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.     

Dominant effects of tubulin overexpression in Saccharomyces cerevisiae.

The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.     

Coupling actin dynamics to the endocytic process in Saccharomyces cerevisiae

Summary. Endocytosis is an essential eukaryotic process that, in many systems, has been reported to require a functional actin cytoskeleton. The process of endocytosis is critical for controlling the protein–lipid composition of the plasma membrane and uptake of nutrients as well as pathogens and also plays an important role in regulation of cell signalling. While several distinct pathways for endocytosis have been characterised, all of these require remodelling of the cell cortex. The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognised for many years in budding yeast and is increasingly supported by studies in mammalian cells. Current evidence suggests that cortical patches are sites of endocytosis in Saccharomyces cerevisiae and that these sites are composed of sequentially forming protein complexes. Distinct stages in complex formation are characterised by the presence of different activators of F-actin polymerisation. Disassembly of the complexes is also essential for the endocytosis to proceed. Mutants lacking the kinases Ark1 and Prk1 accumulate actin and endocytic machinery in a single large clump in cells. Phosphorylation of endocytic proteins including Sla1p is proposed to cause their removal from the complex and allow later stages of the invagination process to occur. Dephosphorylation of endocytic components may then allow subsequent reincorporation into new sites of endocytic complex assembly. Correspondence and reprints: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.     

Dissection of septin actin interactions using actin overexpression in Saccharomyces cerevisiae

Although many proteins can be overexpressed several fold without much effect on cell viability and morphology, some become toxic upon a slight increase in their intracellular level. This is particularly true for cytoskeletal proteins and has proven useful in the past for studying the cytoskeleton. In yeast, actin and tubulin are examples of proteins that cannot be overexpressed without affecting cell viability. Here, we have analysed the effect of actin overexpression in Saccharomyces cerevisiae. We show that actin overexpression interferes differently with distinct aspects of actin function. For example, two- to fourfold overexpression of actin did not affect the establishment of actin polarity, whereas it abrogated its maintenance. Also, actin structures that are barely visible in wild-type cells could be observed upon actin overexpression. This allowed us to identify a new ring-like actin structure genetically distinguishable from the actomyosin contractile ring. Formation of this actin structure upon actin overexpression was dependent on the septin cytoskeleton, the poorly understood cytokinetic protein Hof1 and the Arp2/3 complex. In contrast to the actomyosin ring, the ring formed upon actin overexpression required neither Myo1 nor formins for assembly. Therefore, we propose that Hof1 acts as a linker between actin and septins. Furthermore, we found that, in the absence of actin overexpression, a novel, Hof1-dependent actin belt is formed at the bud neck of anaphase cells. The physiological role of this belt might be related to that of the similar structure observed in dividing fission yeast.     

Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae

We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.     

Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis.

We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.     

Calcium control of Saccharomyces cerevisiae actin assembly.

Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo.     

Requirement of Bni5 phosphorylation for bud morphogenesis in Saccharomyces cerevisiae

In budding yeast, G2/M transition is tightly correlated with bud morphogenesis regulated by Swe1 and septin that plays as a scaffold to recruits protein components. BNI5 isolated as a suppressor for septin defect is implicated in septin organization and cytokinesis. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we show that Bni5 phosphorylation is required for mitotic entry regulated by Swe1 pathway. Bni5 modification was evident from late mitosis to G1 phase, and CIP treatment in vitro of affinity-purified Bni5 removed the modification, indicative of phosphorylation on Bni5. The phosphorylation-deficient mutant of BNI5 (bni5-4A) was defective in both growth at semi-restrictive temperature and suppression of septin defect. Loss of Bni5 phosphorylation resulted in abnormal bud morphology and cell cycle delay at G2 phase, as evidenced by the formation of elongated cells with multinuclei. However, deletion of Swe1 completely eliminated the elongated-bud phenotypes of both bni5 deletion and bni5-4A mutants. These results suggest that the bud morphogenesis and mitotic entry are positively regulated by phosphorylation-dependent function of Bni5 which is under the control of Swe1 morphogenesis pathway.     

Tubulin and actin topology during zygote formation of Saccharomyces cerevisiae

The topology of tubulin and actin during mating of Saccharomyces cerevisiae was analysed by fluorescence microscopy with the monoclonal anti-tubulin antibody Tu01 and rhodamine-labelled phalloidin. Preconjugatory cells displayed an asymmetric distribution of the microtubule and actin cytoskeleton and an overall polarization of the cells preceding cell fusion. Prior to karyogamy, the haploid spindle pole bodies were associated with abundant cytoplasmic microtubules. Budding zygotes revealed the same tubulin and actin patterns as vegetative cells. Treatment of the mating mixture with the microtubule inhibitor nocodazole (10 micrograms ml-1) did not prevent polarization and fusion of haploids, zygote formation and emergence of the first zygotic bud. In marked contrast, the migration of the nucleus in preconjugatory cells as well as nuclear migration and fusion within the zygotes was unequivocally blocked by the action of the drug. It is suggested that the problem of the morphogenesis of mating should be approached by considering interactions at the cell periphery.     

Proteomic characterization of a wild-type wine strain of Saccharomyces cerevisiae

Saccharomyces cerevisiae is the optimal eukaryotic model system to study mammalian biological responses. At the same time Saccharomyces cerevisiae is also widely utilized as a biotechnological tool in the food industry. Enological Saccharomyces cerevisiae strains have been so far routinely analyzed for their microbiological aspects. Nevertheless, wine yeasts are gaining an increasing interest in the last years since they strongly affect both the vinification process and the organoleptic properties of the final product wine. The protein repertoire is responsible of such features and, consequently, 2D-PAGE can be an useful tool to evaluate and select optimal wine yeast strains. We present here the first proteomic map of a wild-type wine Saccharomyces cerevisiae strain selected for the guided fermentation of very high quality wines.     

Trypanosoma cruzi: distribution of fluorescently labeled tubulin and actin in epimastigotes

Cytoskeletal components were visualized in epimastigote forms of Trypanosoma cruzi by double immunofluorescence microscopy using monospecific antibodies against tubulin and against actin. Intense staining of the flagellum and the edges of the cell body was observed when the cells were stained with anti-tubulin, reflecting the presence of the basal bodies, the flagellar axoneme and the subpellicular microtubules. A less intense staining was seen in the cell body of epimastigotes stained with anti-actin. However, an intense staining was observed with this antibody in the flagellum, in a pattern similar to that observed with anti-tubulin. It is suggested that the paraxial structure, which is formed by a complex array of 6-nm-thick microfilaments is composed, at least in part, of actin.     

An electron microscopic study of bud development in Saccharomycodes ludwigii and Saccharomyces cerevisiae

Summary Examination of sectioned cells fixed in KMnO₄ has shown that the wall of the first bud of a cell of Saccharomycodes ludwigii arises as an extension of the main wall of the parent, while in subsequent buds it develops by extension of the half-septum remaining at a previous detachment scar. Septa are formed by the deposition of wall material on each side of an electron transparent plate which develops centripetally. Structural changes occur in the marginal region of the septum prior to rupture of the main wall and the separation of cells at the surface of the septum-plate. The broken walls remain as annular rings around the scars following the successive development of buds at both apices of the cell.In Saccharomyces cerevisiae the bud wall arises as a direct extension of the parent wall or as an extension of an additional inner layer developed locally.The two types of bud origin are compared in the two yeasts and a comparison is also made with the development of buds, fission cells, conidia and germ tubes in other organisms.     

Saccharomyces cerevisiae forms actin ring structures in sporulation, similarly to Zygosaccharomyces rouxii

Yeast filamentous actin (F-actin) exists mainly as patches and cables. Previously, we investigated the behavior of F-actin during sporulation of Zygosaccharomyces rouxii and found a novel actin ring localized around the spore periphery in zygotic asci at a late stage of sporulation. To clarify whether the actin rings are also formed in sporulation in the model yeast Saccharomyces cerevisiae, we observed the distribution of F-actin in sporulating S. cerevisiae by rhodamine-phalloidin staining and confocal laser scanning microscopy. Ringlike actin structures were detected at the peripheral regions of S. cerevisiae spores in globose asci. When asci of S. cerevisiae were induced to become zygotic, actin rings were more obvious than those in globose asci. These results indicate that S. cerevisiae forms characteristic actin ring structures at a late stage of sporulation, similarly to Z. rouxii.     

Novel proteins linking the actin cytoskeleton to the endocytic machinery in Saccharomyces cerevisiae

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.     

Xylulose fermentation by mutant and wild-type strains of Zygosaccharomyces and Saccharomyces cerevisiae

Anaerobic xylulose fermentation was compared in strains of Zygosaccharomyces and Saccharomyces cerevisiae, mutants and wild-type strains to identify host-strain background and genetic modifications beneficial to xylose fermentation. Overexpression of the gene (XKS1) for the pentose phosphate pathway (PPP) enzyme xylulokinase (XK) increased the ethanol yield by almost 85% and resulted in ethanol yields [0.61 C-mmol (C-mmol consumed xylulose)⁻¹] that were close to the theoretical yield [0.67 C-mmol (C-mmol consumed xylulose)⁻¹]. Likewise, deletion of gluconate 6-phosphate dehydrogenase (gnd1Δ) in the PPP and deletion of trehalose 6-phosphate synthase (tps1Δ) together with trehalose 6-phosphate phosphatase (tps2Δ) increased the ethanol yield by 30% and 20%, respectively. Strains deleted in the promoter of the phosphoglucose isomerase gene (PGI1) – resulting in reduced enzyme activities – increased the ethanol yield by 15%. Deletion of ribulose 5-phosphate (rpe1Δ) in the PPP abolished ethanol formation completely. Among non-transformed and parental strains S. cerevisiae ENY. WA-1A exhibited the highest ethanol yield, 0.47 C-mmol (C-mmol consumed xylulose)⁻¹. Other non-transformed strains produced mainly arabinitol or xylitol from xylulose under anaerobic conditions. Contrary to previous reports S. cerevisiae T23D and CBS 8066 were not isogenic with respect to pentose metabolism. Whereas, CBS 8066 has been reported to have a high ethanol yield on xylulose, 0.46 C-mmol (C-mmol consumed xylulose)⁻¹ (Yu et al. 1995), T23D only formed ethanol with a yield of 0.24 C-mmol (C-mmol consumed xylulose)⁻¹. Strains producing arabinitol did not produce xylitol and vice versa. However, overexpression of XKS1 shifted polyol formation from xylitol to arabinitol. Received: 2 July 1999 / Accepted in revised form: 12 October 1999     

Genetic determination of polygalacturonase production in wild-type and laboratory strains of Saccharomyces cerevisiae

The genetic determination of polygalacturonase (PG) production in Saccharomyces cerevisiae was studied by biochemical and classical genetic techniques. Crosses of PG⁺ strains with PG^– strains showed that in the haploid wild-type-derived strain, two structural genes were involved in the production of a hydrolysis halo on plates with polygalacturonic acid. However, in the case of PG⁺ laboratory strain IM1-8b, the phenotype was controlled by only one structural gene although the analysis of PG^– IM1-8b mutants demonstrated the existence of at least two complementation groups. All these genetic results were assessed biochemically by means of cation-exchange chromatography. Two enzymes were separated in the wild-type strain, and only one in the laboratory strain. The three enzymes had different K _m values, molecular masses, and optimal pHs for activity. Received: 24 October 1996 / Accepted: 17 December 1996     

Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number.

Microtubule organization in the cytoplasm is in part a function of the number and length of the assembled polymers. The intracellular concentration of tubulin could specify those parameters. Saccharomyces cerevisiae strains constructed with moderately decreased or increased copy numbers of tubulin genes provide an opportunity to study the cellular response to a steady-state change in tubulin concentration. We found no evidence of a mechanism for adjusting tubulin concentrations upward from a deficit, nor did we find a need for such a mechanism: cells with no more than 50% of the wild-type tubulin level were normal with respect to a series of microtubule-dependent properties. Strains with increased copies of both alpha- and beta-tubulin genes, or of alpha-tubulin genes alone, apparently did down regulate their tubulin levels. As a result, they contained greater than normal concentrations of tubulin but much less than predicted from the increase in gene number. Some of this down regulation occurred at the level of protein. These strains were also phenotypically normal. Cells could contain excess alpha-tubulin protein without detectable consequences, but perturbations resulting in excess beta-tubulin genes may have affected microtubule-dependent functions. All of the observed regulation of levels of tubulin can be explained as a response to toxicity associated with excess tubulin proteins, especially if beta-tubulin is much more toxic than alpha-tubulin.     

Regulation of allantoate transport in wild-type and mutant strains of Saccharomyces cerevisiae.

Accumulation of intracellular allantoin and allantoate is mediated by two distinct active transport systems in Saccharomyces cerevisiae. Allantoin transport (DAL4 gene) is inducible, while allantoate uptake is constitutive (it occurs at full levels in the absence of any allantoate-related compounds from the culture medium). Both systems appear to be sensitive to nitrogen catabolite repression, feedback inhibition, and trans-inhibition. Mutants (dal5) that lack allantoate transport have been isolated. These strains also exhibit a 60% loss of allantoin transport capability. Conversely, dal4 mutants previously described are unable to transport allantoin and exhibit a 50% loss of allantoate transport. We interpret the pleiotropic behavior of the dal4 and dal5 mutations as deriving from a functional interaction between elements of the two transport systems.