RESUMEN
Vesicle delivery of Cdc42 has been proposed as an important mechanism for generating and maintaining Cdc42 polarity at the plasma membrane. This mechanism requires the density of Cdc42 on secretory vesicles to be equal to or higher than the plasma membrane polarity cap. Using a novel method to estimate Cdc42 levels on post-Golgi secretory vesicles in intact yeast cells, we: (1) determined that endocytosis plays an important role in Cdc42's association with secretory vesicles (2) found that a GFP-tag placed on the N-terminus of Cdc42 negatively impacts this vesicle association and (3) quantified the surface densities of Cdc42 on post-Golgi vesicles which revealed that the vesicle density of Cdc42 is three times more dilute than that at the polarity cap. This work suggests that the immediate consequence of secretory vesicle fusion with the plasma membrane polarity cap is to dilute the local Cdc42 surface density. This provides strong support for the model in which vesicle trafficking acts to negatively regulate Cdc42 polarity on the cell surface while also providing a means to recycle Cdc42 between the cell surface and internal membrane locations.
Asunto(s)
Membrana Celular/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vesículas Secretoras/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genéticaRESUMEN
Nearly every cell type exhibits some form of polarity, yet the molecular mechanisms vary widely. Here we examine what we term 'chemical systems' where cell polarization arises through biochemical interactions in signaling pathways, 'mechanical systems' where cells polarize due to forces, stresses and transport, and 'mechanochemical systems' where polarization results from interplay between mechanics and chemical signaling. To reveal potentially unifying principles, we discuss mathematical conceptualizations of several prototypical examples. We suggest that the concept of local activation and global inhibition - originally developed to explain spatial patterning in reaction-diffusion systems - provides a framework for understanding many cases of cell polarity. Importantly, we find that the core ingredients in this framework - symmetry breaking, self-amplifying feedback, and long-range inhibition - involve processes that can be chemical, mechanical, or even mechanochemical in nature.
Asunto(s)
Polaridad Celular , Citoesqueleto/química , Mecanotransducción Celular , Actinas/química , Animales , Transporte Biológico , Membrana Celular/química , Membrana Celular/enzimología , Citoesqueleto/enzimología , Drosophila/química , Drosophila/enzimología , Proteínas de Drosophila/química , Activación Enzimática , Retroalimentación Fisiológica , Glucógeno Sintasa Quinasa 3/química , Polimerizacion , Unión Proteica , Estrés Mecánico , Levaduras/química , Levaduras/enzimología , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/químicaRESUMEN
Cell fusion is the key event of fertilization that gives rise to the diploid zygote and is a nearly universal aspect of eukaryotic biology. In the yeast Saccharomyces cerevisiae, several mutants have been identified that are defective for cell fusion, and yet the molecular mechanism of this process remains obscure. One obstacle has been that genetic screens have mainly focused on mating-specific factors, whereas the process likely involves housekeeping proteins as well. Here we implicate Cdc42p, an essential protein with roles in multiple aspects of morphogenesis, as a core component of the yeast cell fusion pathway. We identify a point mutant in the Rho-insert domain of CDC42, called cdc42-138, which is specifically defective in cell fusion. The cell fusion defect is not a secondary consequence of ineffective signaling or polarization. Genetic and morphological data show that Cdc42p acts at a late stage in cell fusion in concert with a key cell fusion regulator, Fus2p, which contains a Dbl-homology domain. We find that Fus2p binds specifically with activated Cdc42p, and binding is blocked by the cdc42-138 mutation. Thus, in addition to signaling and morphogenetic roles in mating, Cdc42p plays a role late in cell fusion via activation of Fus2p.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Polaridad Celular , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Genes Fúngicos , Guanosina Trifosfato/metabolismo , Factor de Apareamiento , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Electrónica de Transmisión , Modelos Moleculares , Péptidos/metabolismo , Feromonas/metabolismo , Mutación Puntual , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genéticaRESUMEN
Rho family GTPases are critical regulators in determining and maintaining cell polarity. In Saccharomyces cerevisiae, Rho3 and Cdc42 play important but distinct roles in regulating polarized exocytosis and overall polarity. Cdc42 is highly polarized during bud emergence and is specifically required for exocytosis at this stage. In contrast, Rho3 appears to play an important role during the isotropic growth of larger buds. Using a novel monoclonal antibody against Rho3, we find that Rho3 localizes to the cell surface in a dispersed pattern which is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we demonstrate that a small region at the N terminus is necessary and sufficient to confer Rho3 localization and function onto Cdc42. Analysis of this domain reveals two essential elements responsible for distinguishing function. First, palmitoylation of a cysteine residue by the Akr1 palmitoyltransferase is required both for the switch of function and the switch of localization properties of this domain. Second, two basic residues distal to the palmitoylation site are required for regulating binding affinity with the Exo70 and Sec3 effectors. This demonstrates the importance of localization and effector binding in determining how these GTPases evolved specific functions at distinct stages of polarized growth.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Polaridad Celular , Exocitosis , Genes Fúngicos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rho/química , Proteínas de Unión al GTP rho/genéticaRESUMEN
Many cell types can spontaneously polarize even in the absence of specific positional cues. In budding yeast, this symmetry-breaking polarization depends on a scaffold protein called Bem1p. A recent study defines Bem1p's molecular function during symmetry breaking.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Polaridad Celular/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Modelos Biológicos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genética , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Quinasas p21 Activadas/metabolismoRESUMEN
BACKGROUND: In 1952, Alan Turing suggested that spatial patterns could arise from homogeneous starting conditions by feedback amplification of stochastic fluctuations. One example of such self-organization, called symmetry breaking, involves spontaneous cell polarization in the absence of spatial cues. The conserved GTPase Cdc42p is essential for both guided and spontaneous polarization, and in budding yeast cells Cdc42p concentrates at a single site (the presumptive bud site) at the cortex. Cdc42p concentrates at a random cortical site during symmetry breaking in a manner that requires the scaffold protein Bem1p. The mechanism whereby Bem1p promotes this polarization was unknown. RESULTS: Here we show that Bem1p promotes symmetry breaking by assembling a complex in which both a Cdc42p-directed guanine nucleotide exchange factor (GEF) and a Cdc42p effector p21-activated kinase (PAK) associate with Bem1p. Analysis of Bem1p mutants indicates that both GEF and PAK must bind to the same molecule of Bem1p, and a protein fusion linking the yeast GEF and PAK bypasses the need for Bem1p. Although mammalian cells lack a Bem1p ortholog, they contain more complex multidomain GEFs that in some cases can directly interact with PAKs, and we show that yeast containing an artificial GEF with similar architecture can break symmetry even without Bem1p. CONCLUSIONS: Yeast symmetry-breaking polarization involves a GEF-PAK complex that binds GTP-Cdc42p via the PAK and promotes local Cdc42p GTP-loading via the GEF. By generating fresh GTP-Cdc42p near pre-existing GTP-Cdc42p, the complex amplifies clusters of GTP-Cdc42p at the cortex. Our findings provide mechanistic insight into an evolutionarily conserved pattern-forming positive-feedback pathway.
Asunto(s)
Polaridad Celular , Factores de Intercambio de Guanina Nucleótido/fisiología , Saccharomyces cerevisiae/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/fisiología , Quinasas p21 Activadas/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Modelos Biológicos , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Quinasas p21 Activadas/química , Quinasas p21 Activadas/metabolismoRESUMEN
We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.
Asunto(s)
Actinas/metabolismo , Estructuras de la Membrana Celular/metabolismo , Fusión de Membrana/fisiología , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiología , Vacuolas/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Actinas/química , Actinas/genética , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Estructuras de la Membrana Celular/química , Estructuras de la Membrana Celular/genética , Colorantes Fluorescentes/farmacología , Eliminación de Gen , Fusión de Membrana/efectos de los fármacos , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Pirenos/farmacología , Proteínas R-SNARE/química , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Vacuolas/química , Vacuolas/genética , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genéticaAsunto(s)
Proteínas de Saccharomyces cerevisiae/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Polaridad Celular , Endocitosis , Modelos Moleculares , Estructura Terciaria de Proteína , Ratas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismoRESUMEN
The small GTP-binding protein Cdc42, the guanine nucleotide exchange factor Scd1, the p21-activated kinase Shk1, and the adaptor protein Scd2 are involved in the Cdc42-dependent signaling cascade in fission yeast. In the present study, we analyzed the Cdc42 binding and scaffolding activities of Scd2 by co-precipitation assays. We found that two SH3-containing regions, amino acid residues 1-87 (CB1 (Cdc42-binding region 1)) and 110-266 (CB2), of Scd2 can bind to the GTP-bound form of Cdc42. CB2 is cryptic because of the intramolecular binding between the SH3 domain in CB2 (SH3(C)) and the PX domain and binds to Cdc42 only when the Scd2 PB1 domain binds to the PC motif-containing region (residues 760-872) of Scd1. This CB2.Cdc42 association, which would stabilize the open configuration of Scd2, enables the SH3(C) domain to bind to the polyproline motif of Shk1. We also found that the GTP-bound form of Cdc42 binds to the CRIB motif of Shk1 more strongly than to Scd2. Thus, Scd2 functions as a scaffold to form a protein complex, and the GTP-bound Cdc42 might be transferred effectively from the upstream activator Scd1 to the downstream effector Shk1 via Scd2.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Unión Competitiva , Proteínas de Ciclo Celular/metabolismo , Guanosina Trifosfato/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Quinasas p21 Activadas , Dominios Homologos srcRESUMEN
Genome sequencing has been completed for multiple organisms, and pilot proteomic analyses reported for yeast and higher eukaryotes. This work has emphasized the facts that proteins are frequently engaged in multiple interactions, and that governance of protein interaction specificity is a primary means of regulating biological systems. In particular, the ability to deconvolute complex protein interaction networks to identify which interactions govern specific signaling pathways requires the generation of biological tools that allow the distinction of critical from noncritical interactions. We report the application of an enhanced Dual Bait two-hybrid system to allow detection and manipulation of highly specific protein-protein interactions. We summarize the use of this system to detect proteins and peptides that target well-defined specific motifs in larger protein structures, to facilitate rapid identification of specific interactors from a pool of putative interacting proteins obtained in a library screen, and to score specific drug-mediated disruption of protein-protein interaction.
Asunto(s)
Péptidos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos/genética , Animales , Clonación Molecular , Bases de Datos de Proteínas , Genoma , Genoma Fúngico , Genoma Humano , Humanos , Datos de Secuencia Molecular , Péptidos/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proyectos Piloto , Proteínas/química , Proteínas/genética , Proteoma/química , Proteoma/genética , Proteoma/metabolismo , Proteína de Retinoblastoma/química , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genética , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismoRESUMEN
The Cdc42p GTPase controls polarized growth and cell cycle progression in eukaryotes from yeasts to mammals, and its precise subcellular localization is essential for its function. To examine the cell cycle-specific targeting of Cdc42p in living yeast cells, a green fluorescent protein (GFP)-Cdc42 fusion protein was used. In contrast to previous immunolocalization data, GFP-Cdc42p was found at the plasma membrane around the entire cell periphery and at internal vacuolar and nuclear membranes throughout the cell cycle, and it accumulated or clustered at polarized growth sites, including incipient bud sites and mother-bud neck regions. These studies also showed that C-terminal CAAX and polylysine domains were sufficient for membrane localization but not for clustering. Time-lapse fluorescence microscopy showed that GFP-Cdc42p clustered at the incipient bud site prior to bud emergence and at the mother-bud neck region postanaphase as a diffuse, single band and persisted as two distinct bands on mother and daughter cells following cytokinesis and cell separation. Initial clustering occurred immediately prior to actomyosin ring contraction and persisted postcontraction. These results suggest that Cdc42p targeting occurs through a novel mechanism of membrane localization followed by cell cycle-specific clustering at polarized growth sites.
Asunto(s)
Saccharomyces cerevisiae/metabolismo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Actomiosina/metabolismo , Secuencia de Aminoácidos , División Celular , Membrana Celular/metabolismo , Polaridad Celular , Proteínas Fluorescentes Verdes , Membranas Intracelulares/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Modelos Biológicos , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genéticaRESUMEN
Membrane fusion reactions have been considered to be primarily regulated by Rab GTPases. In the model system of homotypic vacuole fusion in the yeast Saccharomyces cerevisiae, we show that Cdc42p, a member of the Rho family of GTPases, has a direct role in membrane fusion. Genetic evidence suggested a relationship between Cdc42p and Vtc1p/Nrf1p, a central part of the vacuolar membrane fusion machinery. Vacuoles from cdc42 temperature-sensitive mutants are deficient for fusion at the restrictive temperature. Specific amino acid changes on the Cdc42p protein surface in these mutants define the putative interaction domain that is crucial for its function in membrane fusion. Affinity-purified antibodies to this domain inhibited the in vitro fusion reaction. Using these antibodies in kinetic analyses and assays for subreactions of the priming, docking and post-docking phase of the reaction, we show that Cdc42p action follows Ypt7p-dependent tethering, but precedes the formation of trans-SNARE complexes. Thus, our data define an effector binding domain of Cdc42p by which it regulates the docking reaction of vacuole fusion.
Asunto(s)
Adenosina Trifosfatasas , Ácido Egtácico/análogos & derivados , Proteínas Fúngicas/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vacuolas/fisiología , Proteínas de Transporte Vesicular , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/fisiología , Alelos , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Quelantes/farmacología , Ácido Egtácico/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Sustancias Macromoleculares , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares , Transporte de Proteínas , Proteínas Recombinantes de Fusión/fisiología , Proteínas SNARE , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/fisiologíaRESUMEN
The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of "effector-loop" mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.
Asunto(s)
Mutación , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Alelos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dosificación de Gen , Genes Fúngicos , Genes Reporteros , Prueba de Complementación Genética , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Fenotipo , Plásmidos , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/fisiología , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genéticaRESUMEN
The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved CDC42 homolog from P. marneffei named cflA. By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct cflA function is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression of cflA alleles in Aspergillus nidulans prevented conidiation.
Asunto(s)
Polaridad Celular , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Penicillium/crecimiento & desarrollo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rho , Secuencia de Aminoácidos , Northern Blotting , Microscopía Fluorescente , Datos de Secuencia Molecular , Morfogénesis , Penicillium/genética , Penicillium/ultraestructura , Homología de Secuencia de Aminoácido , Transformación Genética , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismoRESUMEN
In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.
Asunto(s)
Genes Esenciales/genética , Morfogénesis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , División Celular , Polaridad Celular , Quitina/metabolismo , Epistasis Genética , Genes Fúngicos/genética , Haploidia , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Nitrógeno/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genéticaRESUMEN
We present the 1.9 A resolution crystal structure of the catalytic domain of Gyp1p, a specific GTPase activating protein (GAP) for Ypt proteins, the yeast homologues of Rab proteins, which are involved in vesicular transport. Gyp1p is a member of a large family of eukaryotic proteins with shared sequence motifs. Previously, no structural information was available for any member of this class of proteins. The GAP domain of Gyp1p was found to be fully alpha-helical. However, the observed fold does not superimpose with other alpha-helical GAPs (e.g. Ras- and Cdc42/Rho-GAP). The conserved and catalytically crucial arginine residue, identified by mutational analysis, is in a comparable position to the arginine finger in the Ras- and Cdc42-GAPs, suggesting that Gyp1p utilizes an arginine finger in the GAP reaction, in analogy to Ras- and Cdc42-GAPs. A model for the interaction between Gyp1p and the Ypt protein satisfying biochemical data is given.
Asunto(s)
Dominio Catalítico , Proteínas Activadoras de GTPasa/química , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión al GTP rab/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arginina/química , Cristalografía por Rayos X , Proteínas Activadoras de GTPasa/metabolismo , Guanilil Imidodifosfato/química , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Alineación de Secuencia , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/química , Proteínas de Unión al GTP rab/química , Proteínas Activadoras de ras GTPasa/químicaRESUMEN
Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae. Cytological and genetic analyses of these cdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo. In addition, mapping the functional data for each cdc42 mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily. This region overlaps with a region (alpha5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions.