Structural Requirements for Function of Yeast GGAs in Vacuolar Protein Sorting, α-Factor Maturation, and Interactions with Clathrin

The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are ubiquitous adaptor-like proteins that associate with the cytoplasmic face of the trans-Golgi network (TGN) (4, 16, 22, 37, 47). Three GGAs exist in humans (GGA1, GGA2, and GGA3), one each exists in Drosophila melanogaster and Caenorhabditis elegans, and two exist in the budding yeast Saccharomyces cerevisiae (Gga1p and Gga2p). The GGAs are monomeric but have a multidomain structure consisting of VHS (Vps27, Hrs, and STAM), GAT (GGA and TOM), hinge, and GAE (gamma-adaptin ear) domains. Biochemical and immunocytochemical analyses have revealed that each of the four GGA domains serves a specific function. The VHS domain of the human GGAs functions as a recognition module for acidic cluster-dileucine sorting signals contained within the cytosolic tails of sortilin (31) and the cation-independent (CI) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) that sort lysosomal hydrolases to lysosomes (38, 56). The GAT domain of human and yeast GGAs mediates interactions with the GTP-bound form of members of the ARF (ADP-ribosylation factor) family of proteins (4, 16, 55). GAT-ARF interactions are responsible for the regulated recruitment of the GGAs from the cytosol to the Golgi complex (4, 16, 39). The hinge domain of all the GGAs contains putative clathrin-binding motifs composed of acidic and bulky hydrophobic amino acids (16, 39). For the human GGAs, these motifs have been shown to mediate interactions with clathrin in vitro and to promote recruitment of clathrin to the TGN in vivo (39). Finally, the GAE domain of the human GGAs binds proteins such as γ-synergin and rabaptin-5, which may function to regulate assembly of GGA-containing coats or formation of coated vesicular carriers (22, 47). These properties of the human GGA domains indicate that they may mediate ARF-dependent recruitment of clathrin to the TGN in order to sort intracellular cargo receptors from the TGN to the endosomal system. Despite the detailed characterization of the properties of GGA domains, the significance of these properties for the function of the GGAs in vivo remains to be assessed. The existence of two GGAs in yeast provides an opportunity to perform analyses of the physiological roles of the GGAs in an organism easily amenable to genetic manipulation. In yeast, biosynthetic protein sorting from the Golgi complex to the vacuole, the equivalent of the mammalian lysosome, is mediated by two principal routes (for reviews, see references 7, 12, 26, and 29). The alkaline phosphatase (ALP) pathway sorts vacuolar proteins such as ALP and the t-SNARE Vam3p from the Golgi complex to the vacuole directly. Formation of Golgi complex-derived carrier vesicles and transport through this pathway require the adaptor protein (AP) complex AP-3 (14, 46) and the VPS (vacuolar protein sorting) gene products Vps41p/Vam2p, Vps39p/Vam6p (30), and Vps1p (32, 49), a member of the dynamin family of proteins (49). In contrast, the carboxypeptidase Y (CPY) pathway sorts vacuolar proteins including CPY and proteinase A (PrA) and subunits of the vacuolar ATPase from the Golgi complex to the vacuole via a prevacuolar endosomal compartment (PVC). Sorting of both CPY and PrA through this pathway is mediated by interactions with the transmembrane receptor Vps10p, which delivers these proteins to the PVC prior to recycling back to the Golgi complex (13, 27). Vesicle formation and transport in the CPY pathway involve the coat-scaffolding protein clathrin (10, 45), the putative aminophospholipid translocase Drs2p (11), and the synaptojanin-like proteins Inp52p and Inp53p (1, 33), as well as numerous VPS gene products, including Vps41p/Vam2p, Vps39p/Vam6p (30), and Vps1p (1, 49). Mutations in factors operating in these respective pathways result, to differing degrees, in impaired vacuolar sorting and defects in vacuole biogenesis. The yeast GGAs appear to play important, redundant roles in biosynthetic sorting to the vacuole based on studies of a mutant gga1Δ gga2Δ strain containing disruptions of both yeast GGA genes. This mutant is defective for transport of pro-CPY to the vacuole and missorts pro-CPY to the periplasmic space (16, 22, 55). The gga1Δ gga2Δ mutant was also found to be defective in sorting of the syntaxin Pep12p from the Golgi complex to late endosomes (2). The presence of a vacuolar morphology defect in gga1Δ gga2Δ cells has, however, been debated, with one study reporting abnormal morphology (22) and another showing no morphological differences between wild-type and gga1Δ gga2Δ strains (55). As is evident from the well-defined phenotypes arising from mutations in the yeast GGA genes, these proteins appear to play an important role in biosynthetic protein sorting. To expand our understanding of the GGAs' function and to assess the relative importance of the different GGA domains in vivo, we have performed a structure-function analysis of yeast Gga1p and Gga2p. First, we elaborate on the gga1Δ gga2Δ mutant phenotype by reporting defects in sorting of additional vacuolar proteins and abnormal vacuolar morphology and a strong defect in maturation of the mating pheromone α-factor. We then analyze the functional requirement of individual GGA domains and find that the VHS, GAT, and hinge domains are important for GGA-mediated pro-CPY sorting and pro-α-factor processing, while the GAE domain appears less important. In addition, we show that, like their human counterparts, the yeast GGAs are capable of binding clathrin via acidic-bulky-hydrophobic motifs in their hinge domains. We also present evidence that these clathrin-binding motifs contribute to GGA-mediated sorting in vivo. Finally, mutational analysis of the Gga2p VHS domain identifies a highly conserved sequence important for this protein's function.