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Vol. 19, Issue 11, 4826-4836, November 2008

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Yeast Golgi-localized, -Ear–containing, ADP-Ribosylation Factor-binding Proteins Are but Adaptor Protein-1 Is Not Required for Cell-free Transport of Membrane Proteins from the Trans-Golgi Network to the Prevacuolar Compartment

Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109

Submitted May 14, 2007; Revised August 20, 2008; Accepted August 29, 2008
Monitoring Editor: Akihiko Nakano

Golgi-localized, -Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2 membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2 mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome.

Address correspondence to: Robert S. Fuller (bfuller{at}umich.edu)

Abbreviations used: aa, amino acid; ALP, alkaline phosphatase; AP-1, adaptor protein-1; C-tail, cytosolic tail; CPY, carboxypeptidase Y; DPAP, dipeptidyl aminopeptidase; GAT, GGA and TOM1; GGA, Golgi-localized, -ear-containing, ADP-ribosylation factor binding protein; GST, glutathione transferase; HA, hemagglutinin; IP, immunoprecipitation; K-V, Kex2-Vps10p chimera; MPR, mannose-6-phosphate receptor; MSS, medium-speed supernatant; PI4P, phosphatidylinositol 4-phosphate; PSHA, Pep12Ste13HA fusion substrate; PVC, prevacuolar compartment; TGN, trans-Golgi network; VHS, Vps27, Hrs, and Stam.