Site-specific proteases and antisera to the amino terminus of villin have been used to show that villin is organized into seven protease-resistant domains. Six are contained in the amino-terminal Mr 87000 villin core, a Ca2"-regulated actin-severing fragment, whereas the carboxylterminal domain includes the villin "headpiece," a fragment involved in bundling of actin filaments. Ca2" inhibits proteolytic cleavage between domains in the amino-terminal half of villin. The protein sequence of villin deduced from a single cDNA clone contains a conserved sequence that is repeated six times and is found in each domain of the villin core. The conserved repeats are found in other actin-severing proteins but not in the villin headpiece. Our results suggest that actin-severing proteins are organized around a common Mr 14,000-17,000 domain.Villin (M, 95,000) is a major cytoskeletal protein in microvilli from brush-border cells of intestine and kidney (1). Binding studies have shown that villin crosslinks actin filaments into bundles at low Ca2" concentrations, but at high (greater than micromolar) Ca2" concentrations, villin caps and severs actin filaments to short lengths (1, 2). Limited proteolysis of native villin generates large amino-terminal fragments, the villin core (Mr 87,000) (3, 4) and 44T (Mr 44,000) (5), that retain Ca2+-regulated actin-severing properties and a carboxyl-terminal Mr 8700 fragment (villin "headpiece") that is required for the actin-crosslinking activity (3, 4). Further proteolysis of 44T produces a Mr 14,000 amino-terminal fragment (44T-14T), which suggests the presence of smaller domains (6). Similarities in sequences, actin-binding activities, and patterns of proteolytic cleavage suggest that villin is related to gelsolin, a Mr 85,000 actin-severing protein found in vertebrate cells and sera (reviewed in refs. 7
and 8).Gelsolin is also cleaved in half by proteases, but unlike villin the amino-terminal half of gelsolin displays Ca22-insensitive actin-severing activity (9-12). Further proteolysis generates fragments whose sizes suggest that both halves of gelsolin contains small (Mr 14,000) and large (Mr 30,000) domains.Villin and gelsolin are structurally and functionally similar to fragmin (13,14) and severin (15,16) We have been studying the relationship between villin structure and function to explain the role of Ca2+ in regulating actin-severing and -bundling properties. In the work reported here, we used amino-terminal-specific antisera and site-specific proteolysis to show that villin is organized into seven structural domains and that Ca2+ alters the conformation of the amino-terminal half of villin. Analysis of the villin protein sequence deduced from cDNA § showed the presence of a 37-to 51-residue conserved sequence that is repeated six times in the villin core and gelsolin and three times in fragmin and severin. The repeats map within the six domains of the villin core, suggesting that the domains of actin-severing proteins are homologous.
MATERIALS AND METHODSProteins. Vil...
