Gga/Clathrin‐Dependent Transport from the TGN to the Late Endosome/Prevacuolar Compartment

Abstract

Localization of membrane proteins (Kex2p, Vps10p) to the trans Golgi network (TGN) in yeast requires vesicular transport between the TGN and endosomes. While Vps10p follows a direct pathway between the TGN and the late endosome/prevacuolar compartment (PVC), Kex2p partitions between this pathway and an indirect pathway through the early endosome. Using cell‐free reconstitution of TGN‐PVC transport [Abazeed, M.E., et al. (2005) J. Biol. Chem., 280:4442–50], we now find that the direct pathway requires the Gga1/2 clathrin adapators, in addition to clathrin, the dynamin homolog Vps1p, the Vps21p GTPase, the SM protein Vps45p and the PVC syntaxin Pep12p, but is independent of AP‐1 2‐hybrid analysis identifies sites in the Kex2p and Vps10p C‐tails that bind the Gga2p VHS domain. Mutation of the Gga binding site in Kex2p alters Kex2p localization as judged by a sensitive mating assay. To study TGN‐PVC transport in vivo, we are employing quantitative fluorescence resonance energy transfer (FRET) microscopy using donor and acceptor C‐terminal fluorescent protein fusions created in haploids by integrative recombination of Venus YFP and mCherry RFP. Microscopic analysis utilizes “Fast 4D” FRET stoichiometry combined with algorithms for mathematical elimination of autofluorescence. Using this method we have visualized the real‐time interaction between Gga2p and the Arf1 GTPase. Support was from NIH RO1 GM50957.