Rapid, efficient auxin-inducible protein degradation in Candida pathogens.

A variety of inducible protein degradation (IPD) systems have been developed as powerful tools for protein functional characterization. IPD systems provide a convenient mechanism for rapid inactivation of almost any target protein of interest. Auxin-inducible degradation (AID) is one of the most common IPD systems and has been established in diverse eukaryotic research model organisms. Thus far, IPD tools have not been developed for use in pathogenic fungal species. Here, we demonstrate that the original AID and the second generation, AID2, systems work efficiently and rapidly in the human pathogenic yeasts, Candida albicans and Candida glabrata. We developed a collection of plasmids that support AID system use in laboratory strains of these pathogens. These systems can induce >95% degradation of target proteins within minutes. In the case of AID2, maximal degradation was achieved at low nanomolar concentrations of the synthetic auxin analog 5-adamantyl-indole-3-acetic acid. Auxin-induced target degradation successfully phenocopied gene deletions in both species. The system should be readily adaptable to other fungal species and to clinical pathogen strains. Our results define the AID system as a powerful and convenient functional genomics tool for protein characterization in fungal pathogens. IMPORTANCE Life-threatening fungal infections are an escalating human health problem, complicated by limited treatment options and the evolution of drug resistant pathogen strains. Identification of new targets for therapeutics to combat invasive fungal infections, including those caused by Candida species, is an urgent need. In this report, we establish and validate an inducible protein degradation methodology in Candida albicans and Candida glabrata that provides a new tool for protein functional characterization in these, and likely other, fungal pathogen species. We expect this tool will ultimately be useful for the identification and characterization of promising drug targets and factors involved in virulence and drug resistance.

Eng2, a new player involved in feedback loop regulation of Cdc42 activity in fission yeast.

Cell polarity and morphogenesis are regulated by the small GTPase Cdc42. Even though major advances have been done in the field during the last years, the molecular details leading to its activation in particular cellular contexts are not completely understood. In fission yeast, the ß(1,3)-glucanase Eng2 is a "moonlighting protein" with a dual function, acting as a hydrolase during spore dehiscence, and as component of the endocytic machinery in vegetative cells. Here, we report that Eng2 plays a role in Cdc42 activation during polarized growth through its interaction with the scaffold protein Scd2, which brings Cdc42 together with its guanine nucleotide exchange factor (GEF) Scd1. eng2Δ mutant cells have defects in activation of the bipolar growth (NETO), remaining monopolar during all the cell cycle. In the absence of Eng2 the accumulation of Scd1 and Scd2 at the poles is reduced, the levels of Cdc42 activation decrease, and the Cdc42 oscillatory behavior, associated with bipolar growth in wild type cells, is altered. Furthermore, overexpression of Eng2 partially rescues the growth and polarity defects of a cdc42-L160S mutant. Altogether, our work unveils a new factor regulating the activity of Cdc42, which could potentially link the polarity and endocytic machineries.

Glucanases and Chitinases.

In many yeast and fungi, ß-(1,3)-glucan and chitin are essential components of the cell wall, an important structure that surrounds cells and which is responsible for their mechanical protection and necessary for maintaining the cellular shape. In addition, the cell wall is a dynamic structure that needs to be remodelled along with the different phases of the fungal life cycle or in response to extracellular stimuli. Since ß-(1,3)-glucan and chitin perform a central structural role in the assembly of the cell wall, it has been postulated that ß-(1,3)-glucanases and chitinases should perform an important function in cell wall softening and remodelling. This review focusses on fungal glucanases and chitinases and their role during fungal morphogenesis.

A new toolkit for gene tagging in Candida albicans containing recyclable markers.

Gene manipulation and epitope tagging are essential tools for understanding the molecular function of specific genes. The opportunistic human pathogen Candida albicans is a diploid fungus that utilizes a non-canonical genetic code. Since selection markers available in this organism are scarce, several tools based on recyclable markers have been developed for gene disruption, such as the Clox system. This system relies on the Cre recombinase, which recycles selection markers flanked by loxP sites with high efficiency, facilitating single marker or multi-marker recycling. However, PCR-based modules for epitope tagging, such the pFA-modules, mainly use limited non-recyclable auxotrophic markers. To solve this problem, we have used a Gibson assembly strategy to construct a set of new plasmids where the auxotrophic markers of the pFA vectors were swapped with five recyclable marker modules of the Clox system, enhancing the versatility of the pFA plasmids. This new toolkit, named pFA-Clox, is composed of 36 new vectors for gene disruption and epitope tagging (GFP, 3xGFP, mCherry, 3xHA, 5xmyc and TAP). These plasmids contain the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting functional analysis of laboratory strains as well as clinical isolates of C. albicans. In summary, we have adapted the Clox system to the pFA-backbone vectors. Thus, the set of primers used for the amplification of previously published pFA modules can also be utilized in this new pFA-Clox system. Therefore, this new toolkit harbors the advantages of both systems, allowing accelerated gene modification strategies that could reduce time and costs in strain construction for C. albicans.

Signalling through the yeast MAPK Cell Wall Integrity pathway controls P-body assembly upon cell wall stress.

Post-transcriptional control of mRNA is a key event in the regulation of gene expression. From yeast to human cells, P-bodies are cytoplasmic RNA-protein aggregates that play an essential role in this process, particularly under stress conditions. In this work, we show that in the model yeast Saccharomyces cerevisiae cell wall stress induces the formation of these structures. This effect is dependent on multiple elements in the Cell Wall Integrity (CWI) MAPK signalling pathway, a signal transduction cascade responsible for the maintenance of cell integrity under adverse environmental conditions. Remarkably, P-body assembly requires the catalytic activity of the MAPK of the pathway, Slt2/Mpk1. In accordance with the control exerted by this signalling pathway, the timing of P-body formation is similar to that of the activation of the CWI pathway. Noticeably, mRNAs whose expression is regulated by this pathway localize in P-bodies after the cell is exposed to stress following a temporal pattern coincident with CWI pathway activation. Moreover, when these mRNAs are overexpressed in a mutant background unable to form visible P-bodies, the cells show hypersensitivity to agents that interfere with cell wall integrity, supporting that they play a role in the mRNA lifecycle under stress conditions.

The anillin-related Int1 protein and the Sep7 septin collaborate to maintain cellular ploidy in Candida albicans.

Variation in cell ploidy is a common feature of Candida albicans clinical isolates that are resistant to the antifungal drug fluconazole. Here, we report that the anillin-related protein Int1 interacts with septins for coupling cytokinesis with nuclear segregation. Loss of Int1 results in a rapid disassembly of duplicated septin rings from the bud neck at the onset of actomyosin ring contraction. Strikingly, this has no major impact on cytokinesis and septum formation. However, Int1 genetically interacts with the Sep7 septin, maintaining the diffusion barrier at the bud neck and guarantying a faithful nuclear segregation. Indeed, int1ΔΔ sep7ΔΔ mutant cells, in contrast to int1ΔΔ cdc10ΔΔ, undergo a premature activation of mitotic exit prior to the alignment of the mitotic spindle with the division axis, producing large multinucleated cells. Some of these multinucleated cells arise from trimeras similar to those observed upon fluconazole exposure. Finally, the defects in nuclear segregation could be in part due to the inability to maintain the Lte1 mitotic exit activator at the cortex of the daughter cell. These results suggest that Int1 and Sep7 play a role in maintaining genome stability by acting as a diffusion barrier for Lte1.

Regulation of Ace2-dependent genes requires components of the PBF complex in Schizosaccharomyces pombe.

The division cycle of unicellular yeasts is completed with the activation of a cell separation program that results in the dissolution of the septum assembled during cytokinesis between the 2 daughter cells, allowing them to become independent entities. Expression of the eng1(+) and agn1(+) genes, encoding the hydrolytic enzymes responsible for septum degradation, is activated at the end of each cell cycle by the transcription factor Ace2. Periodic ace2(+) expression is regulated by the transcriptional complex PBF (PCB Binding Factor), composed of the forkhead-like proteins Sep1 and Fkh2 and the MADS box-like protein Mbx1. In this report, we show that Ace2-dependent genes contain several combinations of motifs for Ace2 and PBF binding in their promoters. Thus, Ace2, Fkh2 and Sep1 were found to bind in vivo to the eng1(+) promoter. Ace2 binding was coincident with maximum level of eng1(+) expression, whereas Fkh2 binding was maximal when mRNA levels were low, supporting the notion that they play opposing roles. In addition, we found that the expression of eng1(+) and agn1(+) was differentially affected by mutations in PBF components. Interestingly, agn1(+) was a major target of Mbx1, since its ectopic expression resulted in the suppression of Mbx1 deletion phenotypes. Our results reveal a complex regulation system through which the transcription factors Ace2, Fkh2, Sep1 and Mbx1 in combination control the expression of the genes involved in separation at the end of the cell division cycle.

Eng2 is a component of a dynamic protein complex required for endocytic uptake in fission yeast.

Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2-GFP dynamics did not match the pattern of other endocytic proteins. Eng2-GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3-YFP, a WASP-interacting protein, interacted with Eng2 by co-immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module.

The NDR/LATS kinase Cbk1 controls the activity of the transcriptional regulator Bcr1 during biofilm formation in Candida albicans.

In nature, many microorganisms form specialized complex, multicellular, surface-attached communities called biofilms. These communities play critical roles in microbial pathogenesis. The fungal pathogen Candida albicans is associated with catheter-based infections due to its ability to establish biofilms. The transcription factor Bcr1 is a master regulator of C. albicans biofilm development, although the full extent of its regulation remains unknown. Here, we report that Bcr1 is a phosphoprotein that physically interacts with the NDR kinase Cbk1 and undergoes Cbk1-dependent phosphorylation. Mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to alanine markedly impaired Bcr1 function during biofilm formation and virulence in a mouse model of disseminated candidiasis. Cells lacking Cbk1, or any of its upstream activators, also had reduced biofilm development. Notably, mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to glutamate in cbk1Δ cells upregulated the transcription of Bcr1-dependent genes and partially rescued the biofilm defects of a cbk1Δ strain. Therefore, our data uncovered a novel role of the NDR/LATS kinase Cbk1 in the regulation of biofilm development through the control of Bcr1.

Conserved regulators of the cell separation process in Schizosaccharomyces.

The fission yeasts (Schizosaccharomyces) representing a highly divergent phylogenetic branch of Fungi evolved from filamentous ancestors by gradual transition from mycelial growth to yeast morphology. For the transition, a mechanism had been developed that separates the sister cells after the completion of cytokinesis. Numerous components of the separation mechanism have been characterised in Schizosaccharomycespombe, including the zinc-finger transcription factor Ace2p and the fork-head transcription factor Sep1p. Here we show that both regulators have regions conserved within the genus. The most conserved parts contain the DNA-binding domains whose amino-acid sequences perfectly reflect the phylogenetic positions of the species. The less conserved parts of the proteins contain sequence blocks specific for the whole genus or only for the species propagating predominantly or exclusively as yeasts. Inactivation of either gene in the dimorphic species Schizosaccharomycesjaponicus abolished cell separation in the yeast phase conferring hypha-like morphology but did not change the growth pattern to unipolar and did not cause extensive polar vacuolation characteristic of the true mycelium. Neither mutation affected the mycelial phase, but both mutations hampered the hyphal fragmentation at the mycelium-to-yeast transition. Ace2p(Sj) acts downstream of Sep1p(Sj) and regulates the orthologues of the Ace2p-dependent S.pombe genes agn1(+) (1,3-alpha-glucanase) and eng1(+) (1,3-beta-glucanase) but does not regulate the orthologue of cfh4(+) (chitin synthase regulatory factor). These results and the complementation of the cell separation defects of the ace2(-) and sep1(-) mutations of S.pombe by heterologously expressed ace2(Sj) and sep1(Sj) indicate that the cell separation mechanism is conserved in the Schizosaccharomyces genus.

ß(1,3)-glucanosyl-transferase activity is essential for cell wall integrity and viability of Schizosaccharomyces pombe.

BACKGROUND: The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of ß-glucans. The ß(1,3)-glucan synthase complex synthesizes linear ß(1,3)-glucans, which remain unorganized until they are cross-linked to other ß(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding ß(1,3)-glucanosyl-transferases -gas1(+), gas2(+), gas4(+) and gas5(+)- are present in S. pombe, although their function has not been analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display ß(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast. CONCLUSIONS/SIGNIFICANCE: We conclude that ß(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth.

{beta}-glucanase Eng2 is required for ascus wall endolysis after sporulation in the fission yeast Schizosaccharomyces pombe.

Meiosis is the developmental program by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. When Schizosaccharomyces pombe diploid cells undergo meiosis, they differentiate into asci containing four haploid ascospores that are highly resistant to environmental stress. The formation of the ascospore wall requires the activity of several enzymes involved in the biosynthesis and modification of its components, such as alpha- and beta-glucan synthases. Once the spores are completely mature, the wall of the ascus undergoes an endolytic process that results in the release of ascospores from the ascus, allowing their dispersal into the environment. This process requires the activity of the endo-alpha-1,3-glucanase Agn2. Here, we focus on the characterization of the endo-beta-1,3-glucanase Eng2, which is also required for ascospore release from the ascus. Although Eng2 is present during the mitotic cycle, the protein accumulates after meiosis II. The expression of eng2(+) is required for the efficient release of ascospores, as shown by placing eng2(+) under the control of a repressible promoter. Furthermore, a point mutation that destroys the catalytic activity of the protein results in a phenotype similar to that of the mutant strain. Finally, we demonstrate that exogenous addition of purified Eng2 releases the ascospores from asci generated by an eng2Delta mutant. We propose that Eng2 would act together with Agn2 to completely hydrolyze the ascus wall, thereby assisting in the release of ascospores in S. pombe.

Dbf2 is essential for cytokinesis and correct mitotic spindle formation in Candida albicans.

We have characterized the DBF2 gene, encoding a protein kinase of the NDR family in Candida albicans, and demonstrate that this gene is essential for cell viability. Conditional mutants were constructed by using the MET3 promoter to analyse the phenotype of cells lacking this kinase. The absence of Dbf2 resulted in cells arrested as large-budded pairs that failed to contract the actomyosin ring, a function similar to that described for its Saccharomyces cerevisiae orthologue. In addition to its role in cytokinesis, Dbf2 regulates mitotic spindle organization and nuclear segregation as Dbf2-depleted cells have abnormal microtubules and severe defects in nuclear migration to the daughter cell, which results in a cell cycle block during mitosis. Taken together, these results imply that Dbf2 performs several functions during exit from mitosis and cytokinesis. Consistent with a role in spindle organization, the protein localizes to the mitotic spindle during anaphase, and it interacts physically with tubulin, as indicated by immunoprecipitation experiments. Finally, DBF2 depletion also resulted in impaired true hyphal growth.

Septins localize to microtubules during nutritional limitation in Saccharomyces cerevisiae.

BACKGROUND: In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. RESULTS: Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11) from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. CONCLUSION: Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources.

The Schizosaccharomyces pombe endo-1,3-beta-glucanase Eng1 contains a novel carbohydrate binding module required for septum localization.

Cell separation in Schizosaccharomyces pombe is achieved through the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ring-like structure that surrounds the septum. Correct localization of these hydrolases requires the presence of both the septins and the exocyst. In this work, we show that the glucanase Eng1 contains a region at the C-terminus that acts as a carbohydrate-binding module (CBM) and that it is not present in other members of glycoside hydrolases family 81 (GH81). In vitro, the purified CBM has affinity for beta-1,3-glucan chains with a minimum degree of polymerization of 30 glucose units. Deletion of the CBM results in a protein that is largely defective in complementing the separation defect of eng1Delta mutants. This defect is due to a reduction in the catalytic activity against insoluble substrates and to a defect in targeting of Eng1 to the septum, as the truncated protein localizes to the lateral cell wall of the cell. Thus, the targeting of Eng1 to the primary septum requires not only trans-factors (septins and the exocyst complex) but also a cis-element localized to the C-terminus of the protein.

The beta-1,3-glucanosyltransferase gas4p is essential for ascospore wall maturation and spore viability in Schizosaccharomyces pombe.

Meiosis is the developmental programme by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. The formation of the Schizosaccharomyces pombe ascospore wall requires the co-ordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. During sporogenesis, the beta-1,3-glucan synthase bgs2p synthesizes linear beta-1,3-glucans, which remain unorganized and alkali-soluble until covalent linkages are set up between beta-1,3-glucans and other cell wall components. Several proteins belonging to the glycoside hydrolase family 72 (GH72) with beta-1,3-glucanosyltransferase activity have been described in other organisms, such as the Saccharomyces cerevisiae Gas1p or the Aspergillus fumigatus Gel1p. Here we describe the characterization of gas4(+), a new gene that encodes a protein of the GH72 family. Deletion of this gene does not lead to any apparent defect during vegetative growth, but homozygous gas4Delta diploids show a sporulation defect. Although meiosis occurs normally, ascospores are unable to mature or to germinate. The expression of gas4(+) is strongly induced during sporulation and a yellow fluorescent protein (YFP)-gas4p fusion protein localizes to the ascospore periphery during sporulation. We conclude that gas4p is required for ascospore maturation in S. pombe.

Sep7 is essential to modify septin ring dynamics and inhibit cell separation during Candida albicans hyphal growth.

When Candida albicans yeast cells receive the appropriate stimulus, they switch to hyphal growth, characterized by continuous apical elongation and the inhibition of cell separation. The molecular basis of this inhibition is poorly known, despite its crucial importance for hyphal development. In C. albicans, septins are important for hypha formation and virulence. Here, we used fluorescence recovery after photobleaching analysis to characterize the dynamics of septin rings during yeast and hyphal growth. On hyphal induction, septin rings are converted to a hyphal-specific state, characterized by the presence of a frozen core formed by Sep7/Shs1, Cdc3 and Cdc12, whereas Cdc10 is highly dynamic and oscillates between the ring and the cytoplasm. Conversion of septin rings to the hyphal-specific state inhibits the translocation of Cdc14 phosphatase, which controls cell separation, to the hyphal septum. Modification of septin ring dynamics during hyphal growth is dependent on Sep7 and the hyphal-specific cyclin Hgc1, which partially controls Sep7 phosphorylation status and protein levels. Our results reveal a link between the cell cycle machinery and septin cytoskeleton dynamics, which inhibits cell separation in the filaments and is essential for hyphal morphogenesis.

Cdc15 is required for spore morphogenesis independently of Cdc14 in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae exit from mitosis requires the Cdc14 phosphatase to reverse CDK-mediated phosphorylation. Cdc14 is released from the nucleolus by the Cdc14 early anaphase release (FEAR) and mitotic exit network (MEN) pathways. In meiosis, the FEAR pathway is essential for exit from anaphase I. The MEN component Cdc15 is required for the formation of mature spores. To analyze the role of Cdc15 during sporulation, a conditional mutant in which CDC15 expression was controlled by the CLB2 promoter was used. Cdc15-depleted cells proceeded normally through the meiotic divisions but were unable to properly disassemble meiosis II spindles. The morphology of the prospore membrane was aberrant and failed to capture the nuclear lobes. Cdc15 was not required for Cdc14 release from the nucleoli, but it was essential to maintain Cdc14 released and for its nucleo-cytoplasmic transport. However, cells carrying a CDC14 allele with defects in nuclear export (Cdc14-DeltaNES) were able to disassemble the spindle and to complete spore formation, suggesting that the Cdc14 nuclear export defect was not the cause of the phenotypes observed in cdc15 mutants.

Rho4 GTPase is involved in secretion of glucanases during fission yeast cytokinesis.

Rho GTPases are regulators of signaling pathways that control actin organization and cell polarity processes in all eukaryotic cells. In Schizosaccharomyces pombe, Rho4p is involved in the regulation of septum degradation during cytokinesis. Here we show that Rho4p participates in the secretion of the glucanases Eng1p and Agn1p, which are responsible for the septum degradation. First, eng1+ or agn1+ overexpression suppressed the rho4delta multiseptation phenotype, and simultaneous overproduction of Rho4p and Eng1p or of Rho4p and Agn1p caused a dramatic lysis. Second, Rho4p was not necessary for Eng1p-mediated glucanase activity as measured in cell extracts; however, rho4delta cells have a lower level of (1,3)-beta-D-glucanase activity in the culture medium. Additionally, Eng1- or Agn1-green fluorescent protein did not properly localize to the septum in rho4delta cells grown at 37 degrees C. There was a decreased amount of these enzymes in the cell wall and in the culture medium of rho4delta cells at 37 degrees C. These results provide evidence that Rho4p is involved in the regulation of Eng1p and Agn1p secretion during cytokinesis.

Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene.

To improve our understanding of the factors involved in the osmotic stability of yeast cells, a search for novel conditional Saccharomyces cerevisiae cell lysis mutants was performed. Ten temperature-sensitive (ts) mutant strains of S. cerevisiae were isolated that lyse at the restrictive temperature on hypotonic, but not on osmotically supported medium. The ten mutants fell into four complementation groups: ts1 to ts4. To clone the wild-type gene corresponding to the ts4 mutation, a strategy aimed at complementing the thermosensitive phenotype-using low-copy and high-copy DNA libraries--was followed, but only two extragenic suppressors were identified. Another approach, in which classic genetic methods were combined with the use of yeast artificial chromosomes and traditional cloning procedures, allowed the identification of the NUD1 gene--which codes for a component of the spindle-pole body-as the wild-type gene corresponding to the ts4 mutation. Cloning and sequencing of the defective allele from the chromosome of the mutant cells resulted in the identification of a point mutation that produces a single amino acid change in the protein: a Gly-to-Glu change at position 585 (the nud1-G585E allele). Further analysis revealed that cells carrying this allele show a thermosensitive growth defect. At the restrictive temperature, the cells arrest with large buds, elongated spindles, and duplicated nuclei. In addition, with longer incubation times they are unable to maintain cellular integrity and lyse. Our results have allowed the identification of the first single amino acid mutation in NUD1, and suggest a link between cell cycle progression and cellular integrity.