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Flagellar Radial Spokes Contain a Ca2+-stimulated Nucleoside Diphosphate Kinase
Ramila S. Patel-King,
Oksana Gorbatyuk,
Sachiko Takebe, and
Stephen M. King *
Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3305
Received for publication April 29, 2004.
Revision received May 21, 2004.
Accepted for publication June 1, 2004.
Monitoring Editor: Paul Matsudaira
Abstract:
The radial spokes are required for Ca2+-initiated intraflagellarsignaling, resulting in modulation of inner and outer arm dyneinactivity. However, the mechanochemical properties of this signalingpathway remain unknown. Here, we describe a novel nucleosidediphosphate kinase (NDK) from the Chlamydomonas flagellum. Thisprotein (termed p61 or RSP23) consists of an N-terminal catalyticNDK domain followed by a repetitive region that includes threeIQ motifs and a highly acidic C-terminal segment. We find thatp61 is missing in axonemes derived from the mutants pf14 (lacksradial spokes) and pf24 (lacks the spoke head and several stalkcomponents) but not in those from pf17 (lacking only the spokehead). The p61 protein can be extracted from oda1 (lacks outerdynein arms) and pf17 axonemes with 0.5 M KI, and copurifieswith radial spokes in sucrose density gradients. Furthermore,p61 contains two classes of calmodulin binding site: IQ1 interactswith calmodulin-Sepharose beads in a Ca2+-independent manner,whereas IQ2 and IQ3 show Ca2+-sensitive associations. Wild-typeaxonemes exhibit two distinct NDKase activities, at least oneof which is stimulated by Ca2+. This Ca2+-responsive enzyme,which accounts for 45% of total axonemal NDKase, is missingfrom pf14 axonemes. We found that purified radial spokes alsoexhibit NDKase activity. Thus, we conclude that p61 is an integralcomponent of the radial spoke stalk that binds calmodulin andexhibits Ca2+-controlled NDKase activity. These observationssuggest that nucleotides other than ATP may play an importantrole in the signal transduction pathway that underlies the regulatorymechanism defined by the radial spokes.
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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. 
