Vps74p, a member of the GOLPH3 protein family, binds directly to coatomer and the cytoplasmic tails of a subset of Golgi‐resident glycosyltransferases to mediate their Golgi retention. We identify a cluster of arginine residues at the N ‐terminal end of GOLPH3 proteins that are necessary and sufficient to mediate coatomer binding. While loss of coatomer binding renders Vps 74p non‐functional for glycosyltransferase retention, the Golgi membrane‐binding capabilities of the mutant protein are not significantly reduced. We establish that the oligomerization status and phosphatidylinositol‐4‐phosphate‐binding properties of Vps 74p largely account for the membrane‐binding capacity of the protein and identify an Arf 1p– Vps 74p interaction as a potential contributing factor in Vps 74p Golgi membrane association .

Golgi-resident glycosyltransferases are a family of enzymes that sequentially modify glycoproteins in a subcompartment-specific manner. These type II integral membrane proteins are characterized by a short cytoplasmically exposed amino-terminal tail and a luminal enzymatic domain. The cytoplasmic tails play a role in the localization of glycosyltransferases, and coat protein complex I (COPI) vesicle-mediated retrograde transport is also involved in their Golgi localization. However, the tails of these enzymes lack known COPI-binding motifs. Here, we found that Vps74p bound to a pentameric motif present in the cytoplasmic tails of the majority of yeast Golgi-localized glycosyltransferases, as well as to COPI. We propose that Vps74p maintains the steady-state localization of Golgi glycosyltransferases dynamically, by promoting their incorporation into COPI-coated vesicles.

Using nuclear magnetic resonance spectroscopy, we establish that the N-terminal domain of the yeast vacuolar R-SNARE Nyv1p adopts a longin-like fold similar to those of Sec22b and Ykt6p. Nyv1p is sorted to the limiting membrane of the vacuole via the adaptor protein (AP)3 adaptin pathway, and we show that its longin domain is sufficient to direct transport to this location. In contrast, we found that the longin domains of Sec22p and Ykt6p were not sufficient to direct their localization. A YXX Phi-like adaptin-dependent sorting signal (Y(31)GTI(34)) unique to the longin domain of Nyv1p mediates interactions with the AP3 complex in vivo and in vitro. We show that amino acid substitutions to Y(31)GTI(34) (Y31Q;I34Q) resulted in mislocalization of Nyv1p as well as reduced binding of the mutant protein to the AP3 complex. Although the sorting of Nyv1p to the limiting membrane of the vacuole is dependent upon the Y(31)GTI(34) motif, and Y31 in particular, our findings with structure-based amino acid substitutions in the mu chain (Apm3p) of yeast AP3 suggest a mechanistically distinct role for this subunit in the recognition of YXX Phi-like sorting signals.