the positioning of the short mitotic spindle. Further phenotypic analysis of Deltacgp1 cells revealed that loss of Cgp1p function led to severe depolarization of the actin cytoskeleton. In addition, cells deleted for CGP1 were hypersensitive to the actin-disrupting compound Latrunculin-A, exhibited strongly reduced polarized localization of the unconventional myosin Myo2p, and showed defects in other actin-related processes, such as shmoo formation and cell wall integrity. Cgp1p was recently identified by several groups as Vps54p, which is a member of the VFT complex that is involved in vesicular protein transport at the level of the late Golgi, acting as a tethering factor. Our data show for the first time that Cgp1p/Vps54p links aspects of vesicular protein transport with the organization of the actin cytoskeleton.

Role of Rho family GTPases in epithelial morphogenesis.

Epithelial cell sheets line the organ and body surfaces and the specialized barrier functions of these epithelia regulate the exchange of substances with the outside environment and between different body compartments. Epithelia play a role in a wide range of physiological processes such as digestion, excretion, and leukocyte trafficking. In addition, during development, some epithelia form transient primitive structures, including the neural tube and somites, which are essential for the development of more complex organs. The establishment and maintenance of epithelial cell polarity is critical for the development and functioning of multicellular organisms (Nelson 2000). A multi-step model for the establishment of cell polarity has been proposed by Drubin and Nelson (1996). Cell polarity is initiated by a spatial cue, such as generated by cell–cell contact sites. This cue is interpreted and marked by the formation of signaling complexes that relay the spatial information to the actin cytoskeleton. Localized actin assembly then leads to the formation of a targeting patch, which functions to reinforce the initial cue. Subsequently, this cue can further be propagated via a reorganization of the microtubule cytoskeleton, which in turn causes a redistribution of the membrane trafficking apparatus. In addition to actin cytoskeletal dynamics and vesicle trafficking, epithelial morphogenesis also depends on cell–substrate and cell–cell adhesion. Members of the Rho family of GTPases play essential roles in each of these processes (for reviews, see Hall 1998; Kaibuchi et al. 1999a,b; Braga 2000; Ellis and Mellor 2000; Schwartz and Shattil 2000; Ridley 2001a,b) and therefore it is not surprising to see that Rho GTPases have emerged as critical players at multiple stages of epithelial morphogenesis. In this review we will discuss the involvement of Rho family members in the development and maintenance of epithelial morphology and highlight recent advances in our understanding of the roles of these GTPases in the establishment of epithelial polarity. We will also discuss the participation of these GTPases in epithelial remodeling during wound-healing and epithelial-mesenchymal transitions. As other members of the Ras superfamily, Rho GTPases cycle between a GDP-bound (inactive) state and a GTP-bound (active) state. In the active state, these GTPases relay signals from growth factors, cytokines, and adhesion molecules to regulate a wide range of biological processes, including actin cytoskeleton organization, transcriptional regulation, and vesicle trafficking (Van Aelst and D’Souza-Schorey 1997; Hall 1998). The nucleotide state of Rho family proteins is controlled by three classes of regulatory proteins: guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs) (Boguski and McCormick 1993). GEFs catalyze the exchange of GDP for GTP by facilitating the release of GDP and transient stabilization of the nucleotide-free protein. GAPs promote the intrinsic GTP hydrolyzing activity of Rho proteins, thereby enhancing their conversion to the GDP-bound form. GDIs preferentially bind to GDP-bound GTPases and prevent spontaneous and GEF-catalyzed release of nucleotide, thereby maintaining the GTPases in the inactive state. Although activation of Rho GTPases in response to extracellular signals in principle could occur either via the activation of GEFs or inhibition of GAPs and GDIs, studies on oncogenic forms of GEFs suggest that nucleotide exchange is the rate-limiting step in GTPase activation. The localized activation of GEFs is likely to be of critical importance in polarity establishment and morphogenesis. Localized control of GEFs and GTPases has been extensively characterized in the budding yeast Saccharomyces cerevisiae. Polarized growth is important at several stages of the budding yeast life cycle, including bud formation during vegetative growth and shmoo formation during mating. Recent genetic and biochemical analyses of the roles of the GTPase Cdc42 and its GEF Cdc24 in these processes has led to a model in which GEF activity is regulated in four distinct steps: GEF recruitment to the plasma membrane and subsequent activation, stabilization by adaptor proteins, and termination of signaling by GEF inactivation (Gulli and Peter 2001). Less is known about the regulation of GEFs in 3Corresponding author. E-MAIL vanaelst@cshl.org; FAX (516) 367-8815. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.978802.

Relative dependence of different outputs of the Saccharomyces cerevisiae pheromone response pathway on the MAP kinase Fus3p.

Fus3p and Kss1p act at the end of a conserved signaling cascade that mediates numerous cellular responses for mating. To determine the role of Fus3p in different outputs, we isolated and characterized a series of partial-function fus3 point mutants for their ability to phosphorylate a substrate (Ste7p), activate Ste12p, undergo G1 arrest, form shmoos, select partners, mate, and recover. All the mutations lie in residues that are conserved among MAP kinases and are predicted to affect either enzyme activity or binding to Ste7p or substrates. The data argue that Fus3p regulates the various outputs assayed through the phosphorylation of multiple substrates. Different levels of Fus3p function are required for individual outputs, with the most function required for shmoo formation, the terminal output. The ability of Fus3p to promote shmoo formation strongly correlates with its ability to promote G1 arrest, suggesting that the two events are coupled. Fus3p promotes recovery through a mechanism that is distinct from its ability to promote G1 arrest and may involve a mechanism that does not require kinase activity. Moreover, catalytically inactive Fus3p inhibits the ability of active Fus3p to activate Ste12p and hastens recovery without blocking G1 arrest or shmoo formation. These results raise the possibility that in the absence of sustained activation of Fus3p, catalytically inactive Fus3p blocks further differentiation by restoring mitotic growth. Finally, suppression analysis argues that Kss1p contributes to the overall pheromone response in a wild-type strain, but that Fus3p is the critical kinase for all of the outputs tested.

KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes.

The KNR4 gene, originally isolated by complementation of a K9 killer-toxin-resistant mutant displaying reduced levels of both 1,3-beta-glucan and 1,3-beta-glucan synthase activity, was recloned from a YCp50 genomic library as a suppressor of Saccharomyces cerevisiae calcofluor-white-hypersensitive (cwh) mutants. In these mutants, which were characterized by increased chitin levels, the suppressor effect of KNR4 resulted, for some of them, in a lowering of polymer content to close to wild-type level, with no effect on the contents of beta-glucan and mannan. In all cases, this effect was accompanied by a strong reduction in mRNA levels corresponding to CHS1, CHS2 and CHS3, encoding chitin synthases, without affecting expression of FKS1 and RHO1, two genes encoding the catalytic subunit and a regulatory component of 1,3-beta-glucan synthase, respectively. Overexpression of KNR4 also inhibited expression of CHS genes in wild-type strains and in two other cwh mutants, whose sensitivity to calcofluor white was not suppressed by this gene. The physiological relevance of the KNR4 transcriptional effect was addressed in two different ways. In a wild-type strain exposed to alpha-factor, overexpression of this gene inhibited CHS1 induction and delayed shmoo formation, two events which are triggered in response to the pheromone, whereas it did not affect bud formation and cell growth in a chs1 chs2 double mutant. A chimeric protein made by fusing green fluorescent protein to the C terminus of Knr4p which fully complemented a knr4delta mutation was found to localize in patches at presumptive bud sites in unbudded cells and at the incipient bud site during bud emergence. Taken together, these results demonstrate that KNR4 has a regulatory role in chitin deposition and in cell wall assembly. A mechanism by which this gene affects expression of CHS genes is proposed.

A small conserved domain in the yeast Spa2p is necessary and sufficient for its polarized localization.

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother-daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (a2p omolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150-amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.

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.

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.

Mating-type-specific cell cycle arrest and shmoo formation by ? pheromone of Saccharomyces cerevisiae in Hansenula wingei

The cell cycle of a (5) mating type cells of Hansenula wingei was arrested in the G1 phase by α pheromone of Saccharomyces cerevisiae but not by α(21) pheromone of H. wingei, although both the α pheromones are known to induce sexual agglutination ability of a mating type cells of H. wingei. Cells of α mating type of H. wingei became shmooed or arrested in the G1 phase in response to neither a pheromone of H. wingei nor α pheromone of S. cerevisiae.

Sexual conjugation in yeast. Cell surface changes in response to the action of mating hormones.

In the yeast Saccharomyces cerevisiae, sexual conjugation between haploid cells of opposite mating type results in the formation of a diploid zygote. When treated with fluorescently labeled concanavalin A, a zygote stains nonuniformly, with the greatest fluorescence occurring at the conjugation bridge between the two haploid parents. In the mating mixture, unconjugated haploid cells often elongate to pear-shaped forms ("shmoos") which likewise exhibit asymmetric staining with the most intense fluorescence at the growing end. Shmoo formation can be induced in cells of one mating type by the addition of a hormone secreted by cells of the opposite mating type; such shmoos also stain asymmetrically. In nearly all cases, the nonmating mutants that were examined stained uniformly after incubation with the appropriate hormone. Asymmetric staining is not observed with vegetative cells, even those that are budded. These results suggest that, before and during conjugation, localized cell surface changes occur in cells of both mating types; the surface alterations facilitate fusion and are apparently mediated by the hormones in a manner that is mating-type specific.