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Eukaryotic Cell, February 2004, p. 72-81, Vol. 3, No. 1
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.1.72-81.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Flagellar Radial Spoke Protein 2 Is a Calmodulin Binding Protein Required for Motility in Chlamydomonas reinhardtii

Pinfen Yang,1 Chun Yang,1 and Winfield S. Sale2*

Department of Biology, Marquette University, Milwaukee, Wisconsin 53233,1 Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 303222

Received 6 October 2003/ Accepted 12 December 2003

Genetic and morphological studies have revealed that the radial spokes regulate ciliary and flagellar bending. Functional and biochemical analysis and the discovery of calmodulin in the radial spokes suggest that the regulatory mechanism involves control of axonemal protein phosphorylation and calcium binding to spoke proteins. To identify potential regulatory proteins in the radial spoke, in-gel kinase assays were performed on isolated axonemes and radial spoke fractions. The results indicated that radial spoke protein 2 (RSP2) can bind ATP and transfer phosphate in vitro. RSP2 was cloned and mapped to the PF24 locus, a gene required for motility. Sequencing revealed that pf24 contains a point mutation converting the first ATG to ATA, resulting in only trace amounts of RSP2 and confirming the RSP2 mapping. Surprisingly, the sequence does not include signature domains for conventional kinases, indicating that RSP2 may not perform as a protein kinase in vivo. However, the predicted RSP2 protein sequence contains Ca2+-dependent calmodulin binding motifs and a GAF domain, a domain found in diverse signaling proteins for binding small ligands including cyclic nucleotides. As predicted from the sequence, recombinant RSP2 binds calmodulin in a calcium-dependent manner. We postulate that RSP2 is a regulatory subunit of the radial spoke involved in localization of calmodulin for control of motility.

* Corresponding author. Mailing address: Department of Cell Biology, Whitehead Biomedical Research Building, Emory University School of Medicine, 615 Michael St., Atlanta, GA 30322. Phone: (404) 727-6265. Fax: (404) 727-6256. E-mail: win{at}cellbio.emory.edu.

Eukaryotic Cell, February 2004, p. 72-81, Vol. 3, No. 1
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.1.72-81.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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