Saccharomyces cerevisiae mutants that have a post-Golgi block in the exocytic pathway accumulate 100-nm vesicles carrying secretory enzymes as well as plasma membrane and cell-wall components. We have separated the vesicle markers into two groups by equilibrium isodensity centrifugation. The major population of vesicles contains Bg12p, an endoglucanase destined to be a cell-wall component, as well as Pma1p, the major plasma membrane ATPase. In addition, Snc1p, a synaptobrevin homologue, copurifies with these vesicles. Another vesicle population contains the periplasmic enzymes invertase and acid phosphatase. Both vesicle populations also contain exoglucanase activity; the major exoglucanase normally secreted from the cell, encoded by EXG1, is carried in the population containing periplasmic enzymes. Electron microscopy shows that both vesicle groups have an average diameter of 100 nm. The late secretory mutants sec1, sec4, and sec6 accumulate both vesicle populations, while neither is detected in wild-type cells, early sec mutants, or a sec13 sec6 double mutant. Moreover, a block in endocytosis does not prevent the accumulation of either vesicle species in an end4 sec6 double mutant, further indicating that both populations are of exocytic origin. The accumulation of two populations of late secretory vesicles indicates the existence of two parallel routes from the Golgi to the plasma membrane.

Exocytic vesicles that accumulate in a temperature-sensitive sec6 mutant at a restrictive temperature can be separated into at least two populations with different buoyant densities and unique cargo molecules. Using a sec6 mutant background to isolate vesicles, we have found that vacuolar protein sorting mutants that block an endosome-mediated route to the vacuole, including vps1, pep12, vps4, and a temperature-sensitive clathrin mutant, missort cargo normally transported by dense exocytic vesicles, such as invertase, into light exocytic vesicles, whereas transport of cargo specific to the light exocytic vesicles appears unaffected. Immunoisolation experiments confirm that missorting, rather than a changed property of the normally dense vesicles, is responsible for the altered density gradient fractionation profile. The vps41Δ and apl6Δ mutants, which block transport of only the subset of vacuolar proteins that bypasses endosomes, sort exocytic cargo normally. Furthermore, a vps10Δ sec6 mutant, which lacks the sorting receptor for carboxypeptidase Y (CPY), accumulates both invertase and CPY in dense vesicles. These results suggest that at least one branch of the yeast exocytic pathway transits through endosomes before reaching the cell surface. Consistent with this possibility, we show that immunoisolated clathrin-coated vesicles contain invertase.

The branching of exocytic transport routes in both yeast and mammalian cells has complicated studies of the late secretory pathway, and the mechanisms involved in exocytic cargo sorting and exit from the Golgi and endosomes are not well understood. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route do not exhibit a strong secretory phenotype and are therefore difficult to isolate. In a genetic screen designed to isolate such mutants, we identified a novel conserved protein, Avl9p, the absence of which conferred lethality in a vps1⌬ apl2⌬ strain background (lacking a dynamin and an adaptor-protein complex 1 subunit). Depletion of Avl9p in this strain resulted in secretory defects as well as accumulation of Golgi-like membranes. The triple mutant also had a depolarized actin cytoskeleton and defects in polarized secretion. Overexpression of Avl9p in wild-type cells resulted in vesicle accumulation and a post-Golgi defect in secretion. Phylogenetic analysis indicated evolutionary relationships between Avl9p and regulators of membrane traffic and actin function. INTRODUCTIONMembrane vesicle-mediated intracellular transport of proteins and lipids is a fundamental process in all eukaryotic cells. Membrane transport pathways are essential for cell growth and division as well as for maintaining normal cell homeostasis of nondividing cells. The mechanisms of transport carrier formation and fusion of these carriers with their target membranes are well conserved among eukaryotes, so relatively simple organisms can serve as useful models for studying these processes. The yeast Saccharomyces cerevisiae has proven to be an especially valuable tool in the study of membrane traffic, and the majority of the components of the secretory machinery shared by all eukaryotic cells were originally identified in yeast (Novick et al., 1980;Bankaitis et al., 1986;Rothman and Stevens, 1986;Robinson et al., 1988).The first extensive yeast genetic screen for mutants that block the exocytic pathway identified many of the components involved in endoplasmic reticulum-to-Golgi transport and vesicle fusion with the plasma membrane, but only 2 of the 23 genes identified in this screen blocked anterograde transport from the Golgi (Novick et al., 1980;Guo et al., 2000;Lee et al., 2004). As in mammalian cells, there are multiple exocytic transport routes from the yeast Golgi, so cargo in a blocked pathway can be missorted and secreted by an alternate route (Harsay and Bretscher, 1995;Harsay and Schekman, 2002). This has made it difficult to identify mutants that have a defect in exocytic transport from the Golgi, and relatively little is known about the molecular machinery involved at this transport step.Although small (ϳ40 -100 nm) vesicles are the best-characterized transport intermediates in the secretory pathway, larger tubular structures also transport cargo. In mammalian cells, tubular vesicles appear to be the most abundant class of exocytic carriers from the Golgi (Hir...