P4-ATPase requirement for AP-1/clathrin function in protein transport from the trans-Golgi network and early endosomes.
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 drs2Delta cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.
Liu
Ke
K
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634, USA.
Surendhran
Kavitha
K
Nothwehr
Steven F
SF
Graham
Todd R
TR
D000483:Alleles; D001692:Biological Transport; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D005089:Exocytosis; D015966:Gene Expression Regulation, Fungal; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry; D008954:Models, Biological; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
D002966:Clathrin; C104014:DRS2 protein, S cerevisiae; D008051:Lipid Bilayers; D029701:Saccharomyces cerevisiae Proteins; D018808:Transcription Factor AP-1; D049452:Green Fluorescent Proteins; D000252:Calcium-Transporting ATPases