A conserved clathrin assembly motif essential for synaptic vesicle endocytosis.
Although clathrin assembly by adaptor proteins (APs) plays a major role in the recycling of synaptic vesicles, the molecular mechanism that allows APs to assemble clathrin is poorly understood. Here we demonstrate that AP180, like AP-2 and AP-3, binds to the N-terminal domain of clathrin. Sequence analysis reveals a motif, containing the sequence DLL, that exists in multiple copies in many clathrin APs. Progressive deletion of these motifs caused a gradual reduction in the ability of AP180 to assemble clathrin in vitro. Peptides from AP180 or AP-2 containing this motif also competitively inhibited clathrin assembly by either protein. Microinjection of these peptides into squid giant presynaptic terminals reversibly blocked synaptic transmission and inhibited synaptic vesicle endocytosis by preventing coated pit formation at the plasma membrane. These results indicate that the DLL motif confers clathrin assembly properties to AP180 and AP-2 and, perhaps, to other APs. We propose that APs promote clathrin assembly by cross-linking clathrin triskelia via multivalent interactions between repeated DLL motifs in the APs and complementary binding sites on the N-terminal domain of clathrin. These results reveal the structural basis for clathrin assembly and provide novel insights into the molecular mechanism of clathrin-mediated synaptic vesicle endocytosis.
Morgan
J R
JR
Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Prasad
K
K
Hao
W
W
Augustine
G J
GJ
Lafer
E M
EM
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics; D002417:Cattle; D002478:Cells, Cultured; D002966:Clathrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000187:drug effects / Q000378:metabolism; D049832:Decapodiformes; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008845:Microinjections; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D020349:Surface Plasmon Resonance; D009435:Synaptic Transmission / Q000187:drug effects / Q000235:genetics; D013572:Synaptic Vesicles / Q000378:metabolism*
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D008565:Membrane Proteins; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D010750:Phosphoproteins; C439023:clathrin assembly protein AP180