Intermediate Filament Protein Domain Interactions as Revealed by Two-hybrid Screens (*)
- From the Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521
- §Supported by an Established Investigator Award from the American Heart Association(1989-1994). To whom correspondence should be addressed: Dept. of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine ML0521, 231 Bethesda Ave., Cincinnati, OH 45267-0521. Tel.: 513-558-3614; Fax: 513-558-4454.
Abstract
All intermediate filament proteins possess three distinct domains: heads, rod and tail, and subdomains within the rod called
helices 1A, 1B, 2A, and 2B. Subunit packing within a filament is a consequence of interactions among these domains. Several
such interactions are known, but probably many more contribute to stabilizing filament structure. We examined a number of
such potential interactions using the yeast two-hybrid system. Domains or subdomains of murine vimentin, a Type III intermediate
filament protein, were fused with either the DNA-binding or trans-activating domain of GAL4, a transcription factor. Interaction
between the vimentin domains/subdomains functionally reconstituted GAL4, thereby activating transcription of a GAL1-LacZ reporter gene. The oligomeric 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 interactions, among which was a strong homotypic binding of helix 2B to form tetramers. They also
demonstrated a lack of interaction among others expected to do so based on current structural models. From these results we
deduced which of the candidates for interactions, suggested by current models, were true protein-protein interactions and
which represented nearest-neighbors only. Thus, the A
and A
modes of molecular alignment identified by Steinert et al. (Steinert, P. M., Marekov, L. N., Fraser, R. D. B., and Parry, D. A. D.(1993) J. Mol. Biol. 230, 436-452) are probably true interactions, whereas the A
and A
modes may describe adjacent but non-interacting molecules.
Footnotes
-
↵* This work was supported in part by Grant CB104 from the American Cancer Society and National Institutes of Health Grant AR35973. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- IF
-
intermediate filament: MUG, 4-methylumbelliferyl β-D-galactopyranoside
- DB
-
DNA binding domain
- TA
-
transcription activating domain.
-
- Received September 12, 1995.
- Revision received November 10, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
