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Vol. 15, Issue 8, 3891-3902, August 2004

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Flagellar Radial Spokes Contain a Ca²⁺-stimulated Nucleoside Diphosphate Kinase

Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3305

Submitted April 29, 2004; Revised May 21, 2004; Accepted June 1, 2004
Monitoring Editor: Paul Matsudaira

The radial spokes are required for Ca²⁺-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca²⁺-independent manner, whereas IQ2 and IQ3 show Ca²⁺-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca²⁺. This Ca²⁺-responsive enzyme, which accounts for 45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca²⁺-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes.

Abbreviations used: MBP, maltose-binding protein; NDK, nucleoside diphosphate kinase; RSP, radial spoke protein; UTR, untranslated region.

^* Corresponding author. E-mail address: steve{at}king2.uchc.edu.

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