Type V myosin focuses the polarisome and shapes the tip of yeast cells.

The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and the fusion of secretory vesicles in yeasts and filamentous fungi. Polarisomes are compact, spotlike structures at the growing tips of their respective cells. The molecular forces that control the form and size of this microcompartment are not known. Here we identify a complex between the polarisome subunit Pea2 and the type V Myosin Myo2 that anchors Myo2 at the cortex of yeast cells. We discovered a point mutation in the cargo-binding domain of Myo2 that impairs the interaction with Pea2 and consequently the formation and focused localization of the polarisome. Cells carrying this mutation grow round instead of elongated buds. Further experiments and biophysical modeling suggest that the interactions between polarisome-bound Myo2 motors and dynamic actin filaments spatially focus the polarisome and sustain its compact shape.

A time-resolved interaction analysis of Bem1 reconstructs the flow of Cdc42 during polar growth.

Cdc42 organizes cellular polarity and directs the formation of cellular structures in many organisms. By locating Cdc24, the source of active Cdc42, to the growing front of the yeast cell, the scaffold protein Bem1, is instrumental in shaping the cellular gradient of Cdc42. This gradient instructs bud formation, bud growth, or cytokinesis through the actions of a diverse set of effector proteins. To address how Bem1 participates in these transformations, we systematically tracked its protein interactions during one cell cycle to define the ensemble of Bem1 interaction states for each cell cycle stage. Mutants of Bem1 that interact with only a discrete subset of the interaction partners allowed to assign specific functions to different interaction states and identified the determinants for their cellular distributions. The analysis characterizes Bem1 as a cell cycle-specific shuttle that distributes active Cdc42 from its source to its effectors. It further suggests that Bem1 might convert the PAKs Cla4 and Ste20 into their active conformations.

Cdc24 interacts with septins to create a positive feedback loop during bud site assembly in yeast.

Yeast cells select the position of their new bud at the beginning of each cell cycle. The recruitment of septins to this prospective bud site is one of the critical events in a complex assembly pathway that culminates in the outgrowth of a new daughter cell. During recruitment, septin rods follow the high concentration of Cdc42GTP that is generated by the focused localization of the Cdc42 guanine-nucleotide-exchange factor Cdc24. We show that, shortly before budding, Cdc24 not only activates Cdc42 but also transiently interacts with Cdc11, the septin subunit that caps both ends of the septin rods. Mutations in Cdc24 that reduce affinity to Cdc11 impair septin recruitment and decrease the stability of the polarity patch. The interaction between septins and Cdc24 thus reinforces bud assembly at sites where septin structures are formed. Once the septins polymerize to form the septin ring, Cdc24 is found at the cortex of the bud and directs further outgrowth from this position.

The APSES protein Sok2 is a positive regulator of sporulation in Ashbya gossypii.

Ashbya gossypii is a homothallic, flavinogenic, filamentous ascomycete that starts overproduction of riboflavin and fragments its mycelium quantitatively into spore producing sporangia at the end of a growth phase. Mating is not required for sporulation and the standard homothallic laboratory strain is a MATa strain. Here we show that ectopic expression of Saccharomyces cerevisiae MATα2 in A. gossypii completely suppresses sporulation, inhibits riboflavin overproduction and downregulates among others AgSOK2. AgSok2 belongs to a fungal-specific group of (APSES) transcription factors. Deletion of AgSOK2 strongly reduces riboflavin production and blocks sporulation. The initiator of meiosis, AgIME1, is a transcription factor essential for sporulation. We characterized the AgIME1 promoter region required for complementation of the Agime1 mutant. Reporter assays with AgIME1 promoter fragments fused to lacZ showed that AgSok2 does not control AgIME1 transcription. However, global transcriptome analysis identified two other essential regulators of sporulation, AgIME2 and AgNDT80, as potential targets of AgSok2. Our data suggest that sporulation and riboflavin production in A. gossypii are under mating type locus and nutritional control. Sok2, a target of the cAMP/protein kinase A pathway, serves as a central positive regulator to promote sporulation. This contrasts Saccharomyces cerevisiae where Sok2 is a repressor of IME1 transcription.

The cell polarity proteins Boi1p and Boi2p stimulate vesicle fusion at the plasma membrane of yeast cells.

Eukaryotic cells can direct secretion to defined regions of their plasma membrane. These regions are distinguished by an elaborate architecture of proteins and lipids that are specialized to capture and fuse post-Golgi vesicles. Here, we show that the proteins Boi1p and Boi2p are important elements of this area of active exocytosis at the tip of growing yeast cells. Cells lacking Boi1p and Boi2p accumulate secretory vesicles in their buds. The essential PH domains of Boi1p and Boi2p interact with Sec1p, a protein required for SNARE complex formation and vesicle fusion. Sec1p loses its tip localization in cells depleted of Boi1p and Boi2p but overexpression of Sec1p can partially compensate for their loss. The capacity to simultaneously bind phospholipids, Sec1p, multiple subunits of the exocyst, Cdc42p and the module for generating active Cdc42p identify Boi1p and Boi2p as essential mediators between exocytosis and polar growth.

SPLIFF: A Single-Cell Method to Map Protein-Protein Interactions in Time and Space.

Protein interactions occur at certain times and at specific cellular places. The past years have seen a massive accumulation of binary protein-protein interaction data. The rapid increase of this context-free information necessitates robust methods to monitor protein interactions with temporal and spatial resolution in single cells. We have developed a simple split-ubiquitin-based method (SPLIFF) that uses the ratio of two fluorescent reporters as a signal for protein-protein interactions. One protein of the pair of interest is attached to the linear fusion of mCherry, the C-terminal half of ubiquitin, and GFP (mCherry-Cub-GFP). The other potential binding partner is expressed as a C-terminal fusion to the N-terminal half of ubiquitin (Nub). Upon co-expression the interaction between the two proteins of interest induces the reassociation of Nub and Cub to the native-like ubiquitin. GFP is subsequently cleaved from the C-terminus of Cub and degraded whereas the red-fluorescent mCherry stays attached to the Cub-fusion protein. We first implemented this method in the model yeast Saccharomyces cerevisiae. One fusion protein is expressed in cells of the a-mating type and the complementary fusion protein in cells of the α-mating type. Upon mixing, both cell types fuse and the Nub- and Cub-fusion proteins are free to interact. The red and green fluorescence is monitored by two-channel fluorescence time-lapse microcopy. The moment of cell fusion defines the start of the analysis. The calculated ratio of green to red fluorescence allows mapping the spatiotemporal interaction profiles of the investigated proteins in single cells.

A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip.

The cortical endoplasmic reticulum (cER) of yeast underlies the plasma membrane (PM) at specific contact sites to enable a direct transfer of information and material between both organelles. During budding, directed movement of cER to the young bud followed by subsequent anchorage at its tip ensures the faithful inheritance of this organelle. The ER membrane protein Scs2p tethers the cER to the PM and to the bud tip through so far unknown receptors. We characterize Epo1p as a novel member of the polarisome that interacts with Scs2p exclusively at the cell tip during bud growth and show that Epo1p binds simultaneously to the Cdc42p guanosine triphosphatase-activating protein Bem3p. Deletion of EPO1 or deletion of BEM3 in a polarisome-deficient strain reduces the amount of cER at the tip. This analysis therefore identifies Epo1p as a novel and important component of the polarisome that promotes cER tethering at sites of polarized growth.

A fluorescent reporter for mapping cellular protein-protein interactions in time and space.

We introduce a fluorescent reporter for monitoring protein-protein interactions in living cells. The method is based on the Split-Ubiquitin method and uses the ratio of two auto-fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two-channel time-lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio-temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran-GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein-protein interactions identified from large-scale screens can be effectively followed up by high-resolution single-cell analysis.

Detecting protein-protein interactions with the Split-Ubiquitin sensor.

A detailed understanding of a cellular process requires the knowledge about the interactions between its protein constituents. The Split-Ubiquitin technique allows to monitor and detect interactions of very diverse proteins, including transcription factors and membrane-associated proteins. The technique is based on unique features of ubiquitin, the enzymes of the ubiquitin pathway, and the reconstitution of a native-like ubiquitin from its N- and C-terminal fragments. Using Ura3p as a reporter for the reconstitution of the ubiquitin fragments, methods are presented that enable to screen in yeast for interaction partners of a given protein with either a randomly generated expression library or a defined but more limited array of protein fusions.

Characterization of α-factor pheromone and pheromone receptor genes of Ashbya gossypii.

The genome of Ashbya gossypii contains homologs of most of the genes that are part of the Saccharomyces cerevisiae pheromone-signal transduction cascade. However, we currently lack understanding of a potential sexual cycle for this pre-whole genome duplication hemiascomycete. The sequenced strain bears three identical copies encoding MATa. We show that the syntenic A. gossypii homolog of MFα1 (AFL062w) does not encode a mature α-factor peptide, but identified another gene, AAR163c, which encodes a candidate α-specific mating pheromone and is thus reannotated as AgMFα2. The expression of the AgSTE2α-factor receptor in an Scste2 S. cerevisiae MATa strain resulted in dosage-dependent growth arrest upon exposure to A. gossypiiα-factor, which indicated that the pheromone response was effectively coupled to the S. cerevisiae signal transduction cascade. Comparison of α-pheromones and α-pheromone receptors showed greater conservation between Eremothecium cymbalariae and S. cerevisiae than between A. gossypii and E. cymbalariae. We constructed A. gossypii strains deleted for the STE2 and STE3 pheromone receptors. These strains showed no phenotypic abnormalities and an ste2, ste3 double mutant is still able to sporulate. The deletion of STE12 as the downstream target of pheromone signalling, however, led to a hypersporulation phenotype.

A constraint network of interactions: protein-protein interaction analysis of the yeast type II phosphatase Ptc1p and its adaptor protein Nbp2p.

We used a generally applicable strategy to collect and structure the protein interactions of the yeast type II protein phosphatase Ptc1p and its binding partner Nbp2p. The procedure transformed primary unstructured protein interaction data into an ensemble of alternative interaction states. Certain combinations of proteins are allowed in different network configurations. Nbp2p serves as the network hub and brings seven kinases in close contact to Ptc1p. As a consequence, the deletion of NBP2 affects several cellular processes including organelle inheritance and the responses to mating hormone, cell wall stress and high osmolarity; it also impairs the proper execution of the morphogenetic program. Our constraint interaction map provides a basis for understanding a subset of the observed phenotypes and assigns the Ptc1p-Nbp2p module a role in synchronizing the associated kinases during the cell cycle.

An Ashbya gossypii cts2 mutant deficient in a sporulation-specific chitinase can be complemented by Candida albicans CHT4.

The Candida albicans genome encodes four chitinases, CHT1, CHT2, CHT3 and CHT4. All four C. albicans chitinase-encoding genes are non-essential. The corresponding proteins belong to two groups in which Cht1, Cht2 and Cht3 are more similar to Saccharomyces cerevisiae Cts1, while Cht4 is more similar to ScCts2. In the filamentous fungus Ashbya gossypii, a CTS2 homolog (ACL166w) was identified as the sole chitinase gene. The AgCts2 is 490 aa in Length and shows 42.3% overall identity to ScCts2 (511 aa) and 33.2% identity to CaCht4 (388 aa). The A. gossypii cts2 deletion mutant showed no growth retardation or vegetative morphogenetic defects. However, upon sporulation Agcts2 mutants revealed a defect in spore formation. Expression of AgCts2 using a lacZ reporter gene was only found in the centre of a mycelium corresponding to the sporogenous part of a colony. The mutant spore phenotype of Agcts2 could be complemented by either AgCTS2, the S. cerevisiae CTS2, or the C. albicans CHT4 gene when expressed by either the AgCTS2 or the AgTEF1 promoter.

A molecular toolbox for manipulating Eremothecium coryli.

The genus Eremothecium contains dimorphic and filamentous fungal species, most notably Eremothecium sinecaudum (Holleya sinecauda), a dimorphic plant pathogen, which was isolated from mustard seeds, and Eremothecium gossypii (Ashbya gossypii), a filamentous fungus, which is well known for its ability to produce riboflavin. In this study, we present the initial molecular characterization of another Eremothecium species classified as Eremothecium coryli. E.coryli is a dimorphic fungus. We have developed, based on previously described reagents, a transformation system for E. coryli using kanMX and NATMX3 as dominant selectable marker genes on freely replicating plasmids conferring resistance to the antibiotics G418 and nourseothricin, respectively. As reporter genes we could introduce lacZ and GFP, which were controlled either by the AgTEF1 promoter or by regulatable MET promoters derived from the A. gossypii and Saccharomyces cerevisiae MET3 genes. These newly established tools will allow a detailed comparison of different growth modes in filamentous or dimorphic species within the genus Eremothecium.

Use of MET3 promoters for regulated gene expression in Ashbya gossypii.

A central tool for gene function analysis is the construction mutant strains. This can be done conveniently in A. gossypii using PCR-based tools. The deletion of essential genes can be performed since initial transformants are sheltered in a heterokaryotic mycelium, which contains nuclei with both wild type and mutant alleles. The analysis of mutant phenotypes in A. gossypii is regularly started by germinating spores, which contain only one nucleus. Thus, selection can be used to identify mutant germ cells and germlings. However, such an analysis yields only mutant mycelia if the deleted gene is not essential. We describe the use of the regulatable Saccharomyces cerevisiae and A. gossypii MET3 promoters as novel tools to regulate gene expression in A. gossypii. Conditional expression was tested using GFP and lacZ-reporter genes. Regulation of MET3 promoters was found to be dependent on methionine but not on cysteine and down-regulation to about 1/10 of the initial expression levels was achieved. We used the A. gossypii WAL1 and CYK1 genes as models to demonstrate that MET3 promoters could regulate the expression of these genes and reveal their mutant phenotypes depending on the presence or absence of methionine. Finally, we show that the AgMET3 promoter contains two Cpf1-binding sites and that AgCPF1 can complement the S. cerevisiae cpf1 methionine auxotrophy.

Candida albicans Rho-type GTPase-encoding genes required for polarized cell growth and cell separation.

Rho proteins are essential regulators of morphogenesis in eukaryotic cells. In this report, we investigate the role of two previously uncharacterized Rho proteins, encoded by the Candida albicans RHO3 (CaRHO3) and CaCRL1/CaRHO4 genes. The CaRHO3 gene was found to contain one intron. Promoter shutdown experiments using a MET3 promoter-controlled RHO3 revealed a strong cell polarity defect and a partially depolarized actin cytoskeleton. Hyphal growth after promoter shutdown was abolished in rho3 mutants even in the presence of a constitutively active ras1(G13V) allele, and existing germ tubes became swollen. Deletion of C. albicans RHO4 indicated that it is a nonessential gene and that rho4 mutants were phenotypically different from rho3. Two distinct phenotypes of rho4 cells were elongated cell morphology and an unexpected cell separation defect generating chains of cells. Colony morphology of crl1/rho4 resulted in a growth-dependent smooth (long cell cycle length) or wrinkled (short cell cycle length) phenotype. This phenotype was additionally dependent on the rho4 cell separation defect and was also found in a Cacht3 chitinase mutant that shows a strong cytokinesis defect. The overexpression of the endoglucanase encoding the ENG1 gene, but not CHT3, suppressed the cell separation defect of crl1/rho4 but could not suppress the cell elongation phenotype. C. albicans Crl1/Rho4 and Bnr1 both localize to septal sites in yeast and hyphal cells but not to the hyphal tip. Deletion of RHO4 and BNR1 produced similar morphological phenotypes. Based on the localization of Rho4 and on the rho4 mutant phenotype, we propose a model in which Rho4p may function as a regulator of cell polarity, breaking the initial axis of polarity found during early bud growth to promote the construction of a septum.

New pFA-cassettes for PCR-based gene manipulation in Candida albicans.

Several modules for efficient PCR-based gene disruption have recently been introduced in Candida albicans. These are based on auxotrophic marker genes for deficient strains derived from SC5314/CAI4. Commonly used protocols for the transformation C. albicans are based either on the lithium acetate procedure or on electroporation also used for Saccharomyces cerevisiae. Here we present our updated arsenal of pFA-modules that now include the heterologous marker genes HIS1 from C. dubliniensis and LEU2 from C. maltosa (Noble and Johnson 2005) and the dominant selection marker ca SAT1 (Reuss et al. 2004). We also introduce the Ashbya gossypii TEF1 -promoter as a strong constitutive promoter. With these new elements an enlarged collection of pFA-marker and pFA-marker-promoter modules were generated containing 17 new modules. In addition, N-terminal tagging with GFP-(GA) 6 and epitope-tagging modules using the 6 x-HIS-tag were constructed. This adds to the previous modules that only enabled C-terminal GFP-tagging of genes (Gola et al. 2003). In total 29 pFA-modules are currently freely available from our lab which - together with an update on the diagnostic verification procedure - further enlarge the C. albicans molecular toolbox and enhance our capabilities to use PCR-based gene alteration methods in C. albicans.

Candida albicans CHT3 encodes the functional homolog of the Cts1 chitinase of Saccharomyces cerevisiae.

Chitin synthesis and chitin degradation play an important role in cellular morphogenesis and influence the cell shape of fungal organisms. The Candida albicans genome contains four chitinase genes, CHT1, CHT2, and CHT3, which are homologous to the Saccharomyces cerevisiae CTS1 gene and C. albicans CHT4, which is homologous to S. cerevisiae CTS2. To determine which of the C. albicans CHT genes represents the functional homolog of the S. cerevisiae CTS1 gene we constructed mutants of these genes and characterized the resulting phenotypes using morphological assays such as in vivo time lapse microscopy and enzymatic assays to determine the chitinase activity. Deletion of CaCHT1 and CaCHT2 provided no phenotypic alterations in liquid culture but resulted in increased hyphal growth on solid media. Deletion of CaCHT3 generated chains of unseparated cells in the yeast growth phase strongly resembling the cts1 deletion phenotype of S. cerevisiae cells. Expression of CHT3 under control of the regulatable MAL2-promoter in C. albicans resulted in the reversion of the cell separation defect when cells were grown in maltose. Cht3, but not Cht2 when expressed in S. cerevisiae was also able to reverse the cell separation defect of the S. cerevisiae c ts1 deletion strain. Measurements of chitinase activity from yeast cells of C. albicans showed that Cht2 is bound to cells, consistent with it being GPI-anchored while Cht3 is secreted into growth medium; Cht3 is also the principal, observed activity.

New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region.

The use of PCR-based techniques for directed gene alterations has become a standard tool in Saccharomyces cerevisiae. In our efforts to increase the speed of functional analysis of Candida albicans genes, we constructed a modular system of plasmid vectors and successfully applied PCR-amplified functional analysis (FA)-cassettes in the transformation of C. albicans. These cassettes facilitate: (a) gene disruptions; (b) tagging of 3'-ends of genes with green fluorescent protein (GFP); and (c) replacements of endogenous promoters to achieve regulated expression. The modules consists of a core of three selectable marker genes, CaURA3, CaHIS1 and CaARG4. Modules for C-terminal GFP-tagging were generated by adding GFP-sequences flanked at the 5'-end by a (Gly-Ala)3-linker and at the 3'-end by the S. cerevisiae URA3-terminator to these selection markers. Promoter exchange modules consist of the respective marker genes followed by the regulatable CaMAL2 or CaMET3 promoters at their 3'-ends. In order to ensure a reliably high rate of homologous gene targeting, the flanking homology regions required a size of 100 bp of gene-specific sequences, which were provided with the oligonucleotide primers. The use of shorter flanking homology regions produced unsatisfactory results with C. albicans strain BWP17. With these new modules only a minimal set of primers is required to achieve the functional analysis of C. albicans genes and, therefore, provides a basic tool to increase the number of functionally characterized C. albicans genes of this human pathogen in the near future.