A conserved region in the tail domain of vimentin is involved in its assembly into intermediate filaments

Abstract

Although the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg-asp-gly (RDG), of the tail domain incorporated into existing IF networks in vimentin-expressing (vim+) cells, but were assembly-incompetent in cells that did not express IF proteins (vim-). The failure of the RDG mutant vimentins to assemble into filament arrays in vim- cells was reversible by re-introducing a wild-type vimentin cDNA, whereupon both wild-type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process.

Intermediate Filament Protein Domain Interactions as Revealed by Two-hybrid Screens

“…Also of some interest is that neither end domain binds to the full-length vimentin molecule avidly (Table III, rows 9 and 14). Thus, the binding of tail sequences to other parts of the IF protein molecule as a mechanism of modulating filament assembly, suggested by several investigators including ourselves (Eckelt et al, 1992;Kouklis et al, 1991Kouklis et al, , 1993McCormick et al, 1993;Makarova et al, 1994;Rogers et al, 1995), is likely to be a transient or weak interaction, or perhaps one that depends on post-translational modification not found in yeast.…”

“…Additionally, less well recognized interactions involving the head and tail domains also contribute to IF assembly (Birkenberger and Ip, 1990;Eckelt et al, 1992;Herrmann et al, 1992;Makarova et al, 1994;Rogers et al, 1995). It is generally agreed that the head domain of IF proteins is essential for filament assembly, and it has been demonstrated in numerous studies that experimental removal of this domain results in assembly incompetence.…”

“…Involvement of the tail domain is a matter of some controversy. While it seems clear that removal of the tail does not appreciably hinder in vitro assembly of certain IF proteins, in other cases it has been reported to result in structural aberrations in vitro, in abortive assembly in cells (Kaufmann et al, 1985;Quinlan et al, 1989;Makarova et al, 1994;Bader et al, 1991), or in unusual localization of the IF protein to the nucleus (Rogers et al, 1995).…”

Glial Fibrillary Acidic Protein Filaments Can Tolerate the Incorporation of Assembly-compromised GFAP-δ, but with Consequences for Filament Organization and αB-Crystallin Association

“…The GFAP-␦ has a completely different C-terminal tail domain and the consensus view is that the tail domains of type III IF proteins play an important role both in controlling filament width in vitro Herrmann et al, 1996) as well as in establishing proper IF networks in vivo (Eckelt et al, 1992;McCormick et al, 1993;Chen and Liem, 1994;Makarova et al, 1994). Our data show that the presence of GFAP-␦ changes the assembly and filament-filament interactions of the GFAP filaments (Figures 1 and 4).…”

Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly

“…36 This sequence was predicted to form a beta turn, hence the name ''beta site'' 25 and was subsequently identified as a region important for the correct assembly of 10 nm filaments. 24,37 Additional studies by…”