title: Clathrin-independent pathways do not contribute significantly to endocytic flux.
abstract: Several different endocytic pathways have been proposed to function in mammalian cells. Clathrin-coated pits are well defined, but the identity, mechanism and function of alternative pathways have been controversial. Here we apply universal chemical labelling of plasma membrane proteins to define all primary endocytic vesicles, and labelling of specific proteins with a reducible SNAP-tag substrate. These approaches provide high temporal resolution and stringent discrimination between surface-connected and intracellular membranes. We find that at least 95% of the earliest detectable endocytic vesicles arise from clathrin-coated pits. GPI-anchored proteins, candidate cargoes for alternate pathways, are also found to enter the cell predominantly via coated pits. Experiments employing a mutated clathrin adaptor reveal distinct mechanisms for sorting into coated pits, and thereby explain differential effects on the uptake of transferrin and GPI-anchored proteins. These data call for a revision of models for the activity and diversity of endocytic pathways in mammalian cells.
authors.affiliation: Department of Cell Biology, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Vassilis
authors.identifier: http://orcid.org/0000-0002-1731-9910
authors.initials: V
authors.lastname: Bitsikas
authors.affiliation: New England Biolabs, Inc., Ipswich, United States.
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
authors.affiliation: Department of Cell Biology, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Nichols
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D015415:Biomarkers / Q000378:metabolism; D019921:Biotinylation; D019556:COS Cells; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D040901:Proteomics; D011990:Receptors, Transferrin / Q000378:metabolism; D015203:Reproducibility of Results; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: D033962:Adaptor Protein Complex 2; D015415:Biomarkers; D002966:Clathrin; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D011990:Receptors, Transferrin
keywords: cell biology; clathrin; endocytosis; human; membrane transport; vesicle