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

Abstract

The radial spokes are required for Ca 2؉ -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 Ca 2؉ -independent manner, whereas IQ2 and IQ3 show Ca 2؉ -sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca 2؉ . This Ca 2؉ -responsive enzyme, which accounts for ϳ45% 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 Ca 2؉ -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. INTRODUCTIONThe eukaryotic cilium/flagellum is a highly conserved motile organelle built around nine outer doublet microtubules and a central pair of singlet microtubules that form the 9 ϩ 2 axoneme. Individual flagella provide for the motility of single cells such as mammalian sperm and Chlamydomonas, and ciliated epithelia are essential for fluid/mucus transport in many organs. Moreover, nodal cilia are required to define the left-right axis during mammalian development (Supp et al., 1997;Nonaka et al., 1998). Many cells also contain primary cilia that seem to play critical sensory roles during normal tissue function and development . The power for ciliary/flagellar movement is generated by the inner and outer rows of dynein arms that associate with the A tubule of each microtubule doublet and transiently interact with the B tubule of the adjacent doublet to produce a linear force vector that results in the microtubules sliding with respect to each other. This sliding motion is then converted to a flagellar bend by other axonemal components, including the radial spoke and central pair microtubule complexes (see Mitchell (2000) for a review of Chlamydomonas flagellar structure and function).In Chlamydomonas, control of flagellar waveform is mediated through Ca 2ϩ signaling pathways (Bessen et al., 1980;Kamiya and Witman, 1984). Reactivatio...

Radial spoke proteins ofChlamydomonasflagella

“…These studies reveal that the C. reinhardtii radial spoke, which sediments as a 20S particle, contains at least 23 distinct polypeptides, termed radial spoke protein (RSP)1 to RSP23 Yang et al, 2001;Patel-King et al, 2004), with a combined molecular mass of approximately 1200 kDa (Padma et al, 2003). Five of these proteins are located in the spoke head and the rest are in the spoke stalk.…”

“…The human genes encoding these proteins are candidates for causing primary ciliary dyskinesia, a severe inherited disease involving missing or defective axonemal structures, including the radial spokes. 3,4,6,16 (HSP40),20 (calmodulin) Williams et al, 1989;Curry et al, 1992;Yang et al, 2005;Zimmer et al, 1988;King and Patel-King, 1995;Patel-King et al, 2004). The predicted amino acid sequences have provided hints as to the possible functions of these proteins.…”

Axonemal protofilament ribbons, DM10 domains, and the link to juvenile myoclonic epilepsy

“…The first is the nm23-H7 class of nucleoside diphosphate kinase (NDK7) that consists of an N-terminal DM10 domain and two functional catalytic NDK modules. In Chlamydomonas, this protein (termed p40) is tightly associated with the flagellar axoneme and is solubilized only following treatment with 0.5 M KI [Patel-King et al, 2004].…”

NDP Kinase 7 Is a Conserved Microtubule-Binding Protein Preferentially Expressed in Ciliated Cells

“…They are thought to be expressed predominantly in testis (Lacombe et al, 2000;Sadek et al, 2003) and NDK5, 8 (also named Sptrx-2; Sadek et al, 2001) and 9 (also named Txl-2; Sadek et al, 2003) are known to localize to the sperm flagella. An NDK5 homologue is identified in Chlamydomonas flagella, suggesting that it is a conserved flagellar protein (Patel-King et al, 2004). NDK8 and 9 are not conserved in lower organisms such as Chlamydomonas, but they are associated also with respiratory and lung cilia.…”

“…A first step to understanding the specific role of each NDK in cilia and flagella would be to clarify its expression and localization. However, while various NDP kinases have been reasonably well characterized, little information is available about NDK7 except that Patel- King et al (2004) surmised that this protein is tightly associated with the axoneme. They showed that Western blot analyses using an anti-Chlamydomonas NDK5 (p61) antibody detected an approximately 40 kDa axonemal protein (p40) in addition to NDK5 and, from its size, suggested that it might be Chlamydomonas NDK7.…”