IN-VITRO ASSEMBLY PROPERTIES OF VIMENTIN MUTAGENIZED AT THE BETA-SITETAIL MOTIF

The intermediate filament (IF) proteins vimentin, desmin and peripheri n share a 9-residue sequence motif (beta-site) located near the end of their COOH-terminal tail domain. Peptide inhibition experiments have previously suggested that the beta-site is involved in interactions th at limit the lateral growth of IFs and prevent inappropriate filament- filament associations. To investigate this question further, we have c onstructed and expressed, in Escherichia coli, hamster vimentin bearin g different mutations in the beta-site. We show here that a single exc hange of glycine 450 with a valine residue, or an internal deletion of amino acids 444-452, strongly interferes with the normal assembly of IFs under in vitro conditions. These mutants polymerize into irregular fibrils that have a strong tendency to anastomose and laterally aggre gate under isotonic conditions. In contrast, a non-conservative substi tution of arginine 448 for glutamic acid does not significantly interf ere with filament structure and yields subunits that polymerize into l ong, smooth filaments that show a slight aberration in thickness. All mutant proteins are soluble in low salt and form oligomers similar to the ones formed by wild-type vimentin. On the basis of these findings and on related observations, we propose that the tail domain of type I ll IF proteins contains important structural elements involved in late ral protofilament-protofilament interactions.