STRUCTURE AND ASSEMBLY PROPERTIES OF THE INTERMEDIATE FILAMENT PROTEIN VIMENTIN - THE ROLE OF ITS HEAD, ROD AND TAIL DOMAINS

We have investigated the functional role of the non-helical end domain s of vimentin on its assembly properties using truncated Xenopus and h uman recombinant proteins. Removal of the amino-terminal ''head'' doma in yielded a molecule that did not assemble into 10 nm filaments but r emained in a soluble oligomeric particle form with a sedimentation coe fficient considerably smaller than that of wild-type vimentin (Vim(wt) ). In contrast, removal of the carboxy-terminal ''tail'' domain had no obvious effect on the sedimentation characteristics. In particular, s edimentation equilibrium analysis under low ionic strength conditions yielded oligomeric particle species of M(r), 135,000 to 360,000, indis tinguishable from those obtained with Vim(wt). When induced to form fi laments from this state by rapid dilution into filament forming buffer , Vim(wt) and Vim(Delta T) protein generated similar viscosity profile s. However, as determined by scanning transmission electron microscopy , under these conditions Vim(Delta T) formed filaments of heterogeneou s diameter, corresponding to various distinct mass-per-length (MPL) va lues: whereas Vim(wt) yielded MPL values peaking between 40 and 45 kDa /nm, Vim(Delta T) filaments produced histograms which could be fitted by three Gaussian curves peaking between 37 and 131 kDa/nm. In contras t, when dialyzed against, instead of being rapidly diluted into, filam ent forming buffer, Vim(Delta T) gave histograms with one major peak a t about 54 kDa/nm. The MPL heterogeneity observed for Vim(Delta T) was already evident at the earliest stages of assembly. For example, ten seconds after initiation, ''unit-length'' filament segments (58 to 63 nm) were formed with both wt and Delta T proteins, but the diameters w ere considerably larger for Vim(Delta T) compared to Vim(wt) (20(+/-3) nm versus 16(+/-3)nm), indicating a distinct role of the carboxy-term inal tail domain in the width control during unit-length filament form ation. Despite this difference both Vim(Delta T) and Vim(wt) filaments appeared to grow stepwise in a modular fashion from such unit-length filament segments. This suggests that assembly occurred by a principal ly similar mechanism involving the end-on-fusion or annealing of unit- length filaments. (C) 1996 Academic Press Limited.