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dc.contributor.authorCasey, Diane M.
dc.contributor.authorYagi, Toshiki
dc.contributor.authorKamiya, Ritsu
dc.contributor.authorWitman, George B.
dc.date2022-08-11T08:08:55.000
dc.date.accessioned2022-08-23T16:12:11Z
dc.date.available2022-08-23T16:12:11Z
dc.date.issued2003-08-16
dc.date.submitted2008-08-18
dc.identifier.citationJ Biol Chem. 2003 Oct 24;278(43):42652-9. Epub 2003 Aug 14. <a href="http://dx.doi.org/10.1074/jbc.M303064200">Link to article on publisher's site</a>
dc.identifier.issn0021-9258 (Print)
dc.identifier.doi10.1074/jbc.M303064200
dc.identifier.pmid12920131
dc.identifier.urihttp://hdl.handle.net/20.500.14038/33343
dc.description.abstractThe outer dynein arm-docking complex (ODA-DC) targets the outer dynein arm to its correct binding site on the flagellar axoneme. The Chlamydomonas ODA-DC contains three proteins; loss of any one prevents normal assembly of the outer arm, leading to a slow, jerky swimming phenotype. We showed previously that the smallest ODA-DC subunit, DC3, has four EF-hands (Casey, D. M., Inaba, K., Pazour, G. J., Takada, S., Wakabayashi, K., Wilkerson, C. G., Kamiya, R., and Witman, G. B. (2003) Mol. Biol. Cell 14, 3650-3663). Two of the EF-hands fit the consensus pattern for calcium binding, and one of these contains two cysteine residues within its binding loop. To determine whether the predicted EF-hands are functional, we purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of dithiothreitol and Mg2+. The protein bound one calcium ion with an affinity (Kd) of approximately 1 x 10-5 m. Calcium binding was observed only in the presence of dithiothreitol and thus is redox-sensitive. DC3 also bound Mg2+ at physiological concentrations but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium binding activity of the bacterially expressed protein. To investigate the role of the EF-hands in vivo, we transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the Glu to Gln mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains unknown.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12920131&dopt=Abstract ">Link to article in PubMed</a>
dc.relation.urlhttp://dx.doi.org/10.1074/jbc.M303064200
dc.subjectAmino Acid Motifs; Amino Acid Substitution; Animals; Calcium; Calcium-Binding Proteins; Chlamydomonas; Dynein ATPase; Flagella; Magnesium; Mutagenesis, Site-Directed; Oxidation-Reduction; Protein Subunits; Protozoan Proteins; Swimming
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.titleDC3, the smallest subunit of the Chlamydomonas flagellar outer dynein arm-docking complex, is a redox-sensitive calcium-binding protein
dc.typeJournal Article
dc.source.journaltitleThe Journal of biological chemistry
dc.source.volume278
dc.source.issue43
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_sp/187
dc.identifier.contextkey583232
html.description.abstract<p>The outer dynein arm-docking complex (ODA-DC) targets the outer dynein arm to its correct binding site on the flagellar axoneme. The Chlamydomonas ODA-DC contains three proteins; loss of any one prevents normal assembly of the outer arm, leading to a slow, jerky swimming phenotype. We showed previously that the smallest ODA-DC subunit, DC3, has four EF-hands (Casey, D. M., Inaba, K., Pazour, G. J., Takada, S., Wakabayashi, K., Wilkerson, C. G., Kamiya, R., and Witman, G. B. (2003) Mol. Biol. Cell 14, 3650-3663). Two of the EF-hands fit the consensus pattern for calcium binding, and one of these contains two cysteine residues within its binding loop. To determine whether the predicted EF-hands are functional, we purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of dithiothreitol and Mg2+. The protein bound one calcium ion with an affinity (Kd) of approximately 1 x 10-5 m. Calcium binding was observed only in the presence of dithiothreitol and thus is redox-sensitive. DC3 also bound Mg2+ at physiological concentrations but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium binding activity of the bacterially expressed protein. To investigate the role of the EF-hands in vivo, we transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the Glu to Gln mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains unknown.</p>
dc.identifier.submissionpathgsbs_sp/187
dc.contributor.departmentDepartment of Cell Biology
dc.contributor.departmentGraduate School of Biomedical Sciences
dc.source.pages42652-9


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