Correspondence to G.B. Witman: george.witman{at}umassmed.edu
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations used: FAP, flagellar associated protein; IFT, intraflagellar transport; InsP3, inositol 1,4,5-trisphosphate; MS, mass spectrometry; PKD, polycystic kidney disease; PMCA, plasma membrane Ca2+ ATPase.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Although the above approaches can provide very valuable information on proteins associated with cilia and basal bodies, they are a complement to and not a substitute for direct proteomic analyses using mass spectrometry (MS). For example, comparative genomic approaches cannot readily identify genes encoding flagellar proteins, such as kinesins and many signal transduction proteins, that have close homologues in plants, and examination of gene induction during flagellar regeneration is likely to miss many proteins that function in both the flagellum and cytoplasm. In contrast, such proteins can be readily identified by a proteomics approach, which also can uniquely provide an indication of the abundance of a protein and its distribution in the flagellum. A preliminary proteomic analysis of detergent-extracted ciliary axonemes from cultured human bronchial epithelial cells identified 214 proteins (Ostrowski et al., 2002); however, this study was compromised by the presence of other cellular structures in the axonemal preparation, and by limitations in the amount of material available and/or sequence data obtained, with the result that only 89 of the proteins were identified by more than a single peptide. Here, we use MS to identify the proteins in biochemically fractionated C. reinhardtii flagella, which are available in large amounts and in high purity.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The list of flagellar proteins identified by two or more peptides is rich in motor proteins, signal transduction proteins, proteins with predicted coiled-coil domains, and predicted membrane proteins (Table I), and contains a number of proteins whose homologues are associated with disease in humans and model vertebrates. Nearly 90 proteins are highly conserved in humans (BLAST E score 1e-10) but have not been previously characterized in any organism. Individual proteins of interest are described in the Discussion.
|
Assessment of exclusiveness of the dataset
Even though electron microscopy indicated that the flagellar fractions were highly pure, at some level the dataset is likely to contain proteins that are contaminants. The number of peptides found by electrospray MS of complex mixtures roughly correlates with the abundance and size of the proteins from which they are derived (Washburn et al., 2001). Most known flagellar proteins were identified by more than five peptides; notable exceptions include several outer dynein arm subunits, which would not have been present in flagella of the outer dynein armless mutant used for all but the Tergitol-insoluble membrane + axonemes fraction; Tctex1 (one peptide), which is very small; and EB1 (two peptides), which is located only at the tip of the flagellum and therefore is of relatively low abundance. Obvious contaminants in the dataset (tRNA synthetases, ribosomal proteins, and histones) were usually identified by one or two peptides, even though these proteins are highly abundant in the cell body. Thus, the 360 proteins identified by five or more peptides are highly likely to be true flagellar proteins, with possible exceptions including methionine synthase, elongation factors, arginyl-tRNA synthetase, carbonic anhydrase, and two cell wall proteins. However, even some of the latter proteins may function in the flagellum: methionine synthase gene expression is induced by flagellar adhesion (Kurvari et al., 1995); elongation factor 1a binds calmodulin and localizes to Tetrahymena cilia (Ueno et al., 2003); and arginyl-tRNA synthetase is reported to modify protein substrates to allow degradation via the ubiquitin pathway (Ferber and Ciechanover, 1987), several other components of which were found in the flagellum. Several glycolytic enzymes also are present in this data subset and are likely to function in the flagellum (see Discussion).
The 292 proteins identified by two to four peptides should be considered candidate flagellar proteins as this group contains both known flagellar proteins and likely contaminants. Within this group, the genes of 19 out of 51 (37%) randomly chosen proteins that were not previously reported to be in the flagellum were found to be induced at least 1x by deflagellation (i.e., their transcript levels doubled; see Induction of genes by deflagellation section). Flagellar proteins would include these plus known flagellar proteins plus some whose genes are not induced by deflagellation, so that the percent of true flagellar proteins in this subset is likely to be substantially higher than 37%. The group of proteins identified by single peptides was not analyzed in detail but likely contains low abundance and small flagellar proteins along with contaminants.
Distribution of proteins in flagellar fractions
In the subset of proteins identified by five or more peptides, the peptides of 3/4 of the proteins were present predominantly in the KCl extract and extracted axoneme fractions and thus probably represent axonemal components (Fig. 3 A). As expected, this includes the previously identified inner dynein arm proteins, central pair proteins, and radial spoke proteins. The peptides of
1/4 of the proteins were abundant predominantly in the membrane + matrix fraction; this included all proteins known to be subunits of IFT motors or particles. Within the subset of proteins identified by two to four peptides, the proteins were approximately equally distributed between the membrane + matrix and axonemal fractions (Fig. 3 B). Therefore, the majority of proteins found in the flagellar proteome are in the axonemal fractions. The difference in the distribution of proteins in the two data subsets suggests that the membrane + matrix fraction contains a larger proportion of low abundance proteins than the axonemal fractions. Fig. 3 C shows a hierarchical cluster diagram based on the number and distribution of peptides in the different fractions for proteins identified by 15 or more peptides. Proteins known to be associated with IFT, the outer dynein arm, or the inner dynein arm grouped closely together, indicating that the analysis is likely to have predictive value. Thus, novel proteins that group with these proteins may be new constituents of these structures. Proteins of previously unidentified structures or processes may similarly group with each other.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Motor and signal transduction proteins
In addition to the very large number of known dynein subunits and three previously known kinesin subunits, a previously uncharacterized kinesin heavy chain (C_70192) was identified. Phylogenetic analysis did not support definitive assignment of this protein to a specific kinesin family. Peptides from this protein were found primarily in the axonemal fractions and cluster analysis (Fig. 3 C) grouped it with radial spoke and central pair components. This may be the unidentified flagellar kinesin detected by immunological methods (Fox et al., 1994). No myosin heavy chains were found.
The flagellar proteome contains over 90 putative signal transduction proteins. Based on Interpro domain analyses and homology searches, the proteins identified by two or more peptides include 21 protein kinases and 11 protein phosphatases, the vast majority of which have not previously been reported to be in the flagellum. These findings are consistent with the fact that a large number of flagellar proteins are phosphorylated (Piperno et al., 1981), and with the roles of protein phosphorylation and dephosphorylation in the control of flagellar motility (Porter and Sale, 2000), signaling (Pan and Snell, 2000), and probably assembly (Rosenbaum and Witman, 2002). We also found 27 EF hand-containing proteins that are potential Ca2+-binding proteins, consistent with the well-established role of Ca2+ in the control of flagellar behavior (Witman, 1993) and signaling (Bloodgood, 1992; Praetorius and Spring, 2001). 10 proteins are predicted to have IQ (calmodulin-binding) domains. An InsP3 receptor was identified by 33 unique peptides in the membrane + matrix fraction, suggesting that it is a very abundant flagellar component. 10 small GTPases were identified, including five RAB subfamily members, four ARF subfamily members, and RAN. A RAB GDP-dissociation inhibitor that controls the GDPGTP exchange reaction also is present. Two predicted 14-3-3 proteins, which mediate signal transduction by binding to phosphoserine-containing proteins, were identified. Six ion pumps or channels, including a homologue of human polycystin 2 (see Homologues of vertebrate disease proteins) and three predicted PMCAs (C_370080, C_550076, C_700061), were present. The proteome also contained a group of four closely related proteins (all encoded by scaffold 152) that are composed of 8 to 12 transmembrane helixes and a PAS domain; PAS domains are thought to be sensory motifs that are involved in detecting diverse stimuli ranging from light or oxygen to redox state and small ligands (Taylor and Zhulin, 1999). We also confirmed the presence of a second type of PAS domain-containing protein, the blue-light receptor phototropin (Huang et al., 2004), in the axonemal fraction. The identification of so many signal transduction proteins underscores the importance of signaling in the function of motile as well as nonmotile cilia, and should greatly facilitate studies to understand signaling mechanisms in these organelles (Pazour and Witman, 2003).
Nucleotide production and metabolism
Flagellar motility, assembly and signal transduction require large amounts of ATP and GTP, and the flagellum contains numerous proteins that are likely to be involved in maintaining adequate levels of these nucleotides. Two adenylate kinases and a nucleoside diphosphate kinase have been characterized in the past year (Patel-King et al., 2004; Wirschell et al., 2004; Zhang and Mitchell, 2004). We found two additional nucleoside diphosphate kinases (C_1090038, C_1230002), two additional adenylate kinases (C_750038, C_10320001), and two guanylate kinases (C_160167, C_3300001), four of which are associated with the axoneme.
Interestingly, the flagellar proteome also contains all enzymes of the late glycolytic pathway required for the conversion of fructose 1,6-bisphosphate to pyruvate, which could generate ATP for use in the flagellum (Fig. 6). Consistent with glycolysis occurring in the flagellum, we also found two isoforms of malate dehydrogenase, which could be involved in a malateoxaloacetate shuttle to regenerate the NAD consumed by the glycolytic pathway. It might be argued that, because the early steps of the glycolytic pathway are reported to occur primarily in the chloroplast and the later steps in the cytoplasm in C. reinhardtii (Klein, 1986), the cytoplasmic enzymes might simply have diffused into the flagella but are not flagellar proteins per se. Our data argue against this, because aldolase, also reported to be located primarily in the chloroplast (Klein, 1986), was identified by numerous peptides (Fig. 6). We also did not find phosphoenolpyruvate carboxylase, a cytoplasmic marker (Klein, 1986), or any enzymes of pyrimidine biosynthesis, which should be abundant in the cytoplasm. Moreover, peptides derived from glyceraldehyde-3-phosphate dehydrogenase were found almost exclusively in the axonemal fractions, indicating that this enzyme is anchored to the axoneme, and peptides derived from enolase were found in both membrane + matrix and axonemal fractions, indicating that some of the flagellar enolase is similarly assembled into the axoneme. These results suggest that there is a mechanism to exclude soluble cytoplasmic proteins from the flagellum, and that the glycolytic enzymes are true flagellar components. The localization of glycolytic enzymes to cilia may be a way to maintain a constant ATP/ADP ratio along the length of the cilium. Dynein activity is sensitive to ATP/ADP ratios (Yagi, 2000; unpublished data). If ATP were available to the cilium only via diffusion from the cell body, the ATP/ADP ratios would vary along the length of the organelle, and dynein function would be compromised. The presence of glycolytic enzymes in cilia is likely to be widespread; glyceraldehyde-3-phosphate dehydrogenase is a component of the mammalian sperm flagellum and is essential for sperm motility and fertilization (Miki et al., 2004), and photoreceptor rod outer segments, which are developmentally derived from cilia, are reported to contain the entire glycolytic pathway as a way of maintaining the high levels of ATP needed for the formation of cGMP in this compartment (Hsu and Molday, 1994).
|
The flagellar proteome also contains homologues (C_410060 and C_610051) of hydin and napa, which are respectively mutated in the hy3 and hyh mouse models for hydrocephalus (Davy and Robinson, 2003; Chae et al., 2004; Hong et al., 2004); the hydin gene also is a strong candidate for causing hydrocephalus in humans (Davy and Robinson, 2003). Hydrocephalus is the abnormal accumulation of cerebral spinal fluid in the ventricles of the brain. The fact that hydin and napa homologues are both present in the C. reinhardtii flagellar proteome indicates that cilia probably are involved in the development of hydrocephalus caused by defects in these proteins.
Homologues of a C. reinhardtii inner dynein arm heavy chain and a central pair protein previously were shown to cause male sterility when defective in the mouse (Neesen et al., 2001; Sapiro et al., 2002). The C. reinhardtii flagellar proteome contains homologues of additional proteins associated with male sterility in the mouse. These include C_20334, a homologue of the Pacrg (Parkin-coregulated gene) protein (Lorenzetti et al., 2004), and C_2260011, a homologue of Pp1cc encoding a protein phosphatase 1 catalytic subunit (Varmuza et al., 1999). In addition, C_1620015 and C_10830001 are homologues of an Arf-like protein encoded by Arl4, which results in reduced sperm count when defective in the mouse (Schurmann et al., 2002), and C_60170 is a close homologue of a protein kinase encoded by Mak, which causes reductions in litter size and sperm motility when missing (Shinkai et al., 2002). The fact that homologues of these proteins are in the C. reinhardtii flagellum indicates that the proteins are likely to exert their effects as components of the mammalian sperm flagellum.
All three of the PMCAs in the C. reinhardtii flagellar proteome are closely related to the product of the mouse wriggle/deafwaddler gene, which causes deafness and balance problems when defective. This raises the intriguing possibility that the mammalian wriggle/deafwaddler gene product may function in the kinocilium of the inner ear.
Ciliary targeting sequences
Myristoylation of NH2-terminal glycine residues plays a role in directing proteins without membrane spanning domains to the ciliary membrane in Leishmania and Trypanosoma (Godsel and Engman, 1999; Tull et al., 2004). To determine if the myristoylation motif was enriched in ciliary proteins, all 19,832 predicted C. reinhardtii proteins were analyzed using a myristoylation prediction algorithm (Maurer-Stroh et al., 2004). The program predicted that 103 proteins (0.5%) were myristoylated. Of these, 18, representing 2.7% of all flagellar proteins, were found in the flagellar proteome. Thus, proteins predicted to be myristoylated are enriched in the flagellum. These proteins were found primarily in the membrane + matrix fraction and represented 8% of all such proteins (Table II). Interestingly, this motif may also function in ciliary targeting in mammals because cystin, the gene product of the mouse cystic kidney disease gene Cpk, also contains this motif and is localized to cilia (Hou et al., 2002). A number of the mammalian homologues of the potentially myristoylated C. reinhardtii proteins also contain the myristoylation motif. For example, human ARF4, 5, and 6 are predicted to be likely myristoylated whereas several of the others are predicted to be potentially myristoylated. Mammalian protein phosphatase 2c (but not the other protein phosphatase 2c isoforms) is predicted to be myristoylated as is mammalian type II cGMP-dependent protein kinase (but not type I), suggesting that protein phosphatase 2c
and type II cGMP-dependent protein kinase are localized to mammalian cilia.
|
Comparison with other broad studies of C. reinhardtii gene expression
Two recently published studies also examined expression levels of large numbers of C. reinhardtii genes in response to deflagellation. Li et al. (2004) used real time PCR to examine 103 genes that had been chosen based on the observation that they were conserved in ciliated species but not in nonciliated species. 33 of these also were examined by us and the results for these are very similar in the two datasets. Stolc et al. (2005) used a gene chip to examine genome-wide expression patterns including all of the genes we identified except FAP41. The Stolc et al. dataset has a high rate of false negatives such that only 54% of genes previously shown to be induced by deflagellation were found to be induced 100% (equivalent to 1x induction in our study) or greater. Thus, the lack of induction as measured by the gene chip cannot be interpreted as evidence for nonciliary roles. However, the Stolc et al. (2005) dataset has a very low false positive rate, as indicated by the fact that, within a subset of 45 potential flagellar genes that we examined by real-time PCR analysis and that Stolc et al. reported to be induced at least 100%, 40 were found to be induced at least 1x in our analysis. The exceptions were C_1140006, C_200160, and the genes encoding Hsp70, calmodulin, and Ran. Of 220 genes found to be induced at least 100% by Stolc et al. (2005), 155 encode proteins identified in the flagellar proteome by two or more peptides and an additional 18 encode proteins identified by one peptide. The remaining 47 genes may encode flagellar proteins not detected in our analysis, encode proteins that are involved in flagellar regeneration but not incorporated into the flagellum, encode transcripts that are not translated, or be false positives.
Conserved uncharacterized proteins
Remarkably, the flagellar proteome contains 87 proteins that have conserved homologues in humans (BLAST Expect 1e-10), but whose homologues have not yet been characterized in any organism. 60 out of 69 (87%) of these genes examined were induced by deflagellation, suggesting that they function primarily in cilia. Only eight of these were previously identified by Ostrowski et al. (2002). This confirms that the protein composition of cilia has not been well characterized, and highlights the need for further research on these proteins.
Conclusions
Our analysis of the C. reinhardtii flagellar proteome has identified many proteins of known function not previously ascribed to cilia and flagella, as well as many other proteins whose functions and cellular locations were completely unknown before now. The results indicate that cilia and flagella are far more complex than previously believed. The findings provide a starting point for future studies to elucidate the roles of these proteins in the assembly and function of these ubiquitous cell organelles, to understand why defects in some of these proteins lead to human diseases, and to clarify the origin of cilia during the evolution of the eukaryotic cell.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of protein digests and MS
The entire gel lane for each fraction was cut into 33 slices (Tergitol-insoluble membrane + axonemes fraction) or 4145 slices (oda1 fractions), and proteins were digested "in gel" according to established methods (Lahm and Langen, 2000). Eluted peptides were separated on a LC Packings Ultimate Nanoflow HPLC system as follows: 10 µl of the peptide digest solution (approximately one half of the total digest) was manually injected onto a micro trap column (precolumn cartridge 0.3 mm x 5 mm C18PM; LC Packings) and the trap column was manually washed with 10 µl of 0.1% formic acid before switching in line with the reverse phase separating column (100 µm x 15 cm C18 PepMap; LC Packings). A gradient was developed from 100% solvent A (0.1% formic acid) to 60% solvent B (0.1% formic acid in acetonitrile/water 70:30) in 40 min at a flow rate of 500 nl/min. The outlet of the column was connected to an electrospray needle (20 µm taper tip; New Objective, Inc.). Electrospray MS was performed on a Finnigan LCQ Deca ion trap mass spectrometer (Thermo Electron Corp.). Data-dependent acquisitions were set up according to a triple play experiment program where full MS scans from 400 D-2000 D were on going until an MS signal grew above a specified threshold at which time a high-resolution scan (Zoom Scan) was performed to determine monoisotopic mass and charge state followed by a single MSMS scan. Dynamic exclusion was applied to prevent repeat scans of the same peptide masses. The raw data files were converted into mass peak lists using the LCQ_DTA program and then searched against both the Joint Genome Institute version 2 draft assembly of the C. reinhardtii genome translated in all six frames and the predicted protein models derived from this assembly (http://genome.jgi-psf.org/chlre2/chlre2.home.html). Searches were performed using the Mascot search engine (Matrix Science Ltd.) with 1D mass tolerances for both the parent and fragment masses.
Peptides with Mascot scores >40 were compared to the genome assembly by BLAST to identify the models from which they were derived. In most cases, the peptides could be unambiguously assigned to one model. However, in some cases, such as the tubulins, it was not possible to identify the specific member of the gene family from which the peptide was derived. In other cases, the peptides did not match any predicted model but grouped tightly together, suggesting that this region of the genome encodes an expressed protein that was not predicted (e.g., FAP41). In still other cases, the peptides mapped just outside of predicted genes; if EST data suggested that the gene model should be extended to include the peptide, we associated this peptide with the model.
The peptide sequences of the gene models were compared to the NCBI protein database with BLAST to identify the most closely related proteins in all species and to the predicted proteomes of humans (NCBI build 33) and Arabidopsis (Ath1_pep_20030417.z) to determine whether the proteins were conserved in these two species. The chromosomal map position of the best human match was extracted from the NCBI Gene database. The proteins were also put through Interpro domain analysis (Mulder et al., 2005), the multicoil coiled-coil predictor (Wolf et al., 1997), the TMHMM membrane-spanning helix predictor (Krogh et al., 2001), and the MYR myristoylation predictor (Maurer-Stroh et al., 2004). Cluster analysis was performed with Expander (Sharan et al., 2003). The results of these analyses are available at our web site (http://labs.umassmed.edu/chlamyfp/index.php).
Real time PCR analysis of gene induction
To measure induction of gene expression by deflagellation, C. reinhardtii 137c cells were deflagellated by pH shock, after which the cells synchronously regenerate their flagella, and RNA was isolated 30 min later from the deflagellated cells and from untreated control cells. The RNA was reverse transcribed using Clontech PowerScript Reverse Transcriptase (BD Biosciences) and the relative amount of specific messages in the two samples was determined by real-time PCR using pairs of primers that spanned introns and SYBR green to monitor amplification. The relative amount of the noninduced G protein ß subunit (Schloss, 1990) was measured in each trial and used to correct for slight differences in amount of mRNA in each sample. In our terminology, "0x induction," "1x induction" and "2x induction" indicate no change, a doubling, or a tripling of transcript levels, respectively. Three independent sets of RNA were isolated and analyzed. Primer pairs that spanned introns were picked using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).
On-line supplemental material
Table S1 shows flagellar proteins identified by two or more peptides. Table S2 shows flagellar proteins identified by a single peptide. Table S3 lists known C. reinhardtii flagellar proteins. Figure S1 depicts SDSpolyacrylamide gels used for proteomic analysis. Online supplemental materials are available at http://www.jcb.org/cgi/content/full/jcb.200504008/DC1.
![]() |
Acknowledgments |
---|
This work was supported by grants from the National Institutes of Health (NIH; GM-60992 to G.J. Pazour and GM-30626 to G.B. Witman), by a Worcester Foundation for Biomedical Research Foundation Scholar Award (to G.J. Pazour), and by the Robert W. Booth Fund at the Greater Worcester Community Foundation (to G.B. Witman). The University of Massachusetts Medical School (UMMS) Proteomic Mass Spectrometry Lab is partially supported by NIH DK32520 to the UMMS DERC.
Submitted: 1 April 2005
Accepted: 17 May 2005
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Avidor-Reiss, T., A.M. Maer, E. Koundakjian, A. Polyanovsky, T. Keil, S. Subramaniam, and C.S. Zuker. 2004. Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis. Cell. 117:527539.[CrossRef][Medline]
Bergman, K., U.W. Goodenough, D.A. Goodenough, J. Jawitz, and H. Martin. 1975. Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction. J. Cell Biol. 67:606622.[Abstract]
Bloodgood, R.A. 1992. Calcium-regulated phosphorylation of proteins in the membrane-matrix compartment of the Chlamydomonas flagellum. Exp. Cell Res. 198:228236.[CrossRef][Medline]
Bradley, B.A., J.J. Wagner, and L.M. Quarmby. 2004. Identification and sequence analysis of six new members of the NIMA-related kinase family in Chlamydomonas. J. Eukaryot. Microbiol. 51:6672.[Medline]
Chae, T.H., S. Kim, K.E. Marz, P.I. Hanson, and C.A. Walsh. 2004. The hyh mutation uncovers roles for alpha Snap in apical protein localization and control of neural cell fate. Nat. Genet. 36:264270.[CrossRef][Medline]
Davy, B.E., and M.L. Robinson. 2003. Congenital hydrocephalus in hy3 mice is caused by a frameshift mutation in Hydin, a large novel gene. Hum. Mol. Genet. 12:11631170.
Dentler, W.L. 1990. Linkages between microtubules and membranes in cilia and flagella. Ciliary and Flagellar Membranes. R.A. Bloodgood, editor. Plenum Press, New York. 3164.
Dutcher, S.K. 1995. Flagellar assembly in two hundred and fifty easy-to-follow steps. Trends Genet. 11:398404.[CrossRef][Medline]
Dymek, E.E., P.A. Lefebvre, and E.F. Smith. 2004. PF15p is the Chlamydomonas homologue of the katanin p80 subunit and is required for assembly of flagellar central microtubules. Eukaryot. Cell. 3:870879.
Ferber, S., and A. Ciechanover. 1987. Role of arginine-tRNA in protein degradation by the ubiquitin pathway. Nature. 326:808811.[CrossRef][Medline]
Fox, L., K. Sawin, and W. Sale. 1994. Kinesin-related proteins in eukaryotic flagella. J. Cell Sci. 107:15451550.
Gilula, N.B., and P. Satir. 1972. The ciliary necklace. A ciliary membrane specialization. J. Cell Biol. 53:494509.
Godsel, L.M., and D.M. Engman. 1999. Flagellar protein localization mediated by a calcium-myristoyl/palmitoyl switch mechanism. EMBO J. 18:20572065.
Hong, H.K., A. Chakravarti, and J.S. Takahashi. 2004. The gene for soluble N-ethylmaleimide sensitive factor attachment protein alpha is mutated in hydrocephaly with hop gait (hyh) mice. Proc. Natl. Acad. Sci. USA. 101:17481753.
Hou, X., M. Mrug, B.K. Yoder, E.J. Lefkowitz, G. Kremmidiotis, P. D'Eustachio, D.R. Beier, and L.M. Guay-Woodford. 2002. Cystin, a novel cilia-associated protein, is disrupted in the cpk mouse model of polycystic kidney disease. J. Clin. Invest. 109:533540.
Hsu, S.C., and R.S. Molday. 1994. Glucose metabolism in photoreceptor outer segments. Its role in phototransduction and in NADPH-requiring reactions. J. Biol. Chem. 269:1795417959.
Huang, K., T. Kunkel, and C.F. Beck. 2004. Localization of the blue-light receptor phototropin to the flagella of the green alga Chlamydomonas reinhardtii. Mol. Biol. Cell. 15:36053614.
Kamiya, R. 1988. Mutations at twelve independent loci result in absence of outer dynein arms in Chlamydomonas reinhardtii. J. Cell Biol. 107:22532258.[Abstract]
King, S.M., T. Otter, and G.B. Witman. 1986. Purification and characterization of Chlamydomonas flagellar dyneins. Methods Enzymol. 134:291306.[Medline]
Klein, U. 1986. Compartmentation of glycolysis and of the oxidative pentose-phosphate pathway in Chlamydomonas reinhardtii. Planta. 167:8186.[CrossRef]
Krogh, A., B. Larsson, G. von Heijne, and E.L. Sonnhammer. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305:567580.[CrossRef][Medline]
Kurvari, V., F. Qian, and W.J. Snell. 1995. Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes. Plant Mol. Biol. 29:12351252.[CrossRef][Medline]
Lahm, H.W., and H. Langen. 2000. Mass spectrometry: a tool for the identification of proteins separated by gels. Electrophoresis. 21:21052114.[CrossRef][Medline]
Lefebvre, P.A., and J.L. Rosenbaum. 1986. Regulation of the synthesis and assembly of ciliary and flagellar proteins during regeneration. Annu. Rev. Cell Biol. 2:517546.[CrossRef][Medline]
Li, J.B., J.M. Gerdes, C.J. Haycraft, Y. Fan, T.M. Teslovich, H. May-Simera, H. Li, O.E. Blacque, L. Li, C.C. Leitch, et al. 2004. Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell. 117:541552.[CrossRef][Medline]
Liu, S., W. Lu, T. Obara, S. Kuida, J. Lehoczky, K. Dewar, I.A. Drummond, and D.R. Beier. 2002. A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish. Development. 129:58395846.[CrossRef][Medline]
Lorenzetti, D., C.E. Bishop, and M.J. Justice. 2004. Deletion of the Parkin coregulated gene causes male sterility in the quaking(viable) mouse mutant. Proc. Natl. Acad. Sci. USA. 101:84028407.
Luck, D.J.L., and G. Piperno. 1989. Dynein arm mutants of Chlamydomonas. Cell Movement/Volume I: The Dynein ATPases. F.D. Warner, P. Satir, and I.R. Gibbons, editors. Alan R. Liss, Inc, New York. 4960.
Markey, D.R., and G.B. Bouck. 1977. Mastigoneme attachment in Ochromonas. J. Ultrastruct. Res. 59:173177.[CrossRef][Medline]
Maurer-Stroh, S., M. Gouda, M. Novatchkova, A. Schleiffer, G. Schneider, F.L. Sirota, M. Wildpaner, N. Hayashi, and F. Eisenhaber. 2004. MYRbase: analysis of genome-wide glycine myristoylation enlarges the functional spectrum of eukaryotic myristoylated proteins. Genome Biol. 5:R21.[CrossRef][Medline]
Miki, K., W. Qu, E.H. Goulding, W.D. Willis, D.O. Bunch, L.F. Strader, S.D. Perreault, E.M. Eddy, and D.A. O'Brien. 2004. Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility. Proc. Natl. Acad. Sci. USA. 101:1650116506.
Mulder, N.J., R. Apweiler, T.K. Attwood, A. Bairoch, A. Bateman, D. Binns, P. Bradley, P. Bork, P. Bucher, L. Cerutti, et al. 2005. InterPro, progress and status in 2005. Nucleic Acids Res. 33(Database Issue):D201D205.
Neesen, J., R. Kirschner, M. Ochs, A. Schmiedl, B. Habermann, C. Mueller, A.F. Holstein, T. Nuesslein, I. Adham, and W. Engel. 2001. Disruption of an inner arm dynein heavy chain gene results in asthenozoospermia and reduced ciliary beat frequency. Hum. Mol. Genet. 10:11171128.
Ong, A.C., and D.N. Wheatley. 2003. Polycystic kidney diseasethe ciliary connection. Lancet. 361:774776.[CrossRef][Medline]
Ostrowski, L.E., K. Blackburn, K.M. Radde, M.B. Moyer, D.M. Schlatzer, A. Moseley, and R.C. Boucher. 2002. A proteomic analysis of human cilia: identification of novel components. Mol. Cell. Proteomics. 1:451465.
Pan, J., and W.J. Snell. 2000. Regulated targeting of a protein kinase into an intact flagellum. An aurora/Ipl1p-like protein kinase translocates from the cell body into the flagella during gamete activation in Chlamydomonas. J. Biol. Chem. 275:2410624114.
Patel-King, R.S., O. Gorbatyuk, S. Takebe, and S.M. King. 2004. Flagellar radial spokes contain a Ca2+-stimulated nucleoside diphosphate kinase. Mol. Biol. Cell. 15:38913902.
Pazour, G.J. 2004. Intraflagellar transport and cilia-dependent renal disease: the ciliary hypothesis of polycystic kidney disease. J. Am. Soc. Nephrol. 15:25282536.
Pazour, G.J., and J.L. Rosenbaum. 2002. Intraflagellar transport and cilia-dependent diseases. Trends Cell Biol. 12:551555.[CrossRef][Medline]
Pazour, G.J., and G.B. Witman. 2003. The vertebrate primary cilium is a sensory organelle. Curr. Opin. Cell Biol. 15:105110.[CrossRef][Medline]
Piperno, G., B. Huang, Z. Ramanis, and D. Luck. 1981. Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components. J. Cell Biol. 88:7379.[Abstract]
Porter, M.E., and W.S. Sale. 2000. The 9 + 2 axoneme anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility. J. Cell Biol. 151:F37F42.[CrossRef][Medline]
Porter, M.E., J.A. Knott, S.H. Myster, and S.J. Farlow. 1996. The dynein gene family in Chlamydomonas reinhardtii. Genetics. 144:569585.
Praetorius, H.A., and K.R. Spring. 2001. Bending the MDCK cell primary cilium increases intracellular calcium. J. Membr. Biol. 184:7179.[CrossRef][Medline]
Reinhart, F.D., and R.A. Bloodgood. 1988. Membrane-cytoskeleton interactions in the flagellum: a 240,000 Mr surface-exposed glycoprotein is tightly associated with the axoneme in Chlamydomonas moewusii. J. Cell Sci. 89:521531.[Abstract]
Rosenbaum, J.L., and G.B. Witman. 2002. Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3:813825.[CrossRef][Medline]
Sapiro, R., I. Kostetskii, P. Olds-Clarke, G.L. Gerton, G.L. Radice, and J.F. Strauss III. 2002. Male infertility, impaired sperm motility, and hydrocephalus in mice deficient in sperm-associated antigen 6. Mol. Cell. Biol. 22:62986305.
Sattler, C.A., and L.A. Staehelin. 1974. Ciliary membrane differentiations in Tetrahymena pyriformis. Tetrahymena has four types of cilia. J. Cell Biol. 62:473490.
Schloss, J.A. 1990. A Chlamydomonas gene encodes a G protein ß subunit-like polypeptide. Mol. Gen. Genet. 221:443452.[Medline]
Scholey, J.M. 2003. Intraflagellar transport. Annu. Rev. Cell Dev. Biol. 19:423443.[CrossRef][Medline]
Schurmann, A., S. Koling, S. Jacobs, P. Saftig, S. Krauss, G. Wennemuth, R. Kluge, and H.G. Joost. 2002. Reduced sperm count and normal fertility in male mice with targeted disruption of the ADP-ribosylation factor-like 4 (Arl4) gene. Mol. Cell. Biol. 22:27612768.
Sharan, R., A. Maron-Katz, and R. Shamir. 2003. CLICK and EXPANDER: a system for clustering and visualizing gene expression data. Bioinformatics. 19:17871799.
Shinkai, Y., H. Satoh, N. Takeda, M. Fukuda, E. Chiba, T. Kato, T. Kuramochi, and Y. Araki. 2002. A testicular germ cell-associated serine-threonine kinase, MAK, is dispensable for sperm formation. Mol. Cell. Biol. 22:32763280.
Snell, W.J., J. Pan, and Q. Wang. 2004. Cilia and flagella revealed: from flagellar assembly in Chlamydomonas to human obesity disorders. Cell. 117:693697.[CrossRef][Medline]
Stolc, V., M.P. Samanta, W. Tongprasit, and W.F. Marshall. 2005. Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. Proc. Natl. Acad. Sci. USA. 102:37033707.
Sun, Z., A. Amsterdam, G.J. Pazour, D.G. Cole, M.S. Miller, and N. Hopkins. 2004. A genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney. Development. 131:40854093.
Takada, S., C.G. Wilkerson, K. Wakabayashi, R. Kamiya, and G.B. Witman. 2002. The outer dynein arm-docking complex: composition and characterization of a subunit (oda1) necessary for outer arm assembly. Mol. Biol. Cell. 13:10151029.
Taylor, B.L., and I.B. Zhulin. 1999. PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol. Mol. Biol. Rev. 63:479506.
Tull, D., J.E. Vince, J.M. Callaghan, T. Naderer, T. Spurck, G.I. McFadden, G. Currie, K. Ferguson, A. Bacic, and M.J. McConville. 2004. SMP-1, a member of a new family of small myristoylated proteins in kinetoplastid parasites, is targeted to the flagellum membrane in Leishmania. Mol. Biol. Cell. 15:47754786.
Ueno, H., K. Gonda, T. Takeda, and O. Numata. 2003. Identification of elongation factor-1alpha as a Ca2+/calmodulin-binding protein in Tetrahymena cilia. Cell Motil. Cytoskeleton. 55:5160.[CrossRef][Medline]
Upadhya, P., E.H. Birkenmeier, C.S. Birkenmeier, and J.E. Barker. 2000. Mutations in a NIMA-related kinase gene, Nek1, cause pleiotropic effects including a progressive polycystic kidney disease in mice. Proc. Natl. Acad. Sci. USA. 97:217221.
Varmuza, S., A. Jurisicova, K. Okano, J. Hudson, K. Boekelheide, and E.B. Shipp. 1999. Spermiogenesis is impaired in mice bearing a targeted mutation in the protein phosphatase 1cgamma gene. Dev. Biol. 205:98110.[CrossRef][Medline]
Washburn, M.P., D. Wolters, and J.R. Yates III. 2001. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19:242247.[CrossRef][Medline]
Wirschell, M., G. Pazour, A. Yoda, M. Hirono, R. Kamiya, and G.B. Witman. 2004. Oda5p, a novel axonemal protein required for assembly of the outer dynein arm and an associated adenylate kinase. Mol. Biol. Cell. 15:27292741.
Witman, G.B. 1986. Isolation of Chlamydomonas flagella and flagellar axonemes. Methods Enzymol. 134:280290.[Medline]
Witman, G.B. 1993. Chlamydomonas phototaxis. Trends Cell Biol. 3:403408.[CrossRef][Medline]
Wolf, E., P.S. Kim, and B. Berger. 1997. MultiCoil: a program for predicting two- and three-stranded coiled coils. Protein Sci. 6:11791189.
Yagi, T. 2000. ADP-dependent microtubule translocation by flagellar inner-arm dyneins. Cell Struct. Funct. 25:263267.[CrossRef][Medline]
Zhang, H., and D.R. Mitchell. 2004. Cpc1, a Chlamydomonas central pair protein with an adenylate kinase domain. J. Cell Sci. 117:41794188.