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Vol. 19, Issue 8, 3526-3535, August 2008

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P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

Ke Liu^*,, Kavitha Surendhran^*, Steven F. Nothwehr, and Todd R. Graham^*

*Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634; and Division of Biological Sciences, University of Missouri, Columbia, MO 65211

Submitted January 10, 2008; Revised April 23, 2008; Accepted May 16, 2008
Monitoring Editor: Akihiko Nakano

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2 cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.

Present address: NIH Chemical Genomics Center, Bethesda, MD 20892-3370.

Address correspondence to: Todd R. Graham (tr.graham{at}vanderbilt.edu)

Abbreviations used: ARF, ADP-ribosylation factor; CCV, clathrin-coated vesicles; CW, calcofluor white; GEF, guanine nucleotide exchange factor; GGA, Golgi-localized; -ear–containing, ARF-binding protein; P4-ATPase, type IV P-type ATPase; PE, phosphatidylethanolamine; PS, phosphatidylserine; PVC, prevacuolar compartment.