Characterization of distinct early assembly units of different intermediate filament proteins

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from t he four cytoplasmic sequence homology classes, and compared these values wi th those of the corresponding mature filaments. After two seconds under sta ndard assembly conditions (i.e. 25 mM Tris HCl (pH 7.5), 50 mM NaCl, 37 deg rees C), vimentin, desmin and the neurofilament triplet protein NF-L aggreg ated into similar types of "unit-length filaments" (ULFs), whereas cytokera tins (CKs) 8/18 already yielded long Ifs at this time point, so the ionic s trength had to be reduced. The number of molecules per filament cross-secti on, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 a t two seconds compared to 16 and 21 at one hour. NF-L exhibited correspondi ng values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, w ith major peak values of 32 and 45 at two seconds and 30, 37 and 44 after o ne hour. Desmin formed filaments of distinctly higher mass with 47 molecule s per cross-section, at two seconds and after one hour of assembly. This in dicates that individual typos of IF proteins generate filaments with distin ctly different numbers of molecules per cross-section. Also, the observed s ignificant reduction of apparent filament diameter of ULFs compared to the corresponding mature Ifs is the result of a "conservative" radial compactio n-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subu nits per cross-section. Moreover, the MPL composition of filaments is strik ingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significa ntly increased number of molecules per cross-section (56 and 84) compared t o assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form th at are arrested in this state, indicating that the system is forced into st rong but unproductive interactions between subunits. Similar "dead-end" str uctures were obtained with vimentins mutated to introduce principal alterat ions in subdomains presumed to be of general structural importance, indicat ing that these sequence changes led to new modes of intermolecular interact ions. (C) 1999 Academic Press.