Mutations in genes encoding inner arm dynein heavy chains in Tetrahymena thermophila lead to axonemal hypersensitivity to Ca2+

Calcium-dependent ciliary reversals are seen in ciliated protozoans such as Tetrahymena in response to depolarizing stimuli, but the axonemal mechanisms responsible for this response are not well understood. The model is that the outer arm dyneins (OADs) control the beating frequency while the inner arm dyneins (IADs) regulate ciliary waveform. Since ciliary reversal is a type of waveform change, the model would predict that IAD mutations could affect ciliary reversal. We have used gene disruption techniques to generate several behavioral mutants of Tetrahymena with functional disruptions of various IADs. One such mutant, called KO-6, is missing I1 (the two-headed IAD) and is unable to show ciliary reversals in response to any stimuli due to a loss of axonemal Ca2+ sensitivity [Eur J Cell Biol 80 (2001) 486-497; Cell Motil Cytoskeleton 53 (2002) 281-288.]. In contrast, disruption of 3 one-headed IADs [Liu et al., Cell Motil Cytoskeleton 59 (2004), 201-214] produced mutants, which showed over-responsiveness in bioassays measuring either their depolarization-induced avoiding reactions (AR) in Na+ and Ba2+ solutions or their duration of backward swimming (continuous ciliary reversal or CCR) in K+ solutions. Detergent-extracted and reactivated mutants also showed increased probabilities of CCR at lower Ca2+ concentrations suggesting that the behavioral over-responsiveness of these three mutants in vivo is due to increased axonemal Ca2+ sensitivity. Our data suggest the possibility that the one-headed IADs and the two-headed IAD act antagonistically in vivo and that loss of any one of the one-headed IADs leads to behavioral over-responsiveness due to less resistance to I1-induced reversals.     

Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila

In many organisms, depolarizing stimuli cause an increase in intraciliary Ca2+, which results in reversal of ciliary beat direction and backward swimming. The mechanism by which an increase in intraciliary Ca2+ causes ciliary reversal is not known. Here we show that Tetrahymena cells treated with okadaic acid or cantharidin to inhibit protein phosphatases do not swim backwards in response to depolarizing stimuli. We also show that both okadaic acid and cantharidin inhibit backward swimming in reactivated, extracted cell models treated with Ca2+. In contrast, treatment of whole cells or extracted cell models with protein kinase inhibitors has no effect on backward swimming. These results suggest that a component of the axonemal machinery is dephosphorylated during ciliary reversal. The phosphorylation state of inner arm dynein 1 (I1) was determined before and after cells were exposed to depolarizing conditions that induce ciliary reversal. An I1 intermediate chain is phosphorylated in forward swimming cells but is dephosphorylated in cells treated with a depolarizing stimulus. Our results suggest that dephosphorylation of Tetrahymena inner arm dynein 1 may be an essential part of the mechanism of ciliary reversal in response to increased intraciliary Ca2+.     

Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite.

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca(2+) binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca(2+) binding site suggest that Ca(2+) binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.     

New Axonemal Dynein Heavy Chains from Tetrahymena thermophila

Two dyneins can be extracted from Tetrahymena ciliary axonemes. The 22S dynein contains three heavy chains (HC), sediments at 22S in a sucrose gradient, and makes up the outer arms. The 14S dynein contains two to six HCs, sediments at 14S, and is thought to contribute to formation of the inner arms. We have identified two large proteins that are extracted from Tetrahymena axonemes with high salt and that sediment together at approximately 18S. The two large proteins cleave when subjected to UV light in the presence of ATP and vanadate, suggesting both proteins are dynein HC. Antibodies against one of the 18S HCs do not recognize 22S dynein HCs. Antibodies to 22S dynein HC do not bind appreciably to 18S dynein photocleavage fragments. Taken together, these results indicate that the large proteins that sediment at 18S are axonemal dynein heavy chains.     

Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis

Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000- dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two- dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin.     

Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation

Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10(-6) M Ca(2+). Motility features changed when Ca(2+) concentrations were increased from 10(-6) to 10(-5) M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca(2+). Thus, it is possible that Ca(2+) binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca(2+)-binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by (45)Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca(2+)-binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca(2+)/CaM-dependent manner for regulating the dynein activity. A (32)P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca(2+)/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10(-5) M Ca(2+). It is possible that an increase in Ca(2+) induces Ca(2+)/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme.     

Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase

Photochemical cross-linking of both Tetrahymena and Aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge- membrane complex and the dynein-like membrane-associated ATPase. Electron microscopy was used to ensure that the dynein-like protein did not result from the solubilization of the dynein arms attached to the outer-doublet microtubules. The dynein-like protein has been isolated using sucrose gradients and is similar to axonemal dynein with respect to its sedimentation characteristics nucleotide specificity, and divalent cation requirements. Photochemical cross-linking of ciliary membrane porteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement.     

Characterization of microsome-associated tobacco BY-2 centrins

Centrin - higher plants - MTOCs - microtubules nucleation In most eukaryotic cells, the Ca(2+)-binding protein centrin is associated with structured microtubule-organizing centers (MTOCs) such as centrosomes. In these cells, centrin either forms centrosome-associated contractile fibers, or is involved in centrosome biogenesis. Our aim was to investigate the functions of centrin in higher plant cells which do not contain centrosome-like MTOCs. We have cloned two tobacco BY-2 centrin cDNAs and we show that higher plant centrins define a phylogenetic group of proteins distinct from centrosome-associated centrins. In addition, tobacco centrins were found primarily associated with microsomes and did not colocalize with gamma-tubulin, a known MTOC marker. While the overall level of centrin did not vary during the cell cycle, centrin was prominently detected at the cell plate during telophase. Our results suggest that in tobacco, the major portion of centrin is not MTOC-associated and could be involved in the formation of the cell plate during cytokinesis.     

Tetrahymena calcium-binding protein is indeed a calmodulin

We previously isolated a Ca2+-binding protein from a ciliate, Tetrahymena, and designated it as TCBP (Tetrahymena Ca2+-binding protein). The present paper reports that TCBP, which has two high affinity Ca2+-binding sites (Kd=4.6 X 10(-6) M), could activate porcine brain cyclic nucleotide phosphodiesterase at a concentration of over 10(-6) M free Ca2+, with the same mode of activation as that of authentic (porcine brain) calmodulin. In addition, the amino acid composition of TCBP was essentially the same as that of brain calmodulin. Therefore, we conclude that TCBP as an activator of Tetrahymena guanylate cyclase is indeed a calmodulin.     

Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.     

Characterization of centrin genes from Ochromonas danica (Chrysophyceae) and Scytosiphon lomentaria (Phaeophyceae)

Centrin, the EF‐hand Ca²⁺‐binding protein is localized at the basal apparatus of flagella and in centrioles in many eukaryotic cells. In the present study, centrin genes of the heterokont algae have been clarified for the first time. We isolated and analyzed cDNA and genomic DNA of centrin genes from the crysophycean alga Ochromonas danica Prings (UTEX LB1298) and the brown alga Scytosiphon lomentaria (Lyngbye) Link. The centrin gene of Ochromonas contained an open reading frame of 163 amino acids. The deduced protein, named Odcen, exhibited 85%, 78% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. The centrin genes of Scytosiphon contained an open reading frame of 164 amino acids. The deduced protein, named Slcen, exhibited 84%, 77% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. Both Odcen and Slcen possessed N‐terminal extensions before the conserved amino acid among various centrins, four EF‐hand domains and an aromatic amino acid at the C‐terminus. Southern blot hybridization suggested that the centrin gene occurs as a single copy gene in both Ochromonas and Scytosiphon genomes. Comparison of the sequence of the cDNA and the genomic DNA revealed that the Odcen gene was split into three fragments by introns and Slcen gene consisted of five fragments. The junctions of all introns of both genes conformed to the GT–AG rule. The introns of Slcen gene were considerably long and, as a result, the Slcen gene was approximately seven times longer than Odcen gene.     

Homologues of radial spoke head proteins interact with Ca2+/calmodulin in Tetrahymena cilia

Calmodulin (CaM) is an axonemal component. To examine the pathway of Ca(2+)/CaM signaling in cilia, using Ca(2+)/CaM-affinity column, we identified seven Ca(2+)/CaM-associated proteins from a crude dynein fraction and isolated 62 kDa (p62) and 66 kDa (p66) Ca(2+)/CaM-associated proteins in Tetrahymena cilia. The amino acid sequences deduced from the p62 and p66 cDNA sequences suggested that these proteins were similar to Chlamydomonas radial spoke proteins 4 and 6 (RSP4 and RSP6), components of the radial spoke head, and sea urchin sperm p63, which is a homologue of RSP4/6, and isolated as a key component that affect flagellar bending patterns. Although p62 and p66 do not have a conventional CaM-binding site, those have consecutive sequences which showed high normalized scores (>or= 5) from a CaM target database. These consecutive sequences were also found in RSP4, RSP6, and p63. These radial spoke heads proteins have a high similarity region composed of 15 amino acids between the five proteins. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized along the outer edge of the curved central pair microtubules in axoneme. Therefore, it is possible that the interaction between Ca(2+)/CaM and radial spoke head control axonemal curvature in the ciliary and flagellar waveform.     

Protein phosphatase 2C is involved in the cAMP-dependent ciliary control in Paramecium caudatum

Forward swimming of the Triton-extracted model of Paramecium is stimulated by cAMP. Backward swimming of the model induced by Ca(2+) is depressed by cAMP. Cyclic AMP and Ca(2+) act antagonistically in setting the direction of the ciliary beat. Some ciliary axonemal proteins from Paramecium caudatum are phosphorylated in a cAMP-dependent manner. In the presence of cAMP, axonemal 29- and 65-kDa polypeptides were phosphorylated by endogenous A-kinase in vitro. These phosphoproteins, however, were not dephosphorylated after in vitro phosphorylation, presumably because of the low endogenous phosphoprotein phosphatase activity associated with isolated axonemes. We purified the protein phosphatase that specifically dephosphorylated the 29- and 65-kDa phosphoproteins from Paramecium caudatum. The molecular weight of the protein phosphatase was 33 kDa. The protein phosphatase had common characteristics as protein phosphatase 2C (PP2C). The characteristics of the protein phosphatase were the same as those of the PP2C from Paramecium tetraurelia (PtPP2C) [Grothe et al., 1998: J. Biol. Chem. 273:19167-19172]. We concluded that the phosphoprotein phosphatase is the PP2C from Paramecium caudatum (PcPP2C). The PcPP2C markedly accelerated the backward swimming of the Triton-extracted model in the presence of Ca(2+). On the other hand, the PcPP2C slightly depressed the forward swimming speed. This indicates that the PP2C plays a role in the cAMP-dependent regulation of ciliary movement in Paramecium caudatum through dephosphorylation of 29- and/or 65-kDa regulatory phosphoproteins by terminating the action of cAMP.     

Regulation of 22S dynein by a 29-kD light chain

Previously, a 29-kD axonemal polypeptide (p29) that copurifies with 22S dynein has been shown to be phosphorylated in a cAMP- and Ca(2+)- sensitive manner, consistent with a role for this molecule in the signal transduction cascade leading to fast forward swimming in Paramecium tetraurelia (Hamasaki, T., K. Barkalow, J. Richmond, and P. Satir. 1991. Proc. Natl. Acad. Sci. USA. 88:7912-7922). This study demonstrates the nature of the relationship between p29 and 22S dynein. Chaotropic agents can be used to separate p29 fractions from 22S dynein. When extracted p29 is exchanged into physiological buffers, it regains the ability to recombine with 22S dynein with an apparent dissociation constant of 25 nM; no recombination is seen with 14S dynein or with unrelated control proteins. p29 from Paramecium will also recombine with Tetrahymena 22 but not 14S dynein. After chymotryptic digestion of 22S dynein, p29 preferentially binds to a single-headed fragment, homologous to the alpha H chain of Tetrahymena 22S dynein. 22S dynein treated in vitro by Paramecium protein kinase A in the presence of cAMP and ATP to phosphorylate p29 translocates bovine brain microtubules significantly (1.53x; p < 0.001) faster than before phosphorylation. Similarly, 22S dynein reconstituted in vitro with thiophosphorylated p29 translocates microtubules significantly (1.31x; p < 0.001) faster than controls reconstituted with nonthiophosphorylated p29. p29 is the only moiety thiophosphorylated in the reconstituted dynein. We conclude that p29 functions as a 22S dynein regulatory light chain in that it alone is sufficient to control the rate of microtubule translocation by changes in its phosphorylation state.     

A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase alpha

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase alpha is an 82-kDa DGK isoform that is activated in vitro by Ca(2+). The DGK alpha regulatory region includes tandem C1 protein kinase C homology domains and Ca(2+)-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca(2+) regulation, we expressed a series of DGK alpha deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca(2+)-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca(2+) to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGK alpha fragment that included both the RVH domain and EF hands. These observations suggested that Ca(2+) elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGK alpha. As with Ca(2+), this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca(2+)-induced DGK alpha activation.     

Binding of axonemal dynein to microtubules comprising the cytoplasmic transport system in insect ovarioles

Dynein isolated from ciliary axonemes of Tetrahymena is shown to bind in a characteristic fashion as arms to microtubules dissected from the nutritive tubes of insect ovarioles. The microtubules in nutritive tubes are associated with the transport of cytoplasmic components along their length, and the significance of their ability to bind axonemal dynein, to the possibility that microtubule/dynein interactions are involved in microtubule-associated movements, generally, is discussed.     

Structure of the N-terminal calcium sensor domain of centrin reveals the biochemical basis for domain-specific function

Centrin is an essential component of microtubule-organizing centers in organisms ranging from algae and yeast to humans. It is an EF-hand calcium-binding protein with homology to calmodulin but distinct calcium binding properties. In a previously proposed model, the C-terminal domain of centrin serves as a constitutive anchor to target proteins, and the N-terminal domain serves as the sensor of calcium signals. The three-dimensional structure of the N-terminal domain of Chlamydomonas rheinhardtii centrin has been determined in the presence of calcium by solution NMR spectroscopy. The domain is found to occupy an open conformation typical of EF-hand calcium sensors. Comparison of the N- and C-terminal domains of centrin reveals a structural and biochemical basis for the domain specificity of interactions with its cellular targets and the distinct nature of centrin relative to other EF-hand proteins. An NMR titration of the centrin N-terminal domain with a fragment of the known centrin target Sfi1 reveals binding of the peptide to a discrete site on the protein, which supports the proposal that the N-terminal domain serves as a calcium sensor in centrin.     

Molecular cloning and evolutionary analysis of the calcium-modulated contractile protein,centrin, in green algae and land plants

Centrin (= caltractin) is a ubiquitous, cytoskeletal protein which is a member of the EF-hand superfamily of calcium-binding proteins. A centrin-coding cDNA was isolated and characterized from the prasinophyte green alga Scherffelia dubia. Centrin PCR amplification primers were used to isolate partial, homologous cDNA sequences from the green algae Tetraselmis striata and Spermatozopsis similis. Annealing analyses suggested that centrin is a single-copy-coding region in T. striata and S. similis and other green algae studied. Centrin-coding regions from S. dubia, S. similis and T. striata encode four colinear EF-hand domains which putatively bind calcium. Phylogenetic analyses, including homologous sequences from Chlamydomonas reinhardtii and the land plant Atriplex nummularia, demonstrate that the domains of centrins are congruent and arose from the two-fold duplication of an ancestral EF hand with Domains 1+3 and Domains 2+4 clustering. The domains of centrins are also congruent with those of calmodulins demonstrating that, like calmodulin, centrin is an ancient protein which arose within the ancestor of all eukaryotes via gene duplication. Phylogenetic relationships inferred from centrin-coding region comparisons mirror results of small subunit ribosomal RNA sequence analyses suggesting that centrin-coding regions are useful evolutionary markers within the green algae.     

Centrin is essential for the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels

Voltage-gated Ca(2+) channels play a critical role in controlling Ca(2+) entry in various cells. Ciliary reversal in Paramecium depends on the Ca(2+) influx through voltage-gated Ca(2+) channels on the ciliary membrane. One of the voltage-gated Ca(2+) channel mutants in Paramecium caudatum, cnrC, neither produces Ca(2+) action potentials nor responds to any depolarizing stimuli. Here, we report that the cnrC(+) gene product is P. caudatum centrin (Pccentrin1p), a member of the Ca(2+)-binding EF-hand protein superfamily. The Pccentrin1p gene of cnrC was found to contain a single-base deletion, a mutation that caused the loss of the fourth EF-hand of Pccentrin1p. Moreover, the wild-type Ca(2+) channel function was impaired by Pccentrin1p gene silencing, leading to the loss of current-evoked Ca(2+) action potentials and stimulated ciliary reversal. These results demonstrate that Pccentrin1p is indispensable for the activity of the voltage-gated Ca(2+) channels that control ciliary reversal in Paramecium.     

Calcium-dependent assembly of centrin-G-protein complex in photoreceptor cells

Photoexcitation of rhodopsin activates a heterotrimeric G-protein cascade leading to cyclic GMP hydrolysis in vertebrate photoreceptors. Light-induced exchanges of the visual G-protein transducin between the outer and inner segment of rod photoreceptors occur through the narrow connecting cilium. Here we demonstrate that transducin colocalizes with the Ca(2+)-binding protein centrin 1 in a specific domain of this cilium. Coimmunoprecipitation, centrifugation, centrin overlay, size exclusion chromatography, and kinetic light-scattering experiments indicate that Ca(2+)-activated centrin 1 binds with high affinity and specificity to transducin. The assembly of centrin-G-protein complex is mediated by the betagamma-complex. The Ca(2+)-dependent assembly of a G protein with centrin is a novel aspect of the supply of signaling proteins in sensory cells and a potential link between molecular translocations and signal transduction in general.