10] Use of yeast sec6 mutant for purification of vesicles containing recombinant membrane proteins

Abstract

Publisher Summary This chapter describes the use of the yeast mutant, sec6-4, that contains a temperature-sensitive mutation in a gene product required for the transport of secretory vesicles from the trans-Golgi network to the plasma membrane. When sec6 yeast are grown at the restrictive temperature of 37°, post-Golgi, plasma membrane-targeted vesicles accumulate, and eventually the cells die; however, cells grow normally at the permissive temperature of 25°. This mutant has the particular advantage that gene expression from an inducible promoter can be initiated concomitant with the arrest of vesicle fusion and membrane protein insertion at the plasma membrane. Thus, gene expression begins at the same time that secretory vesicles become unable to fuse with the plasma membrane, ensuring that the desired gene product accumulates in the membranes of these vesicles. The purification of these vesicles is rapid and simple, thereby facilitating the subsequent characterization of the desired gene product. As the Sec6 protein is known to be involved only in the fusion of these vesicles with the plasma membrane, translocation and processing of proteins in the endoplasmic reticulum and processing in the Golgi are largely unaffected by the sec6 mutation.