RESUMEN
In budding yeast, two classes of post-Golgi secretory vesicles carrying different sets of cargoes typified by Bgl2p and invertase are delivered to the plasma membrane for secretion. The exocyst is implicated in tethering these vesicles to the daughter cell membrane for exocytosis. In this study, we report that mutations in the exocyst component Exo70p predominantly block secretion of the Bgl2p vesicles. Furthermore, a defect in invertase vesicle trafficking caused by vps1Delta or pep12Delta in the exo70 mutant background is detrimental to the cell. The secretion defect in exo70 mutants was most pronounced during the early budding stage, which affected daughter cell growth. The selective secretion block does not occur at the vesicle formation or sorting stage because the exocytic vesicles are properly generated and protein processing is normal in the exo70 mutants. Our study suggests that Exo70p functions primarily at early stages of the cell cycle in Bgl2p vesicle secretion, which is critical for polarized cell growth.
Asunto(s)
Ciclo Celular/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/citología , Vesículas Secretoras/fisiología , Polaridad Celular , Exocitosis/fisiología , Glucano Endo-1,3-beta-D-Glucosidasa/metabolismo , Mutación , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , beta-Fructofuranosidasa/metabolismoRESUMEN
The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity.
Asunto(s)
Proteínas Portadoras/metabolismo , Exocitosis , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Polaridad Celular , Fusión de Membrana , Proteínas de la Membrana/genética , Microscopía Electrónica de Transmisión , Mutación , Unión Proteica , Proteínas SNARE , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Transducción de Señal , Proteínas de Transporte Vesicular/biosíntesis , Proteínas de Unión al GTP rab/fisiología , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
Exocytosis is an essential membrane traffic event mediating the secretion of intracellular protein contents such as hormones and neurotransmitters as well as the incorporation of membrane proteins and lipids to specific domains of the plasma membrane. As a fundamental cell biological process, exocytosis is crucial for cell growth, cell-cell communication, and cell polarity establishment. For most eukaryotic cells exocytosis is polarized. A multiprotein complex, named the exocyst, is required for polarized exocytosis from yeast to mammals. The exocyst consists of eight components: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. They are localized to sites of active exocytosis, where they mediate the targeting and tethering of post-Golgi secretory vesicles for subsequent membrane fusion. Here we review the progress made in the understanding of the exocyst and its role in polarized exocytosis.
Asunto(s)
Secreciones Corporales/fisiología , Membrana Celular/metabolismo , Polaridad Celular/fisiología , Exocitosis/fisiología , Vesículas Secretoras/metabolismo , Animales , Membrana Celular/ultraestructura , Humanos , Sustancias Macromoleculares , Fusión de Membrana/fisiología , Complejos Multiproteicos , Transporte de Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Secretoras/ultraestructuraRESUMEN
The exocyst complex plays an essential role in tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. However, how the exocyst complex is assembled and targeted to sites of secretion is unclear. Here, we have investigated the role of the exocyst component Exo84p in these processes. We have generated an array of temperature-sensitive yeast exo84 mutants. Electron microscopy and cargo protein traffic analyses of these mutants indicated that Exo84p is specifically involved in the post-Golgi stage of secretion. Using various yeast mutants, we systematically studied the localization of Exo84p and other exocyst proteins by fluorescence microscopy. We found that pre-Golgi traffic and polarized actin organization are required for Exo84p localization. However, none of the exocyst proteins controls Exo84p polarization. In addition, Sec3p is not responsible for the polarization of Exo84p or any other exocyst component to the daughter cell. On the other hand, several exocyst members, including Sec10p, Sec15p, and Exo70p, clearly require Exo84p for their polarization. Biochemical analyses of the exocyst composition indicated that the assembly of Sec10p, Sec15p, and Exo70p with the rest of the complex requires Exo84p. We propose that there are at least two distinct regulatory mechanisms for exocyst polarization, one for Sec3p and one for the other members, including Exo84p. Exo84p plays a critical role in both the assembly of the exocyst and its targeting to sites of secretion.