RESUMO
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.
Assuntos
Ciclo Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/citologia , Vesículas Secretórias/fisiologia , Polaridade Celular , Exocitose/fisiologia , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Mutação , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , beta-Frutofuranosidase/metabolismoRESUMO
The exocyst is an octameric protein complex implicated in tethering post-Golgi secretory vesicles at the plasma membrane in preparation for fusion. However, it is not clear how the exocyst is targeted to and physically associates with specific domains of the plasma membrane and how its functions are regulated at those regions. We demonstrate that the N terminus of the exocyst component Sec3 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues in Sec3 that are critical for its binding to the guanosine triphosphate-bound form of Cdc42. Genetic analyses indicate that the dual interactions of Sec3 with phospholipids and Cdc42 control its function in yeast cells. Disrupting these interactions not only blocks exocytosis and affects exocyst polarization but also leads to defects in cell morphogenesis. We propose that the interactions of Sec3 with phospholipids and Cdc42 play important roles in exocytosis and polarized cell growth.
Assuntos
Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Membrana Celular/ultraestrutura , Exocitose/fisiologia , Proteínas de Fluorescência Verde/análise , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Tiazolidinas/farmacologia , Tropomiosina/genética , Proteínas de Transporte VesicularRESUMO
The exocyst is an octameric protein complex implicated in the tethering of post-Golgi secretory vesicles to the plasma membrane before fusion. The function of individual exocyst components and the mechanism by which this tethering complex is targeted to sites of secretion are not clear. In this study, we report that the exocyst subunit Exo70 functions in concert with Sec3 to anchor the exocyst to the plasma membrane. We found that the C-terminal Domain D of Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues on Exo70 that are critical for its interaction with phospholipids and the small GTPase Rho3. Further genetic and cell biological analyses suggest that the interaction of Exo70 with phospholipids, but not Rho3, is essential for the membrane association of the exocyst complex. We propose that Exo70 mediates the assembly of the exocyst complex at the plasma membrane, which is a crucial step in the tethering of post-Golgi secretory vesicles for exocytosis.
Assuntos
Membrana Celular/metabolismo , Complexos Multiproteicos/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Alelos , Modelos Biológicos , Mutação/genética , Fosfatidilinositol 4,5-Difosfato , Fosfotransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Differential glycosylation of natural hLf and rhLf from hLf-transgenic mice, which harbored a 146 Kb BAC insert that includes the intact hLf gene sequence, was studied in the present report. There were significant differences between the immunoblotting results of rhLf and natural hLf, which were denatured with nonreducing SDS sample buffer. The differences disappeared after rhLf and natural hLf samples were digested with N-glycosidase F, respectively. The results showed that there were significant differences (P<0.01) between the glycosylation of natural hLf and rhLf that were purified, respectively, from milk samples of seven hLf-transgenic mouse lines.