RESUMEN
The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease. In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.
Asunto(s)
Endocitosis/fisiología , Factores de Intercambio de Guanina Nucleótido , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Nucleótidos , Orgánulos/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , ATPasas de Translocación de Protón Vacuolares , Secuencia de Aminoácidos , Cromosomas Fúngicos , Clonación Molecular , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Eliminación de Gen , Cinética , Factor de Apareamiento , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Orgánulos/ultraestructura , Péptidos/genética , Péptidos/metabolismo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusión/biosíntesis , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Increased production of secreted proteins in Saccharomyces cerevisiae was achieved by overexpressing the yeast syntaxins. Sso1 or Sso2 protein, the t-SNAREs functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane. Up to four- or six-fold yields of a heterologous secreted protein, Bacillus alpha-amylase, or an endogenous secreted protein, invertase, were obtained respectively when expressing either one of the SSO genes, SSO1 or SSO2, from the ADH1 promoter on a multicopy plasmid. Direct correlation between the Sso protein level and the amount of secreted alpha-amylase was demonstrated by modulating the expression level of the SSO2 gene. Quantitation of the alpha-amylase activity in the culture medium, periplasmic space and cytoplasm suggests that secretion into the periplasmic space is the primary stage at which the SSO genes exert the secretion-enhancing function. Pulse-chase data also support enhanced secretion efficiently obtained by SSO overexpression. Our data suggest that the Sso proteins may be rate-limiting components of the protein secretion machinery at the exocytosis step in yeast.