Vacuolar biogenesis in yeast: Sorting out the sorting proteins

Roughly 10 years ago, a small number of yeast labs isolated a collection of mutants that missort the enzyme carboxypeptidase Y (CPY) in an attempt to unravel vacuolar protein sorting, a process that is closely related to lysosomal enzyme sorting in mammals. They came up with an overwhelming number of VfS genes-more than 50 so far-and the screen is not saturated (see Stack et al., 1995b). How can sense be made of such a large collection? Our current model of the vacuolar transport pathway is illustrated in Figure 1. The CPY receptor, together with its bound ligand, is sorted into vesicles that bud from the late Golgi and fuse with the prevacuole (step i), where the two dissociate, allowing the receptor to be recycled (step 2) while CPY is delivered to the vacuole in a distinct vesicle-mediated process (step 3). This pathway is not only required for vacuolar protein sorting, but is also important for maintaining the late Golgi distribution of a number of membrane proteins, which continuously leave the Golgi and are retrieved from the prevacuolar compartment by retrograde transport (reviewed by Nothwehr and Stevens, 1994). It takes a very large number of proteins to carry out just a single round of vesicle budding, targeting, and fusion, and every transport step requires a distinct (but related) set of proteins that confer specificity. The conservation of vesicle transport machinery at different transport steps and in different organisms has allowed observations drawn from yeast genetics, neuroscience, and biochemistry to contribute to a general model of vesicle targeting and fusion known as the SNARE hypothesis (reviewed by Ferro-Novick and Jahn, 1994). In fact, a number of VPS genes encode proteins predicted to participate in the formation of SNARE complexes at several of the steps in Figure 1, thus accounting in part for the large number of VPS genes. Recent advances have focused considerable attention on an early step in vacuolar protein sorting: the budding of vesicles at the late Golgi that are targeted for the prevacuole. Two particularly intriguing proteins have been implicated in the budding process, the dynamin-like Vpsl p and the yeast lipid kinase Vps34p. Both of these proteins are highly related to mammalian proteins involved in the formation of clathrin-coated vesicles and are therefore likely to represent conserved elements in the budding machinery. By examining the role of Vpsl p and Vps34p in vacuolar protein sorting in yeast, we may gain considerable insight into general mechanisms of vesicle formation that operate in all eukaryotes. Dynamin-like GTPases Function at the Golgi Vpslp is an 80 kDa GTPase that is closely related to the mammalian protein dynamin. Whereas dynamin is required for endocytosis at the plasma membrane of mammalian cells, Vpsl p is not required for endocytosis in yeast but instead is intimately involved in sorting at the yeast Golgi. In cells lacking Vpslp, Golgi membrane proteins are delivered to the vacuole and CPY is secreted from the cell (see Nothwehr and Stevens, 1994). Nothwehr et al. (1995) have demonstrated that in the absence of Vpsl p, Golgi and vacuolar membrane proteins are not incorporated into vesicles bound for the prevacuole but are instead misdirected to the plasma membrane and reach the vacuole only after endocytosis (see blue arrows in Figure 1). This suggests that Vpslp is involved in forming the vesicles that divert vacuolar proteins away from the secretory pathway. An increasing amount of data suggests that Vpslp works together with clathrin to form vesicles at the last compartment of the yeast Golgi. Payne and coworkers have shown that temperature-sensitive clathrin mutants also missort CPY and Golgi membrane proteins to the cell surface soon after shifting to 37OC (for references see