Localization of components involved in protein transport and processing through the yeast Golgi apparatus.

TLDR

In yeast, mutations in SEC7 disrupt protein transit through the Golgi and produce abnormal cisternae, while the Kex2 protease localizes to multiple secretory structures, indicating a complex Golgi network. The study aimed to determine the subcellular localization of Sec7p and Kex2 to delineate the yeast Golgi apparatus. Sec7p was detected in both soluble and membrane‑associated fractions, and its colocalization with Kex2 in double‑labeling experiments confirmed the visualization of the yeast Golgi apparatus.

Abstract

Saccharomyces cerevisiae sec7 mutants exhibit pleiotropic deficiencies in the transit of proteins through the Golgi apparatus, and elaborate an array of Golgi apparatus-like cisternae at a restrictive growth temperature (37 degrees C). The SEC7 gene encodes an essential high-molecular weight protein (227 kD) that is phosphorylated in vivo. In cell lysates, Sec7 protein (Sec7p) is recovered in both sedimentable and soluble fractions. A punctate immunofluorescent pattern of Sec7p-associated structures seen in SEC cells coalesces in sec14 mutant yeast that accumulate exaggerated Golgi cisternae at 37 degrees C. Sec7p may function as a peripheral membrane protein that cycles between a soluble, cytosolic pool and a sedimentable, membrane-associated complex for its essential role in vesicular traffic through the Golgi apparatus. The transmembrane Kex2 protease, which processes precursors of secreted peptides within the yeast secretory pathway, is also localized by indirect immunofluorescence to multiple structures in the yeast cell (Redding, K., and R. Fuller, manuscript submitted for publication). In double-immunofluorescence labeling experiments, significant colocalization of Sec7 and Kex2 proteins was found. Colocalization of the two antigens, one implicated in protein transport through the Golgi apparatus and the other in processing within a late Golgi compartment, supports the conclusion that we have visualized the yeast Golgi apparatus.