Mutant analysis on the body-wall troponin C of C. elegans

Tomohide Takaya, Hiromi Terami, Tetsuya Bando, Hiroaki Kagawa.

Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.

The 14th International C. elegans Meeting (Los Angels, USA), 2003/06/30 (Poster).

Abstract

Troponin C (TnC) is a Ca²⁺-binding component of troponin complex involved in Ca²⁺-regulation of muscle contraction through the thin filament under the control of troponin I (TnI) regulation. TnC has four EF-hands that bind one to four Ca²⁺ depending on the TnC from different animals. In the nematode Caenorhabditis elegans, body-wall type TnC (TnC-1) binds two Ca²⁺ at site II and site IV, and 161 amino acid residues are encoded by pat-10. At the amino acid level, CeTnC-1 showed 45% identity with Drosophila TnC, 34% and 29% identity with rabbit cardiac and skeletal TnCs, respectively. The animal having mutations in pat-10 shows Pat phenotype; paralyzed, arrested elongation at two-fold. Sequence analysis showed that pat-10(st575) animal has two mutations in TnC-1, one is m1: D64 to N and the other is m2: W153 to stop. Using mutated proteins produced in bacteria, we find that D64 is necessary for Ca²⁺-binding at site II and Ca²⁺-dependent conformation change, C-terminal H-helix is essential for Ca²⁺-binding at site IV and interaction with TnI. (Terami et al, J. Cell Biol. (1999) 146, 193-202) To investigate which mutation causes Pat phenotype, we constructed genomic pTnC1-m1 and pTnC1-m2 vectors by using site-directed mutagenesis. We injected each of two vectors into RW3613 (pat-10(st575)/+) animals, and counted the segregation rate of the wild type and Pat phenotypes in F2. Phenotype of transgenic animals with m1/pat-10(st575) was almost the wild type, but m2/pat-10(st575) was still Pat phenotype. This result indicates that m2: W153stop is the real reason of Pat phenotype. These biochemical and genetical studies demonstrate that CeTnC-1 is essential for development and Ca²⁺-dependent muscle contraction regulation. Especially, H-helix, which is necessary for Ca²⁺- and TnI bindings, is important to determine the animal phenotype. To analyze more detailed molecular interactions between TnC and Ca²⁺ and TnI molecules, we constructed some mutants of TnC-1 and analyzed Ca²⁺- and TnI binding ability by the protein overlay assay. We found that the residues of F152 and W153 were the key function for TnC-TnI interaction depending on the Ca²⁺ concentration. We will present the results of phenotype analysis of these transgenic animals.