Mutant analysis of the body wall Troponin C of Caenorhabditis elegans

Mutant analysis of the body wall Troponin C of Caenorhabditis elegans

Tomohide Takaya1, Hiromi Terami2, Tetsuya Bando2, Hiroaki Kagawa1,2.

  1. Department of Biology, Faculty of Science, Okayama University, Okayama, Japan.
  2. Graduate School of Science and Technology, Okayama University, Okayama, Japan.

第3回日本線虫集会 (名古屋), 2002/08/06 (口演).


Troponin C (TnC) is a Ca2+-binding component of troponin complex involved in Ca2+ regulation of muscle contraction through the thin filament. In the nematode Caenorhabditis elegans, a TnC gene, pat-10 that encodes 161 amino acid residues and is expressed in body wall muscles throughout the life of the animal. At the amino acid level, C. elegans TnC showed 45% identity with Drosophila TnC and 34% identity with cardiac TnC of vertebrates. The mutations in pat-10 leads to animals that arrest at two-fold stage in embryogenesis. Sequence analysis showed that the pat-10(st568) mutant has two mutations in TnC; one is D64 to N and the other is W153 to stop. Using mutated proteins produced in bacteria, we found that the D64 site is necessary for Ca2+-dependent conformation change and the C-terminal H-helix is essential for troponin I and Ca2+-bindings. To investigate the phenotypes of animals having each of the two different mutant TnCs (m1: D64N or m2: W153stop), we constructed injection vectors using site-directed mutagenesis. We injected each of the both vectors into pat-10(st568)/+ animals and counted the segratation rate of wild type and Pat phenotype. Transgenic animals with m1/pat-10(st568) were almost similar to the wild type, but transgenic animals with m2/pat-10(st568) showed Pat phenotype. These results confirmed that C. elegans TnC is essential for development, and regulates muscle contraction by Ca2+ binding at site IV and TnI interaction. Biochemical and genetical studies demonstrate that troponin C is essential not only for refulating muscle contraction but also for filament assembly during embryogenesis. We are constructing mutant TnC vectors to analyze more fine molecular interactions between site IV and Ca2+ or between H-helix and troponin I.