Compartmental Organization of Golgi-specific Protein Modification and Vacuolar Protein Sorting Events Defined in a Yeast sec 18 ( NSF ) Mutant

The sec18 and sec23 secretory mutants of Saccharomyces cerevisiae have previously been shown to exhibit temperature-conditional defects in protein transport from the ER to the Golgi complex (Novick, P., S . Ferro, and R. Schekman . 1981 . Cell. 25:461469) . We have found that the Sec18 and Sec23 protein functions are rapidly inactivated upon shifting mutant cells to the nonpermissive temperature (<1 min) . This has permitted an analysis of the potential role these SEC gene products play in transport events distal to the ER. The sec-dependent transport of a-factor (af) and carboxypeptidase Y (CPY) biosynthetic intermediates present throughout the secretory pathway was monitored in temperature shift experiments . We found that Sec18p/NSF function was required sequentially for protein transport from the ER to the Golgi complex, through multiple Golgi compartments and from the Golgi complex to the cell surface. In contrast, Sec23p THE Golgi complex plays an important role in the posttranslational modification of glycoproteins and in the sorting of these proteins to their appropriate destination . In mammalian cells the Golgi complex is divided into at least four spatially and functionally distinct regions : cis, medial, trans, and the trans-Golgi network (TGN)' (Palade, 1975; Farquhar, 1985; Pfeffer and Rothman, 1987) . These Golgi compartments differ in their content of specific enzymes involved in the posttranslational modification of glycoproteins . In addition, lysosomal enzymes appear to be sorted from secreted proteins in the TGN, indicating that protein sorting reactions are also restricted to specific compartments ofthe Golgi complex (Grifths and Simons, 1986; Dahms et al ., 1989) . Although Saccharomyces cerevisiae has become an important model organism for studying the secretory pathway, the compartmental organization of the Drs . T. R. Graham and S. D . Emr's present address is Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093 . 1 . Abbreviations used in this paper: af, a factor ; CPY, carboxypeptidase Y; maf, mature a factor ; NSF, N-ethylmaleimide sensitive factor; TGN, trans-Golgi network ; WIMP, Wickerman minimal praline media ; WiMPYE, WIMP yeast extract . © The Rockefeller University Press, 0021-9525/91/07/207/12 $2.00 The Journal of Cell Biology, Volume 114, Number 2, July 1991207-218 function was required in the Golgi complex, but only for transport of of out of an early compartment . Together, these studies define at least three functionally distinct Golgi compartments in yeast . From cis to trans these -compartments contain : (a) An a 1-6 mannosyltransferase ; (b) an al-3 mannosyltransferase ; and (c) the Kex2 endopeptidase . Surprisingly, we also found that a pool of Golgimodified CPY (p2 CPY) located in a compartment distal to the al-3 mannosyltransferase does not require Sec18p function for final delivery to the vacuole . This compartment appears to be equivalent to the Kex2 compartment as we show that a novel vacuolar CPY af-invertase fusion protein undergoes efficient Kex2dependent cleavage resulting in the secretion of invertase . We propose that this Kex2 compartment is the site in which vacuolar proteins are sorted from proteins destined to be secreted . yeast Golgi complex has not been extensively characterized . Reasons for this include : (a) The wild-type yeast Golgi has been difficult to identify by EM, possibly because it lacks the customary stacked cisternal morphology of the mammalian Golgi complex ; (b) subcellular fractionation of yeast organelles has not yet resulted in the clean separation of Golgi enzyme activities ; and (c) few mutants (sec7and sec14) that affect protein transport through the yeast Golgi have been isolated (Esmon et al ., 1981 ; Novick et al ., 1981) . Recently however, Franzusoff and Schekman (1989) have presented evidence that the enzyme(s) which catalyze al-6 linked mannose addition to yeast glycoproteins appear to be sequestered in an early Golgi compartment that lacks al-3 mannosyltransferase and Kex2 endopeptidase activities (Franzusoff and Schekman, 1989) . In addition, Cunningham and Wickner (1989) have reported that membranes enriched for an cal-3 mannosyltransferase activity and Kex2 endopeptidase activity can be partially separated in a Percoll density gradient (Cunningham and Wickner, 1989) . The implication from these studies is that, like the mammalian Golgi complex, the yeast Golgi complex may be composed of discrete cisternae with specific functions in glycoprotein modification and sorting .