Yeast P4-ATPases Drs2p and Dnf1p are essential cargos of the NPFXD/Sla1p endocytic pathway.
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.
Liu
Ke
K
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634, USA.
Hua
Zhaolin
Z
Nepute
Joshua A
JA
Graham
Todd R
TR
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000252:Calcium-Transporting ATPases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005089:Exocytosis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; C427246:CDC50 protein, S cerevisiae; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C104014:DRS2 protein, S cerevisiae; C091908:END3 protein, S cerevisiae; D005656:Fungal Proteins; D008840:Microfilament Proteins; C111870:PAN1 protein, S cerevisiae; C082265:SLA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; C480601:Dnf1 protein, S cerevisiae