A screen for endocytic motifs.
Sorting signals for cargo selection into coated vesicles are usually in the form of short linear motifs. Three motifs for clathrin-mediated endocytosis have been identified: YXXPhi, [D/E]XXXL[L/I] and FXNPXY. To search for new endocytic motifs, we made a library of CD8 chimeras with random sequences in their cytoplasmic tails, and used a novel fluorescence-activated cell sorting (FACS)-based assay to select for endocytosed constructs. Out of the five tails that were most efficiently internalized, only one was found to contain a conventional motif. Two contain dileucine-like sequences that appear to be variations on the [D/E]XXXL[L/I] motif. Another contains a novel internalization signal, YXXXPhiN, which is able to function in cells expressing a mutant mu2 that cannot bind YXXPhi, indicating that it is not a variation on the YXXPhi motif. Similar sequences are present in endogenous proteins, including a functional YXXXPhiN (in addition to a classical YXXPhi) in cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Thus, the repertoire of endocytic motifs is more extensive than the three well-characterized sorting signals.
Kozik
Patrycja
P
University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.
Francis
Richard W
RW
Seaman
Matthew N J
MN
Robinson
Margaret S
MS
D020816:Amino Acid Motifs; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D060908:CTLA-4 Antigen; D002469:Cell Separation; D004705:Endocytosis; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D050656:Transcription Factor AP-2 / Q000737:chemistry*
D015703:Antigens, CD; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C556707:Ctla4 protein, mouse; D050656:Transcription Factor AP-2