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Elife. 2014 Sep 17;3:e03970. doi: 10.7554/eLife.03970.

Clathrin-independent pathways do not contribute significantly to endocytic flux.

Author information

1
Department of Cell Biology, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
2
New England Biolabs, Inc., Ipswich, United States.

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.

KEYWORDS:

cell biology; clathrin; endocytosis; human; membrane transport; vesicle

PMID:
25232658
PMCID:
PMC4185422
DOI:
10.7554/eLife.03970
[Indexed for MEDLINE]
Free PMC Article

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