GGAs: Roles of the different domains and comparison with AP-1 and clathrin

We have previously identified a novel family of proteins called the GGAs (G olgi-localized, gamma -ear-containing, ADP-ribosylation factor-binding prot eins). These proteins consist of an NH2-terminal VHS domain, followed by a GAT domain, a variable domain, and a gamma -adaptin ear homology domain Stu dies from our own laboratory and others, making use of both yeast and mamma ls cells, indicate that the GGAs facilitate trafficking from the trans-Golg i network to endosomes. Here we have further investigated the function of t he GGAs. We find that GGA-deficient yeast are not only defective in vacuola r protein sorting but they are also impaired in their ability to process al pha -factor. Using deletion mutants and chimeras, we show that the VHS doma in is required for GGA function and that the VHS domain from Vps27p will no t substitute for the GGA VHS domain. In contrast, the gamma -adaptin ear ho mology domain contributes to GGA function but is not absolutely required, a nd full function can be restored by replacing the GGA ear domain with the g amma -adaptin ear domain. Deleting the gamma -adaptin gene together with th e two GGA genes exacerbates the phenotype in yeast, suggesting that they fu nction on parallel pathways. In mammalian cells, the association of GGAs wi th the membrane is extremely unstable, which may account for their absence from purified clathrin-coated vesicles. Double- and triple-labeling immunof luorescence experiments indicate that the GGAs and AP-1 are associated with distinct populations of clathrin-coated vesicles budding from the trans-Go lgi network. Together with results from other studies, our findings suggest that the GGAs act as monomeric adaptors, with the four domains involved in cargo selection, membrane localization, clathrin binding, and accessory pr otein recruitment.