Function of type I and type II keratin head domains: their role in dimer, tetramer and filament formation.

To examine the role of the keratin head region and its subdomains in filament assembly we constructed several deletion mutants of type I and type II keratins and analysed their in vitro IF forming capacity. The delta K8 (1-74) and delta K18 (1-56), mutants formed only soluble oligomers, predominantly tetramers with their heterotypic partners. K8 mutants that retained either the entire (delta K8 (1-64)) or nearly the entire (delta K8 (1-66)) H1 subdomain formed some short and irregular IF-like structures with K18. However, filaments never reached the normal length and more protofilamentous material was observed. Analysis of the soluble complexes in 2 M guanidine-HCl indicated that tetramer formation was impaired in the truncated molecules. The length of the deletion correlated with the degree of tetramer destabilization. These results suggest that the head domain--specifically the H1 subdomain of type II keratins-plays a direct role in IF assembly. Its functions include a stabilization of the tetramer molecule, suggesting a role in directing the alignment of dimers as well as in elongation. We also analysed whether both head domains are required or if either type I or type II head domains alone are sufficient for IF formation. Hybrid molecules carrying their partner keratins head domains (K18 (8 head) and K8 (18 head)) were combined with their wild-type partners and tested for IF-forming ability. Both combinations formed filaments distinct from normal IF. The effect of the 'replaced' head domains was not compensated when both hybrid molecules were combined. Taken together, the results indicate that complete removal of the head domains of either K8 or K18 arrested IF assembly at the state of soluble oligomers. Replacement of the head domains by head domains of the complementary partner partly compensated for the effect. However, regular IF formation could not take place when either the head domain was missing or it was replaced by the partner's keratin head.