Clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif.
We have previously shown that the integral membrane protein CD317 has both a conventional transmembrane domain near its N-terminus and a C-terminal glycosyl-phosphatidylinositol (GPI) anchor. With the possible exception of a minor topological variant of the prion protein, there remain no other convincing examples of a mammalian protein with such a topology. CD317 is localised to cholesterol-rich lipid microdomains ('lipid rafts') in the plasma membrane and is internalised from the cell surface for delivery to a juxta-nuclear compartment (most probably the TGN). We have now investigated the mechanism by which CD317 is internalised and find that this raft-associated integral membrane protein is internalised through a clathrin-dependent pathway, internalisation is dependent upon a novel dual-tyrosine-based motif in the cytosolic domain of CD317, the cytosolic domain of CD317 can interact with the mu subunits of the AP2 and AP1 adaptor complexes, interaction with AP1 is required for delivery of CD317 back to the TGN, and removal of the GPI anchor from CD317 reduces the efficiency of CD317 internalisation. Collectively, these data indicate that CD317 is internalised and delivered back to the TGN by the sequential action of AP2 and AP1 adaptor complexes and that, surprisingly, the clathrin-mediated internalisation of CD317 occurs more efficiently if CD317 is localised to lipid rafts.
Rollason
Ruth
R
Department of Biochemistry, University of Bristol, Bristol, BS8 1T, UK.
Korolchuk
Viktor
V
Hamilton
Clare
C
Schu
Peter
P
Banting
George
G
D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism; D000074010:Bone Marrow Stromal Antigen 2
D033962:Adaptor Protein Complex 2; D015703:Antigens, CD; D002966:Clathrin; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D021122:Protein Subunits; D034741:RNA, Small Interfering; D014443:Tyrosine; C094572:BST2 protein, human; D000074010:Bone Marrow Stromal Antigen 2