Cell-free transport from the trans-golgi network to late endosome requires factors involved in formation and consumption of clathrin-coated vesicles.
Transport between the trans-Golgi network (TGN) and late endosome represents a conserved, clathrin-dependent sorting event that separates lysosomal from secretory cargo molecules and is also required for localization of integral membrane proteins to the TGN. Previously, we reported a cell-free reaction that reconstitutes transport from the yeast TGN to the late endosome/prevacuolar compartment (PVC) and requires the PVC t-SNARE Pep12p. Here, we report that factors required both for formation of clathrin-coated vesicles at the TGN (the Chc1p clathrin heavy chain and the Vps1p dynamin homolog) and for vesicle fusion at the PVC (the Vps21p rab protein and Vps45p SM (Sec1/Munc18) protein) are required for cell-free transport. The marker for TGN-PVC transport, Kex2p, is initially present in a clathrin-containing membrane compartment that is competent for delivery of Kex2p to the PVC. A Kex2p chimera containing the cytosolic tail (C-tail) of the vacuolar protein sorting receptor, Vps10p, is also efficiently transported to the PVC. Antibodies against the Kex2p and Vps10p C-tails selectively block transport of Kex2p and the Kex2-Vps10p chimera. The requirements for factors involved in vesicle formation and fusion, the identification of the donor compartment as a clathrin-containing membrane, and the need for accessibility of C-tail sequences argue that the TGN-PVC transport reaction involves selective incorporation of TGN cargo molecules into clathrin-coated vesicle intermediates. Further biochemical dissection of this reaction should help elucidate the molecular requirements and hierarchy of events in TGN-to-PVC sorting and transport.
Abazeed
Mohamed E
ME
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Blanchette
Jennifer M
JM
Fuller
Robert S
RS
D000483:Alleles; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D043484:Proprotein Convertases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism*
D002966:Clathrin; D005656:Fungal Proteins; D008565:Membrane Proteins; C081224:PEP12 protein, S cerevisiae; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C092934:VPS45 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae; C086866:VPS21 protein, S cerevisiae; D020691:rab GTP-Binding Proteins