Dynamics of clathrin and adaptor proteins during endocytosis.
The endocytic adaptor complex AP-2 colocalizes with the majority of clathrin-positive spots at the cell surface. However, we previously observed that AP-2 is excluded from internalizing clathrin-coated vesicles (CCVs). The present studies quantitatively demonstrate that AP-2 disengages from sites of endocytosis seconds before internalization of the nascent CCV. In contrast, epsin, an alternate adaptor for clathrin at the plasma membrane, disappeared, along with clathrin. This suggests that epsin remains an integral part of the CCV throughout endocytosis. Clathrin spots at the cell surface represent a heterogeneous population: a majority (70%) of the spots disappeared with a time course of 4 min, whereas a minority (22%) remained static for > or =30 min. The static clathrin spots undergo constant subunit exchange, suggesting that although they are static structures, these spots comprise functional clathrin molecules, rather than dead-end aggregates. These results support a model where AP-2 serves a cargo-sorting function before endocytosis, whereas alternate adaptors, such as epsin, actually link cargo to the clathrin coat surrounding nascent endocytic vesicles. These data also support a role for static clathrin, providing a nucleation site for endocytosis.
Rappoport
Joshua Z
JZ
Laboratory of Cellular Biophysics, Rockefeller University, New York, NY 10021, USA.
Kemal
Shahrnaz
S
Benmerah
Alexandre
A
Simon
Sanford M
SM
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D033962:Adaptor Protein Complex 2; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D011993:Recombinant Fusion Proteins; C114154:epsin