Bending a membrane: how clathrin affects budding.
Receptor-mediated endocytosis of ligands, such as transferrin and LDL, is suppressed when clathrin synthesis is blocked by RNA interference in HeLa cells. We have found that domains containing the adapter complex 2 (AP2)-coated vesicle adapter and the endocytic accessory proteins CALM (clathrin assembly lymphoid myeloid leukemia protein), epsin, and eps15/eps15R (EGF receptor pathway substrate 15-related) nevertheless persist at the plasma membrane. They are similar in size and number to those seen in clathrin-expressing cells. Here we characterize these membrane domains by fluorescence and electron microscopy in detail. Fluorescence recovery after photobleaching measurements suggest that the exchange between membrane-bound and free cytosolic AP2 molecules is not significantly influenced by the depletion of clathrin. The AP2 membrane domains are dispersed upon interfering with protein-protein interactions that involve the alpha appendage domain of AP2. Electron microscopy of cellular cortices revealed that the AP2 membrane domains lack any curvature, suggesting that clathrin is essential for driving coated pit invagination. A model for coated vesicle formation, incorporating a mechanism commonly referred to as a "Brownian ratchet," is consistent with our observations.
Hinrichsen
Lars
L
Department of Cell Biology, Center of Anatomy, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
Meyerholz
Anika
A
Groos
Stephanie
S
Ungewickell
Ernst J
EJ
D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D022081:Cell Surface Extensions / Q000378:metabolism*; D002966:Clathrin / Q000172:deficiency / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D021962:Membrane Microdomains / Q000648:ultrastructure; D008954:Models, Biological; D011485:Protein Binding; D013997:Time Factors
D033962:Adaptor Protein Complex 2; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; C114154:epsin