Structural elements of the amino-terminal head domain of vimentin essential for intermediate filament formation in vivo and in vitro.

The biological functions of the non-alpha-helical, N- and C-terminal head and tail domains of intermediate filament (IF) proteins are still ill-defined. Previously, it has been shown that the basic, N-terminal head piece of the type III IF protein vimentin is essential for regular IF assembly and that arginine residues within the N-terminus may be involved. In order to identify particular regions within this domain essential for filament formation and stabilization, N-terminally truncated and arginine substitution forms of vimentin were constructed via site-directed in vitro mutagenesis of murine vimentin cDNA. The de novo filament assembly properties of these modified forms were compared with those of wild-type vimentin after transient expression in vimentin-free, cultured cells. In order to investigate their filament assembly competence in vitro, they were also produced in an E. coli expression system. It could be demonstrated that deletion of the first 10, 13, 17, and 32 amino acid residues, respectively, from the N-terminus of vimentin has an increasingly deleterious effect on filament assembly in vitro and network formation in vivo and that, thus, the highly conserved sequence motif, SSYRRXFGG, located in the N-terminus of various IF proteins and partially or totally removed by the above deletions plays a particularly important role in both activities. These results were confirmed and extended by arginine point mutations in the N-terminal head region, which showed that only one of the two adjacent arginine residues located within the conserved sequence motif is essential for filament assembly and stability in vitro as well as network formation in vivo. The neighboring arginine residues could be replaced by lysine residues without severe effects on the assembly properties of the respective mutant proteins. Distinction between the assembly-promoting potentials of the two arginine residues of the N-terminal doublet was considerably facilitated by a Val389-->Asp substitution toward the carboxy-end of the 2B segment of the vimentin rod domain. The synergistic effect of point mutations in this and the N-terminal region of the vimentin molecule implies the interaction of both protein domains in the process of filament assembly. Mutant vimentin proteins that were characterized by distinct incompetence to assemble into IFs caused a massive collapse of the endogenous vimentin filament system when expressed in mouse skin fibroblasts.