title: Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking.
abstract: The precise functions of most of the proteins that participate in clathrin-mediated intracellular trafficking are unknown. We investigated two such proteins, epsinR and gadkin, using the knocksideways method, which rapidly depletes proteins from the available pool by trapping them onto mitochondria. Although epsinR is known to be an N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-specific adaptor, the epsinR knocksideways blocked the production of the entire population of intracellular clathrin-coated vesicles (CCVs), suggesting a more global function. Using the epsinR knocksideways data, we were able to estimate the copy number of all major intracellular CCV proteins. Both sides of the vesicle are densely covered, indicating that CCVs sort their cargo by molecular crowding. Trapping of gadkin onto mitochondria also blocked the production of intracellular CCVs but by a different mechanism: vesicles became cross-linked to mitochondria and pulled out toward the cell periphery. Both phenotypes provide new insights into the regulation of intracellular CCV formation, which could not have been found using more conventional approaches.
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hirst
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.
authors.forename: James R
authors.initials: JR
authors.lastname: Edgar
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.
authors.forename: Georg H H
authors.initials: GH
authors.lastname: Borner
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
authors.forename: Sam
authors.initials: S
authors.lastname: Li
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.
authors.forename: Daniela A
authors.initials: DA
authors.lastname: Sahlender
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.
authors.forename: Robin
authors.initials: R
authors.lastname: Antrobus
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom.
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Robinson
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: C500885:AP1AR protein, human; D033942:Adaptor Proteins, Vesicular Transport; C475910:CLINT1 protein, human; D002966:Clathrin; D008565:Membrane Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins