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.

Abstract. TGN38/41, an integral membrane protein predominantly localized to the trans-Golgi network, has been shown to cycle to the plasma membrane and return to the TGN within 30 min. (Ladinsky, M. S., and K. E. Howell. 1992. Eur. J. Cell BioL 59:92-105). In characterizing the proteins which associate with TGN38/41, a peripheral 62-kD protein, two forms of rab6 and two other small GTP-binding proteins were identified by coimmunoprecipitation. However, •90% of the 62-kD protein is cytosolic and is associated with the same subset of small GTP-binding proteins. Both the membrane and cytoplasmic complexes were characterized by sizing column fractionation and velocity sedimentation. The membrane complex was ,',,250 kD (11.6 S) consisting of the cytosolic complex and a heterodimer of TGN38/41 (160 kD). The cytosolic complex was '~86 kD (6.1 S) consisting of p62 and one small GTP-binding protein. Preliminary evidence indicates that phosphorylation of the p62 molecule regulates the dissociation of the cytosolic complex from TGN38/41. Functionally the cytosolic p62 complex must bind to TGN38/41 for the budding of exocytic transport vesicles from the TGN as assayed in a cell-free system (Salamero, J., E. S. Sztul, and K. E. Howell. 1990. Proc. Natl. Acad. Sci. USA. 87:7717-7721). Interference with p62, rab6 or TGN38, and TGN41 cytoplasmic domains by immunodepletion or competing peptides completely inhibited the budding of exocytic transport vesicles. These results support an essential role for interaction of the cytosolic p62/rab6 complex with TGN38/41 in budding of exocytic vesicles from the TGN.

No abstract