Identification of a nonapeptide motif in the vimentin head domain involved in intermediate filament assembly.
The assembly of soluble vimentin subunits into intermediate filaments (IFs) is dependent on information located in the amino-terminal domain. Using site-directed mutagenesis of a Xenopus laevis vimentin cDNA and an Escherichia coli production system to obtain pure mutated protein, we have identified, in the head domain, a nine amino acid motif (SSYRRIFGG), evolutionarily conserved from amphibia to man, which plays an important role in the orderly formation of IFs. Exchanges in the central di-arginine and in the two aromatic residues interfere with IF assembly of vimentin in vitro: on assembly under standard assembly conditions (160 mM-NaCl) most of the protein is included in dense aggregates, with a variable and minor proportion of IFs, whereas at lower ionic concentrations short and incomplete IF-like structures are formed. The deletion of the whole motif results in a protein that under standard assembly conditions (e.g. 160 mM-NaCl) predominantly and rapidly precipitates into large aggregates of non-IF material, whereas at lower ionic strength (e.g. 50 mM-NaCl) both IFs and dense aggregates are formed simultaneously. Our results show that the mutated protein can assume different forms at the same time and under the same conditions. This motif alone is insufficient for the formation of normal IFs as demonstrated by a mutant in which the motif has been brought closer to the alpha-helical rod domain by deletion of 55 internal amino acid residues. Corresponding observations have been made, by immunofluorescence microscopy, upon transfection of cultured epithelial cells lacking vimentin IFs. The importance of the head domain motif for the assembly and higher-order arrangement of IFs is discussed.
Herrmann
H
H
Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.
Hofmann
I
I
Franke
W W
WW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D007382:Intermediate Filaments / Q000737:chemistry* / Q000648:ultrastructure; D051379:Mice; D008845:Microinjections; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D014162:Transfection; D014169:Transformation, Bacterial; D014746:Vimentin / Q000737:chemistry* / Q000235:genetics; D014783:Viscosity; D014982:Xenopus laevis
D010446:Peptide Fragments; D014746:Vimentin; D004247:DNA