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J. Biol. Chem., Vol. 280, Issue 6, 4442-4450, February 11, 2005

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Cell-free Transport from the trans-Golgi Network to Late Endosome Requires Factors Involved in Formation and Consumption of Clathrin-coated Vesicles^*

Mohamed E. Abazeed, Jennifer M. Blanchette, and Robert S. Fuller

From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

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.

Received for publication, November 5, 2004

^* This work was supported in part by National Institutes of Health Grants GM50915 and GM39697 (to R. S. F.), a University of Michigan Medical Scientist Training Program Grant GM0786 (to M. E. A.), the Genetics Training Program GM07544 (to M. E. A. and J. M. B.), a University of Michigan Rackham Graduate School predoctoral fellowship (to J. M. B.), and P30 CA46592 to the University of Michigan Comprehensive Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biological Chemistry, 1301 E. Catherine Rd., University of Michigan, Ann Arbor, MI 48109-0606. Tel.: 734-936-9764; Fax: 734-763-7799; E-mail: bfuller{at}umich.edu.

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