INTERMEDIATE FILAMENT PROTEIN DOMAIN INTERACTIONS AS REVEALED BY 2-HYBRID SCREENS

All intermediate filament proteins possess three distinct domains: hea ds, rod and tail, and subdomains within the rod called helices 1A, 1B, 2A, and 2B, Subunit packing within a filament is a consequence of int eractions among these domains, Several such interactions are known, bu t probably many more contribute to stabilizing filament structure. We examined a number of such potential interactions using the yeast two-h ybrid system, Domains or subdomains of murine vimentin, a Type III int ermediate filament protein, were fused with either the DNA-binding or trans-activating domain of GAL4, a transcription factor, Interaction b etween the vimentin domains/subdomains functionally reconstituted GAL4 , thereby activating transcription of a GAL1-LacZ reporter gene. The o ligomeric state at which the interactions took place, i.e, whether the domains/subdomains were dimeric or tetrameric as they interacted, was also determined, These studies revealed a number of interesting inter actions, among which was a strong homotypic binding of helix 2B to for m tetramers, They also demonstrated a lack of interaction among others expected to do so based on current structural models. From these resu lts we deduced which of the candidates for interactions, suggested by current models, were true protein-protein interactions and which repre sented nearest-neighbors only, Thus, the A(11) and A(22) modes of mole cular alignment identified by Steinert ef al. (Steinert, P. Ri,, Marek ov, L, N., Fraser, R. D, B., and Parry, D, A, D. (1993) J, Mel. Biol. 230, 436-452) are probably true interactions, whereas the A(12) and A( CN) modes may describe adjacent but non-interacting molecules.