RESUMO
Cytokinesis in Saccharomyces cerevisiae involves coordination between actomyosin ring contraction and septum formation and/or targeted membrane deposition. We show that Mlc1p, a light chain for Myo2p (type V myosin) and Iqg1p (IQGAP), is the essential light chain for Myo1p, the only type II myosin in S. cerevisiae. However, disruption or reduction of Mlc1p-Myo1p interaction by deleting the Mlc1p binding site on Myo1p or by a point mutation in MLC1, mlc1-93, did not cause any obvious defect in cytokinesis. In contrast, a different point mutation, mlc1-11, displayed defects in cytokinesis and in interactions with Myo2p and Iqg1p. These data suggest that the major function of the Mlc1p-Myo1p interaction is not to regulate Myo1p activity but that Mlc1p may interact with Myo1p, Iqg1p, and Myo2p to coordinate actin ring formation and targeted membrane deposition during cytokinesis. We also identify Mlc2p as the regulatory light chain for Myo1p and demonstrate its role in Myo1p ring disassembly, a function likely conserved among eukaryotes.
Assuntos
Cadeias Pesadas de Miosina/metabolismo , Cadeias Leves de Miosina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Actomiosina/fisiologia , Sequência de Aminoácidos , Sequência Conservada , Genótipo , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/genética , Ligação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Deleção de SequênciaRESUMO
Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane. Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro. We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo. Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80-100 nm in diameter. Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants. Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane.
Assuntos
Actinas/metabolismo , Exocitose/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Saccharomyces cerevisiae/fisiologiaRESUMO
In Saccharomyces cerevisiae, polarized growth depends on interactions between the actin cytoskeleton and the secretory machinery. Here we show that the Rab GTPase-activating proteins (GAPs) Msb3 and Msb4 interact directly with Spa2, a scaffold protein of the "polarisome" that also interacts with the formin Bni1. Spa2 is required for the polarized localization of Msb3 and Msb4 at the bud tip. We also show that Msb3 and Msb4 bind specifically to Cdc42-GDP and Rho1-GDP in vitro and that Msb3 and Rho GDP dissociation inhibitor act independently but oppositely on Cdc42. Finally, we show that Msb3 and Msb4 are involved in Bni1-nucleated actin assembly in vivo. These results suggest that Msb3 and Msb4 regulate polarized growth by multiple mechanisms, directly regulating exocytosis through their GAP activity toward Sec4 and potentially coordinating the functions of Cdc42, Rho1, and Bni1 in the polarisome through their binding to these GTPases. A functional equivalent of the polarisome probably exists in other fungi and mammals.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/química , Actinas/metabolismo , Ciclo Celular , Movimento Celular , Polaridade Celular , Exocitose , GTP Fosfo-Hidrolases/química , Genótipo , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Guanosina Difosfato/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microscopia de Interferência , Modelos Biológicos , Modelos Genéticos , Família Multigênica , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Temperatura , Técnicas do Sistema de Duplo-HíbridoRESUMO
Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae. However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process. Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified. Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion. Both proteins share amino acid sequence homology and structural organization. When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does. In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells. These data strongly suggest that Pxl1p is the "ancient paxillin" in yeast. Deletion of PXL1 does not produce any obvious phenotype. However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner. The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Polaridade Celular , Galinhas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Paxilina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The essential GAB1 gene, which encodes an endoplasmic reticulum (ER)-membrane protein, was identified in a screen for mutants defective in cellular morphogenesis. A temperature-sensitive gab1 mutant accumulates complete glycosylphosphatidylinositol (GPI) precursors, and its temperature sensitivity is suppressed differentially by overexpression of different subunits of the GPI transamidase, from strong suppression by Gpi8p and Gpi17p, to weak suppression by Gaa1p, and to no suppression by Gpi16p. In addition, both Gab1p and Gpi17p localize to the ER and are in the same protein complex in vivo. These findings suggest that Gab1p is a subunit of the GPI transamidase with distinct relationships to other subunits in the same complex. We also show that depletion of Gab1p or Gpi8p, but not Gpi17p, Gpi16p, or Gaa1p causes accumulation of cofilin-decorated actin bars that are closely associated with the perinuclear ER, which highlights a functional interaction between the ER network and the actin cytoskeleton.
Assuntos
Actinas/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Alelos , Sequência de Aminoácidos , Membrana Celular/metabolismo , Polaridade Celular , Clonagem Molecular , Sequência Conservada , Deleção de Genes , Genes Essenciais/genética , Glicosilfosfatidilinositóis/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
The budding yeast Saccharomyces cerevisiae initiates polarized growth or budding once per cell cycle at a specific time of the cell cycle and at a specific location on the cell surface. Little is known about the molecular nature of the temporal and spatial regulatory mechanisms. It is also unclear what factors, if any, among the numerous proteins required to make a bud are involved in the determination of budding frequency. Here we describe a class of cdc42 mutants that produce multiple buds at random locations on the cell surface within one nuclear cycle. The critical mutation responsible for this phenotype affects amino acid residue 60, which is located in a domain required for GTP binding and hydrolysis. This mutation bypasses the requirement for the essential guanine-nucleotide-exchange factor Cdc24p, suggesting that the alteration at residue 60 makes Cdc42p hyperactive, which was confirmed biochemically. This result also suggests that the only essential function of Cdc24p is to activate Cdc42p. Together, these data suggest that the temporal and spatial regulation of polarized growth converges at the level of Cdc42p and that the activity of Cdc42p determines the budding frequency.