Distinct role of TGN-resident clathrin adaptors for Rab5/Vps21p activation in the TGN-endosome trafficking pathway.

Clathrin-mediated vesicle trafficking plays central roles in the post-Golgi transport. In yeast, AP-1 complex and GGA adaptors (GGAs) are predicted to generate distinct transport vesicles at the TGN, and epsin-related Ent3p/Ent5p act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN is crucial for yeast Rab5 (Vps21p)-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGAs are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Vps21p activity remains ambiguous. Here we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Vps21p activation. We were able to show that AP-1 disruption in ent3Δ/5Δ mutant impairs Vps21p-GEF Vps9p transport to the Vps21p compartment, and severely reduces Vps21p activity. Additionally, GGAs, PI4 kinase Pik1p and Rab11 GTPases Ypt31p/32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Vps21p activation in the TGN-endosome trafficking pathway.