title: Functional effects of mutations in the tropomyosin-binding sites of tropomodulin1 and tropomodulin3.
abstract: Tropomodulins (Tmods) interact with tropomyosins (TMs) via two TM-binding sites and cap the pointed ends of TM-coated actin filaments. To study the functional interplay between TM binding and TM-actin filament capping by Tmods, we introduced disabling mutations into the first, second, or both TM-binding sites of full-length Tmod1 (Tmod1-L27G, Tmod1-I131D, and Tmod1-L27G/I131D, respectively) and full-length Tmod3 (Tmod3-L29G, Tmod3-L134D, and Tmod3-L29G/L134D, respectively). Tmod1 and Tmod3 showed somewhat different TM-binding site utilization, but nearly all TM binding was abolished in Tmod1-L27G/I131D and Tmod3-L29G/L134D. Disruption of Tmod-TM binding had a modest effect on Tmod1's ability and no effect on Tmod3's ability to stabilize TM-actin pointed ends against latrunculin A-induced depolymerization. However, disruption of Tmod-TM binding did significantly impair the ability of Tmod3 to reduce elongation rates at pointed ends with α/βTM, albeit less so with TM5NM1, and not at all with TM5b. For Tmod1, disruption of Tmod-TM binding only slightly impaired its ability to reduce elongation rates with α/βTM and TM5NM1, but not at all with TM5b. Thus, Tmod-TM binding has a greater influence on Tmods' ability to inhibit subunit association as compared to dissociation from TM-actin pointed ends, particularly for α/βTM, with Tmod3's activity being more dependent on TM binding than Tmod1's activity. Nevertheless, disruption of Tmod1-TM binding precluded Tmod1 targeting to thin filament pointed ends in cardiac myocytes, suggesting that the functional effects of Tmod-TM binding on TM-coated actin filament capping can be significantly modulated by the in vivo conformation of the pointed end or other factors in the intracellular environment.
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California.
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Lewis
authors.forename: Sawako
authors.initials: S
authors.lastname: Yamashiro
authors.forename: David S
authors.initials: DS
authors.lastname: Gokhin
authors.forename: Velia M
authors.initials: VM
authors.lastname: Fowler
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002642:Chick Embryo; D006801:Humans; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D058105:Polymerization / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D053778:Thiazolidines / Q000494:pharmacology; D051348:Tropomodulin / Q000235:genetics* / Q000378:metabolism*; D014335:Tropomyosin / Q000378:metabolism*
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D050505:Mutant Proteins; D020033:Protein Isoforms; D053778:Thiazolidines; D051348:Tropomodulin; D014335:Tropomyosin; C037067:latrunculin A
keywords: actin; cardiomyocyte; pointed-end capping; tropomudulin; tropomyosin