Intermediate filament protein domain interactions as revealed by two-hybrid screens.
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 to 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 A11 and A22 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 A12 and ACN modes may describe adjacent but non-interacting molecules.
Meng
J J
JJ
Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Ohio 45267-0521, USA.
Khan
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D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques; D014746:Vimentin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014746:Vimentin; D001616:beta-Galactosidase