A putative role for intramolecular regulatory mechanisms in the adaptor function of amphiphysin in endocytosis.
Amphiphysin 1 is a brain-specific protein enriched at the synapse and a major binding partner of several components of the clathrin-mediated endocytic machinery (Proc Natl Acad Sci USA 93 (1996) 331). It interacts with clathrin-coat proteins, dynamin, and membranes (Nat Cell Biol 1 (1999) 33; JBC). A role of amphiphysin in synaptic vesicle recycling is supported by both acute and chronic perturbation studies (Science 276 (1997) 259; Neuron 33 (2002) 789). Here we show that amphiphysin directly stimulates clathrin recruitment onto liposomes in an in vitro assay. Amphiphysin-dependent clathrin-coat recruitment is enhanced by the interaction of amphiphysin with dynamin. We also show that the amphiphysin SH3 domain binds full-length amphiphysin, likely via an internal poly-proline region, and that clathrin recruitment onto liposomes by amphiphysin is enhanced in the presence of the isolated amphiphysin SH3 domain. Expression of a mutant amphiphysin harboring two amino acid substitutions in the SH3 domain, and therefore unable to bind proline-containing motifs, induces an accumulation of large intracellular aggregates including amphiphysin, clathrin, AP-2, and other endocytic proteins, as well as a concomitant block of transferrin endocytosis. Thus, putative intramolecular interactions between the amphiphysin COOH-terminal SH3 domain and its internal poly-proline region may regulate clathrin recruitment onto membranes.
Farsad
Khashayar
K
Howard Hughes Medical Institute and Department of Cell Biology,Yale University School of Medicine, New Haven, CT 06510, USA.
Slepnev
Vladimir
V
Ochoa
Giancarlo
G
Daniell
Laurie
L
Haucke
Volker
V
De Camilli
Pietro
P
Hauke
Volker
V
D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D022163:Clathrin-Coated Vesicles / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats
D033942:Adaptor Proteins, Vesicular Transport; D008051:Lipid Bilayers; D008081:Liposomes; D009419:Nerve Tissue Proteins; C075637:amphiphysin