RESUMEN
Spore formation in Saccharomyces cerevisiae is driven by de novo assembly of new membranes termed prospore membranes. A vesicle-docking complex called the meiosis II outer plaque (MOP) forms on the cytoplasmic faces of the spindle-pole bodies at the onset of meiosis II and serves as the initiation site for membrane formation. In this study, a fluorescence-recovery assay was used to demonstrate that the dynamics of the MOP proteins change coincident with the coalescence of precursor vesicles into a membrane. Proteins within the MOP exchange freely with a soluble pool prior to membrane assembly, but after membranes are formed they remain stably within the MOP. By contrast, constitutive spindle-pole-body proteins display low exchange in both conditions. The MOP component Ady4p plays a role in maintaining the integrity of the MOP complex, but this role differs depending on whether the MOP is associated with docked vesicles or a fully formed membrane. These results suggest an architectural rearrangement of the MOP coincident with vesicle fusion.
Asunto(s)
Membrana Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Esporas Fúngicas/metabolismo , Membrana Celular/ultraestructura , Clonación Molecular , Proteínas del Citoesqueleto/genética , Recuperación de Fluorescencia tras Fotoblanqueo , Meiosis , Fusión de Membrana/genética , Proteínas de la Membrana/genética , Centro Organizador de los Microtúbulos/ultraestructura , Complejos Multiproteicos/metabolismo , Organismos Modificados Genéticamente , Estabilidad Proteica , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia/genética , Esporas Fúngicas/ultraestructura , Transgenes/genéticaRESUMEN
During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Huso Acromático/fisiología , Vesículas Transportadoras/fisiología , Proteínas de Transporte Vesicular/fisiología , Membrana Celular/metabolismo , Membrana Celular/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Transferencia Resonante de Energía de Fluorescencia , Meiosis/fisiología , Fusión de Membrana , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Formation of ascospores in the yeast Saccharomyces cerevisiae is driven by an unusual cell division in which daughter nuclei are encapsulated within de novo-formed plasma membranes, termed prospore membranes. Generation of viable spores requires that cytoplasmic organelles also be captured along with nuclei. In mitotic cells segregation of mitochondria into the bud requires a polarized actin cytoskeleton. In contrast, genes involved in actin-mediated transport are not essential for sporulation. Instead, efficient segregation of mitochondria into spores requires Ady3p, a component of a protein coat found at the leading edge of the prospore membrane. Other organelles whose mitotic segregation is promoted by actin, such as the vacuole and the cortical endoplasmic reticulum, are not actively segregated during sporulation but are regenerated within spores. These results reveal that organellar segregation into spores is achieved by mechanisms distinct from those in mitotic cells.