The aftiphilin/p200/gamma-synergin complex.
Aftiphilin is a protein that was recently identified in database searches for proteins with motifs that interact with AP-1 and clathrin, but its function is unknown. Here we demonstrate that aftiphilin has a second, atypical clathrin binding site, YQW, that colocalizes with AP-1 by immunofluorescence, and that is enriched in clathrin-coated vesicles (CCVs), confirming that it is a bona fide component of the CCV machinery. By gel filtration, aftiphilin coelutes with two other AP-1 binding partners, p200a and gamma-synergin. Antibodies against any one of these three proteins immunoprecipitate the other two, and knocking down any of the three proteins by siRNA causes a reduction in the levels of the other two, indicating that they form a stable complex. Like AP-1-depleted cells, aftiphilin-depleted cells missort a CD8-furin chimera and the lysosomal enzyme cathepsin D. However, whereas AP-1-depleted cells recycle endocytosed transferrin more slowly than untreated cells, aftiphilin-depleted cells accumulate endocytosed transferrin in a peripheral compartment and recycle it more rapidly. These observations show that in general, the aftiphilin/p200/gamma-synergin complex facilitates AP-1 function, but the complex may have additional functions as well, because of the opposing effects of the two knockdowns on transferrin recycling.
Hirst
Jennifer
J
Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, United Kingdom.
Borner
Georg H H
GH
Harbour
Michael
M
Robinson
Margaret S
MS
D033961:Adaptor Protein Complex 1; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004705:Endocytosis; D045683:Furin / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism
D033961:Adaptor Protein Complex 1; D002352:Carrier Proteins; D002966:Clathrin; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C121510:SYNRG protein, human; D018808:Transcription Factor AP-1; D014168:Transferrin; D049452:Green Fluorescent Proteins; C468731:FURIN protein, human; D045683:Furin; D002402:Cathepsin D