Molecular dissection, tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans.
The ryanodine receptor of Caenorhabditis elegans (CeRyR) which contains 5,071 amino acid residues, is encoded by a single gene, ryr-1/unc-68. The unc-68(kh30) mutation, isolated in an animal showing abnormal response to the anesthetic ketamine, has the substitution Ser1444Asn in CeRyR, predicted to be a phosphorylation site. To elucidate the function of the region of CeRyR, and to determine the localization of CeRyR in this animal, ten region-peptides were produced in Escherichia coli by using expression plasmids and eight antisera were raised against these fusion peptides. One antibody against the region corresponding to the kh30 mutation site enabled detection of CeRyR from mutant animals both in Western analysis and in situ. Specificity of this antiserum was demonstrated using Western analysis, which showed the full size and the partial size bands in wild-type and in the Tc1-induced deletion mutant animals, respectively, but no corresponding bands in unc-68 null mutant animals. CeRyR was detected in I-bands of muscle sarcomeres by double immunostaining. CeRyR was found in the body wall, pharyngeal, vulval, anal and sex muscles of adult worms and also found to be present in embryonic muscle, but not in non-muscle cells. Two EF-hand motifs and the C terminus were demonstrated to be Ca(2+) binding regions. On the basis of these results, we propose a model for the functional domains of CeRyR, which agrees well with the model of mammalian skeletal RyR, which is based on proteolysis and cross-linking analysis. We discuss the usefulness and limitations of the molecular dissection approach, which uses peptides and peptide-specific antibodies to determine the local structure and function of individual domains within a large molecule.
Hamada
Tomoyo
T
Division of Biomolecular Sciences, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan. hkagawa@cc.okayama-u.ac.jp
Sakube
Yasuji
Y
Ahnn
Joohong
J
Kim
Do Han
DH
Kagawa
Hiroaki
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D003429:Cross Reactions; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D029742:Caenorhabditis elegans Proteins; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C438601:Unc-68 protein, C elegans; D002118:Calcium