Involvement of the N-terminal polypeptide of vimentin in the formation of intermediate filaments.

The potential to form intermediate filaments of a 54 X 10(3) molecular weight (Mr) polypeptide derived from vimentin by cleavage by the intermediate filament-specific, Ca2+-activated proteinase was investigated. Under physiological conditions of assembly, the breakdown product did not form intermediate filaments. Electron microscopy revealed short, rod-like structures similar to those described by Geisler et al. for a 38 X 10(3) Mr alpha-helical core particle derived from desmin. Since the specific, Ca2+-activated proteinase degrades vimentin preferentially from its N terminus, this result suggests the involvement of the basic, N-terminal polypeptide of vimentin in the assembly of intermediate filaments. This was supported by the observation that arginine inhibits the formation of intermediate filaments from intact vimentin. Whereas lysine had very little effect on the assembly process, guanidinium hydrochloride was effective at the same concentration as arginine. On the basis of these findings, an affinity chromatography method for the identification and isolation of intermediate filament subunit proteins was developed. Beside vimentin, desmin, the 68 X 10(3) Mr neurofilament triplet protein, the glial fibrillary acidic protein and cytokeratins also bound to arginine methylester Sepharose 4B in a salt-stable manner and could be eluted with arginine. The 145 X 10(3) Mr neurofilament triplet protein exhibited reduced binding activity, whereas the 210 X 10(3) Mr subunit did not bind to the affinity matrix. Among the degradation products of vimentin produced by the specific, Ca2+-activated proteinase, only those with molecular weights higher than 40 X 10(3) bound to arginine methylester Sepharose 4B. The same applied to the high molecular weight degradation products of desmin with a protein-resistant 37 X 10(3) Mr polypeptide as the major component. The results suggest that arginine residues of the non-alpha-helical, N-terminal polypeptides of intermediate filament subunit proteins play an important role in filament assembly.