Abstract 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 functional analysis Candida albicans genes, we constructed modular system plasmid vectors and successfully applied PCR‐amplified (FA)‐cassettes transformation C. These cassettes facilitate: (a) disruptions; (b) tagging 3′‐ends genes with green fluorescent protein (GFP); (c) replacements endogenous promoters achieve regulated...

Several modules for efficient PCR-based gene disruption have recently been introduced in Candida albicans. These are based on auxotrophic marker genes deficient strains derived from SC5314/CAI4. Commonly used protocols the transformation C. albicans either lithium acetate procedure or electroporation also Saccharomyces cerevisiae. Here we present our updated arsenal of pFA-modules that now include heterologous HIS1 dubliniensis and LEU2 maltosa (Noble Johnson 2005) dominant selection ca SAT1...

ABSTRACT Rho proteins are essential regulators of morphogenesis in eukaryotic cells. In this report, we investigate the role two previously uncharacterized proteins, encoded by Candida albicans RHO3 (Ca ) and Ca CRL1/ RHO4 genes. The gene was found to contain one intron. Promoter shutdown experiments using a MET3 promoter-controlled revealed strong cell polarity defect partially depolarized actin cytoskeleton. Hyphal growth after promoter abolished rho3 mutants even presence constitutively...

We used a generally applicable strategy to collect and structure the protein interactions of yeast type II phosphatase Ptc1p its binding partner Nbp2p. The procedure transformed primary unstructured interaction data into an ensemble alternative states. Certain combinations proteins are allowed in different network configurations. Nbp2p serves as hub brings seven kinases close contact Ptc1p. As consequence, deletion NBP2 affects several cellular processes including organelle inheritance...

The cortical endoplasmic reticulum (cER) of yeast underlies the plasma membrane (PM) at specific contact sites to enable a direct transfer information and material between both organelles. During budding, directed movement cER young bud followed by subsequent anchorage its tip ensures faithful inheritance this organelle. ER protein Scs2p tethers PM through so far unknown receptors. We characterize Epo1p as novel member polarisome that interacts with exclusively cell during growth show binds...

Eukaryotic cells can direct secretion to defined regions of their plasma membrane. These are distinguished by an elaborate architecture proteins and lipids that specialized capture fuse post-Golgi vesicles. Here, we show the Boi1p Boi2p important elements this area active exocytosis at tip growing yeast cells. Cells lacking accumulate secretory vesicles in buds. The essential PH domains interact with Sec1p, a protein required for SNARE complex formation vesicle fusion. Sec1p loses its...

We introduce a fluorescent reporter for monitoring protein-protein interactions in living cells. The method is based on the Split-Ubiquitin and uses ratio of two auto-fluorescent proteins as signal interaction (SPLIFF). mating haploid yeast cells initiates analysis are followed online by two-channel time-lapse microscopy diploid during their first cell cycle. Using this approach we could with high spatio-temporal resolution visualize differences between microtubule binding protein Stu2p its...

The genome of Ashbya gossypii contains homologs most the genes that are part Saccharomyces cerevisiae pheromone-signal transduction cascade. However, we currently lack understanding a potential sexual cycle for this pre-whole duplication hemiascomycete. sequenced strain bears three identical copies encoding MATa. We show syntenic A. homolog MFα1 (AFL062w) does not encode mature α-factor peptide, but identified another gene, AAR163c, which encodes candidate α-specific mating pheromone and is...

Yeast cells select the position of their new bud at beginning each cell cycle. The recruitment septins to this prospective site is one critical events in a complex assembly pathway that culminates outgrowth daughter cell. During recruitment, septin rods follow high concentration Cdc42GTP generated by focused localization Cdc42 guanine-nucleotide-exchange factor Cdc24. We show that, shortly before budding, Cdc24 not only activates but also transiently interacts with Cdc11, subunit caps both...

The polarisome is a cortical proteinaceous microcompartment that organizes the growth of actin filaments and fusion secretory vesicles in yeasts filamentous fungi. Polarisomes are compact, spotlike structures at growing tips their respective cells. molecular forces control form size this not known. Here we identify complex between subunit Pea2 type V Myosin Myo2 anchors cortex yeast We discovered point mutation cargo-binding domain impairs interaction with consequently formation focused...

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 growth phase. Mating not required for sporulation standard homothallic laboratory strain MATa strain. Here we show ectopic expression Saccharomyces cerevisiae MATα2 in A. completely suppresses sporulation, inhibits downregulates among others AgSOK2. AgSok2 belongs to fungal-specific group (APSES)...

Cdc42 organizes cellular polarity and directs the formation of structures in many organisms. By locating Cdc24, source active Cdc42, to growing front yeast cell, scaffold protein Bem1, is instrumental shaping gradient Cdc42. This instructs bud formation, growth, or cytokinesis through actions a diverse set effector proteins. To address how Bem1 participates these transformations, we systematically tracked its interactions during one cell cycle define ensemble interaction states for each...

Abstract Eukaryotic cells can direct secretion to defined regions of their plasma membrane. These are distinguished by an elaborate architecture proteins and lipids that specialized capture fuse post-Golgi vesicles. Here we show the Boi1p Boi2p important elements this area active exocytosis at tip growing yeast cells. Cells lacking accumulate secretory vesicles in bud. The essential PH domains interact with Sec1p, a protein required for SNARE complex formation vesicle fusion. Sec1p loses its...

Abstract Cdc42 organizes cellular polarity and directs the formation of structures in many organisms. By locating Cdc24, source active Cdc42, to growing edge yeast cell, scaffold protein Bem1 is instrumental shaping gradient Cdc42. This instructs bud formation, growth, or cytokinesis through actions a diverse set effector proteins. To address how participates this transformation we systematically mapped its interactions time space. SPLIFF analysis defined unique ensemble interaction-states...

Abstract Yeast cells select at the beginning of each cell cycle position their new bud. The recruitment septins to this prospective bud site (PBS) is one critical events in a complex assembly pathway that culminates outgrowth daughter cell. septin-rods follow hereby high concentration Cdc42 GTP generated by focused location its GEF Cdc24. We show Cdc24 not only activates but temporarily interacts shortly before budding with Cdc11, subunit caps septin rods both ends. Mutations reduce affinity...

Abstract The polarisome is a cortical proteinaceous micro-compartment that organizes the growth of actin filaments and fusion secretory vesicles in yeasts filamentous fungi. Polarisomes are compact, spot-like structures at growing tips their respective cells. molecular forces control form size this not known. Here we identify complex between subunit Pea2 type V Myosin Myo2 anchors cortex yeast We discovered point mutation cargo-binding domain impairs interaction with consequently formation...