Formation of AP-3 transport intermediates requires Vps41 function

Transport of a subset of membrane proteins to the yeast vacuole requires th e function of the AP-3 adaptor protein complex. To define the molecular req uirements of vesicular transport in this pathway, we used a biochemical app roach to analyse the formation and content of the AP-3 transport intermedia te. A vam3(tsf) (vacuolar t-SNARE) mutant blocks vesicle docking and fusion with the vacuole and causes the accumulation of 50-130-nanometre membrane vesicles, which we isolated and showed by biochemical analysis and immunocy tochemistry to contain both AP-3 adaptors and alkaline phosphatase (ALP) pa thway cargoes. Inactivation of AP-3 or the protein Vps41 blocks formation o f this vesicular intermediate. Vps41 binds to the AP-3 delta-adaptin subuni t, suggesting that they function together in the formation of ALP pathway t ransport intermediates at the late Golgi.