F-actin binding site masked by the intramolecular association of vinculin head and tail domains.
Although vinculin is present at all sites of F-actin attachment to plasma membranes and is required for linkage of myofibrils to sarcolemma, it is unclear how it promotes attachment of actin to membranes. Because biochemical evidence for a direct interaction of vinculin with F-actin is controversial, current models of actin-membrane linkages depict only an indirect role for vinculin, as a tether for alpha-actinin. We demonstrate here that an intramolecular association between the 95K head and 30K tail domains of vinculin masks an F-actin binding site present in the carboxy-terminal tail domain. Cosedimentation and crosslinking assays, and direct visualization by transmission electron microscopy, reveal an interaction between F-actin and a bacterially expressed fusion protein containing amino acids 811-1066 of vinculin, and between F-actin and a proteolytic fragment of vinculin containing amino acids 858-1066. Vinculin itself neither cosediments with nor crosslinks F-actin. The amino-terminal 95K head fragment of vinculin, but not intact vinculin, inhibits both cosedimentation and crosslinking. We propose that assembly of vinculin into an adherens junction involves disruption of the head-tail interaction, revealing a site that mediates microfilament attachment.
Johnson
R P
RP
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Craig
S W
SW
D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D005982:Glutathione Transferase / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D016596:Vinculin / Q000378:metabolism* / Q000648:ultrastructure
D000199:Actins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D005982:Glutathione Transferase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase