Alteration of Tropomyosin-binding Properties of Tropomodulin-1 Affects Its Capping Ability and Localization in Skeletal Myocytes
Document Type
Article
Publication Date
2013
Keywords
Actin, Circular Dichroism (CD), Fluorescence, Site-directed Mutagenesis, Tropomyosin, Actin-capping, Intrinsically Disordered Regions, Protein-protein Interactions, Tropomodulin
Digital Object Identifier (DOI)
https://doi.org/10.1074/jbc.M112.434522
Abstract
Tropomodulin (Tmod) is an actin-capping protein that binds to the two tropomyosins (TM) at the pointed end of the actin filament to prevent further actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying actin filament dependent processes such as muscle contraction and intracellular transport. The capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1–4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of actin pointed-end polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the actin-capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the actin filament depends on its TM-binding affinity.
Background: Tropomodulin is a tropomyosin-dependent actin-capping protein.
Results: Mutations in tropomodulin-1 that reduce its affinity for tropomyosin (R11K, D12N, Q144K) reduced inhibition of actin pointed-end polymerization in vitro and decreased assembly of tropomodulin-1 in skeletal myocytes.
Conclusion: The tropomyosin-binding ability of tropomodulin-1 directly influences its actin filament regulatory activity.
Significance: Creating a tool for studying the roles of different tropomodulin isoforms in living cells.
Rights Information
This work is licensed under a Creative Commons Attribution 4.0 License.
Was this content written or created while at USF?
Yes
Citation / Publisher Attribution
Journal of Molecular Biology, v. 288, issue 7, p. 4899-4907
Scholar Commons Citation
Moroz, Natalia A.; Novak, Stefanie M.; Azevedo, Richardo; Colpan, Mert; Uversky, Vladimir N.; Gregorio, Carol C.; and Kostyukova, Alla S., "Alteration of Tropomyosin-binding Properties of Tropomodulin-1 Affects Its Capping Ability and Localization in Skeletal Myocytes" (2013). Molecular Medicine Faculty Publications. 610.
https://digitalcommons.usf.edu/mme_facpub/610
