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Vol. 18, Issue 2, 487-500, February 2007

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Yeast P4-ATPases Drs2p and Dnf1p Are Essential Cargos of the NPFXD/Sla1p Endocytic Pathway

Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634

Submitted July 10, 2006; Revised November 9, 2006; Accepted November 13, 2006
Monitoring Editor: David Drubin

Drs2p family P-type ATPases (P4-ATPases) are required in multiple vesicle-mediated protein transport steps and are proposed to be phospholipid translocases (flippases). The P4-ATPases Drs2p and Dnf1p cycle between the exocytic and endocytic pathways, and here we define endocytosis signals required by these proteins to maintain a steady-state localization to internal organelles. Internalization of Dnf1p from the plasma membrane uses an NPFXD endocytosis signal and its recognition by Sla1p, part of an endocytic coat/adaptor complex with clathrin, Pan1p, Sla2p/End4p, and End3p. Drs2p has multiple endocytosis signals, including two NPFXDs near the C terminus and PEST-like sequences near the N terminus that may mediate ubiquitin (Ub)-dependent endocytosis. Drs2p localizes to the trans-Golgi network in wild-type cells and accumulates on the plasma membrane when both the Ub- and NPFXD-dependent endocytic mechanisms are inactivated. Surprisingly, the pan1-20 temperature-sensitive mutant is constitutively defective for Ub-dependent endocytosis but is not defective for NPFXD-dependent endocytosis at the permissive growth temperature. To sustain viability of pan1-20, Drs2p must be endocytosed through the NPFXD/Sla1p pathway. Thus, Drs2p is an essential endocytic cargo in cells compromised for Ub-dependent endocytosis. These results demonstrate an essential role for endocytosis in retrieving proteins back to the Golgi, and they define critical cargos of the NPFXD/Sla1p system.

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

Abbreviations used: 5-FOA, 5 fluoroorotic acid; ALP, alkaline phosphatase; CM, conserved motif; cs, cold sensitive; EH, Eps15 homology; GIM, Gea2p interaction motif; PVC, prevacuolar compartment; SHD, Sla1p homology domain; TGN, trans-Golgi network.

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