title: Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation.
abstract: CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Daniela A
authors.initials: DA
authors.lastname: Sahlender
authors.forename: Patrycja
authors.initials: P
authors.lastname: Kozik
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Miller
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Peden
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Robinson
mesh_terms: D000595:Amino Acid Sequence; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D034041:Monomeric Clathrin Assembly Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D021382:Protein Sorting Signals; D021381:Protein Transport; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D002966:Clathrin; D034041:Monomeric Clathrin Assembly Proteins; C439003:PICALM protein, human; D021382:Protein Sorting Signals; D050683:R-SNARE Proteins; D034741:RNA, Small Interfering; C476456:VAMP4 protein, human; C121241:VAMP7 protein, human