In vitro assembly properties of vimentin mutagenized at the beta-site tail motif.
The intermediate filament (IF) proteins vimentin, desmin and peripherin 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 that limit the lateral growth of IFs and prevent inappropriate filament-filament associations. To investigate this question further, we have constructed and expressed, in Escherichia coli, hamster vimentin bearing different mutations in the beta-site. We show here that a single exchange 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 aggregate under isotonic conditions. In contrast, a non-conservative substitution of arginine 448 for glutamic acid does not significantly interfere with filament structure and yields subunits that polymerize into long, 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 III IF proteins contains important structural elements involved in lateral protofilament-protofilament interactions.
Kouklis
P D
PD
Programme of Cell Biology, European Molecular Biology Laboratory, Heidelberg, FRG.
Hatzfeld
M
M
Brunkener
M
M
Weber
K
K
Georgatos
S D
SD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001704:Biopolymers / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012995:Solubility; D014746:Vimentin / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D001704:Biopolymers; D014746:Vimentin; D004247:DNA