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2005-07-07
Proteomic analysis of a eukaryotic cilium
Gregory J. Pazour University of Massachusetts Medical School
Et al.
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Repository Citation Pazour GJ, Agrin NS, Leszyk JD, Witman GB. (2005). Proteomic analysis of a eukaryotic cilium. Open Access Articles. https://doi.org/10.1083/jcb.200504008. Retrieved from https://escholarship.umassmed.edu/oapubs/912
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THE JOURNAL OF CELL BIOLOGY
C Published July5,2005 The onlineversionofthisarticlecontains supplementalmaterial. the membraneoraxoneme,includingproteinsinvolved in tains “matrix”proteinsthatarenottightlyassociatedwitheither genera substructures suchasthedyneinarmsandradialspokesthat membrane ofthecell.Theaxonememotileciliaincludes by aciliarymembranethatiscontinuouswiththeplasma cilium (Fig.1)consistsofamicrotubularaxonemeensheathed sensory antennae(PazourandWitman,2003).Structurally,the cilia andspermtails,move;othersarenonmotileserve as Chagas’ disease,andgiardiosis.Somecilia,suchasairway including thosethatcausemalaria,Africansleepingsickness, essen ganelles andhereafterreferredtointerchangeably,alsoare 2004). Ciliaandflagella,whichareessentiallyidenticalor- Wheatley, 2003;Scholey,Snelletal.,2004;Pazour, mental disorders(PazourandRosenbaum,2002;Ong (PKD), retinaldegeneration,obesitysyndromes,anddevelop- cent evidencehasimplicatedciliainpolycystickidneydisease severe inheriteddisorderprimaryciliarydyskinesia;morere- Defects inciliahumanshavelongbeenknowntocausethe confidence. 97outof101previouslyknownflagellar fied withhighconfidence,and292moremoderate Chlamydomonas reinhardtii tify proteinsinpurifiedflagellafromthegreenalga toiden- not wellknown.Hereweusemassspectrometry oftheseorganelles,theircomposition is and importance ranging fromprotiststohumans.Despitetheubiquity perception,andthelifecyclesofeukaryotes sensory 3 2 1 J.Pazour,Gregory http://www.jcb.org/cgi/doi/10.1083/jcb.200504008 The JournalofCell Biology © kidney InsP3, inositol1,4,5-trisphosphate;MS,massspectrometry; PKD,polycystic used:FAP,flagellarassociatedprotein;IFT,intraflagellartransport; Abbreviations Correspondence toG.B.Witman:[email protected] G.J. PazourandN.Agrincontributed equally tothiswork. Introduction Proteomic analysis of a eukaryotic cilium Proteomic analysisofaeukaryotic Proteomic Mass Spectrometry Laboratory,Proteomic MassSpectrometry UniversityofMassachusettsMedicalSchool,Shrewsbury, MA01545 Program inMolecularMedicine,UniversityofMassachusettsMedicalSchool,Worcester, MA01605 Department ofCellBiology,Department UniversityofMassachusettsMedicalSchool,Worcester, MA01655
TeRceelrUiest rs $8.00 TheRockefellerUniversity Press
tial tothelifecyclesofmanyhumanandanimalparasites,
disease; PMCA,plasmamembraneCa te andcontrolaxonemalbending.Theciliumalsocon-
evolution and play important rolesinmotility,evolution andplayimportant throughout that havebeenhighlyconserved ilia andflagellaarewidespreadcellorganelles
1 NathanAgrin, , Vol. 170,No.1,July4,2005103–113 . 360proteinswereidenti- 2 ATPase. 2 JohnLeszyk, 3 andGeorgeB.Witman Chlamydomonas reinhardtii are farmorecomplexthanpreviouslyestimated. terized inanyorganism.Theresultsindicatethatflagella inhumansbuthavenotbeenpreviouslycharac- served flagellum alsocontainsmanyproteinsthatarecon- The hydrocephalus inhumansandmodelvertebrates. eases suchascystickidneydisease,malesterility, and merous proteinswithhomologuesassociateddis- and signaltransductioncomponents,containsnu- plete dataset.Theflagellarproteomeisrichinmotor com- proteins werefound,indicatingthatthisisavery trophoretic analysesofisolatedflagellafromthegreenalga their proteincomposition.Previoustwo-dimensionalgelelec- and oftheirroleindiseasewillrequireincreasedknowledge maintenance oftheorganelle(RosenbaumandWitman,2002). intrafl bodies, theyareacomplement toandnotasubstitutefordirect able informationonproteins associated withciliaandbasal of ciliaryorflagellarproteins. and thereforelikelytobeinvolved intheassemblyorfunction tion; theyidentified220genesthatwereinducedatleast100% 1995), ofwhichonly more than250proteins(LuckandPiperno,1989;Dutcher, netic studiesoftheflagellum,revealedthatflagellacontain that hasmanyadvantagesforbiochemicalandmolecularge- level ofinductionall approach, Stolcetal.(2005)usedagenechiptoexaminethe encoding flagellarandbasalbodycomponents.Inadifferent present intheformerbutnotlatter;thesearecandidatesfor (2004) identified187and688genes,respectively,thatare ciliated organisms,Avidor-Reissetal.(2004)andLi For example,bycomparingthegenomesofciliatedversusnon- identification nome sequenceshasmadepossibleglobalapproachestothe molecular level.Recently,theavailabilityofcompletege- An understandingofhowciliacarryouttheirfunctions Although theaboveapproaches canprovideveryvalu- agellar transport(IFT),aprocessrequiredforassemblyand http://www.jcb.org/cgi/content/full/jcb.200504008/DC1 Supplemental Material canbefoundat: 2 of genesencodingciliaryandflagellarproteins. C. reinhardtii 100 havebeencharacterizedatthe , ageneticallytractableorganism genesafterdeflagella-
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Figure 1. Flagellar structures. Diagram (A) and electron micrograph (B) of a cross section of a motile flagellum (from C. reinhardtii). (C) Central pair of microtubules; (I) inner dynein arm; (IFT) IFT particle; (M) flagellar membrane; (O) outer dynein arm; (R) radial spoke.
proteomic analyses using mass spectrometry (MS). For exam- ple, 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 re- generation 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 Downloaded from 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 (Os- Figure 2. Flow chart for isolation of flagellar fractions used for MS trowski et al., 2002); however, this study was compromised by www.jcb.org analyses. (A) Electron micrograph of cross sections of isolated flagella. Most the presence of other cellular structures in the axonemal prepa- of the flagella have an intact membrane; in these flagella, the matrix is ration, and by limitations in the amount of material available dense and obscures the axonemal microtubules. In a few of the flagella, the and/or sequence data obtained, with the result that only 89 of membrane has ruptured, releasing the matrix into solution and revealing the
microtubules. No other cell organelles are apparent in the flagellar prepara- on July 8, 2008 the proteins were identified by more than a single peptide. tion. In the initial analysis (left pathway), isolated flagella from wild-type cells Here, we use MS to identify the proteins in biochemically frac- were treated with the detergent Tergitol, which disrupts the flagellar mem- tionated C. reinhardtii flagella, which are available in large branes without dissolving them, and releases the flagellar matrix. The Tergitol-insoluble fraction (B) was then collected by centrifugation and ana- amounts and in high purity. lyzed by MS as described in the Materials and methods. For subsequent analyses (right pathway), flagella were isolated from the outer armless mu- tant oda1 and treated with the detergent Nonidet, which dissolves the mem- Results brane and releases the matrix; the preparation was then centrifuged to yield a supernatant containing the “membrane matrix” fraction and a pellet con- Identification of flagellar proteins taining the demembranated axonemes (C). The axonemes were resuspended To identify the proteins that compose C. reinhardtii flagella, in 0.6 M KCl and the mixture was centrifuged to yield a supernatant contain- ing the “KCl extract” and a pellet containing the “extracted axonemes” (D). these organelles were released from vegetative cells by di- The KCl extraction releases numerous axonemal proteins, including those of bucaine treatment, isolated from the cell bodies by low speed the inner dynein arms and the C2 central microtubule, which are missing in centrifugation and sucrose step gradient fractionation, and the extracted axonemes. The “membrane matrix,” “KCl extract,” and “ex- tracted axonemes” were then analyzed by MS. Bar, 0.2 m. (B) Micrograph harvested by high-speed centrifugation. The membranes of courtesy of M. Wirschell (Emory University, Atlanta, GA). flagella isolated in this way generally remain intact and the ma- trix remains in situ (Fig. 2 A). The purified flagella were then fractionated into a Tergitol-insoluble fraction containing mem- from less abundant proteins prompted us to use flagellar frac- brane and axonemes (Fig. 2 B), or into a Nonidet-soluble frac- tions isolated from an outer dynein arm mutant (oda1-1) for the tion containing membrane matrix proteins, a fraction con- remaining work. The proteins in each of the four fractions were taining proteins released from the Nonidet-demembranated separated by one-dimensional SDS-PAGE, each gel lane cut axonemes by KCl extraction, and a fraction containing the ax- into 33 to 45 slices (Fig. S1), the proteins in each slice digested onemal proteins remaining after KCl extraction (Fig. 2 D). with trypsin, and the resulting peptides eluted, separated by Electron microscopy of isolated flagella and axonemal frac- HPLC, and analyzed by MS–MS using electrospray ionization tions indicates that they are highly pure (Fig. 2). and an LCQ ion trap mass spectrometer. The search engine Initial analysis was performed on the Tergitol-insoluble Mascot was used to find the best matches to the MS–MS fraction isolated from wild-type flagella. However, peptides spectra in the translated C. reinhardtii genome, and the peptides derived from the outer dynein arm were very abundant, and also were searched against a database containing all predicted concerns that these might prevent identification of peptides C. reinhardtii proteins to identify the origins of those that
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Table I. Summary of results arm subunits were detected in that initial sample, so any previ- Proteins identified by five or more peptides 360 ously unidentified outer arm subunits of similar abundance also Proteins identified by two to four peptides 292 are likely to have been detected. Thus, this dataset is likely to Proteins identified by one peptide 482 contain nearly all flagellar proteins. Major groupsa Signal transduction proteins 93 Assessment of exclusiveness of the Motor proteins 41 dataset Nucleotide metabolism 9 Even though electron microscopy indicated that the flagellar Glycolytic enzymes malate dehydrogenase 8 Conserved uncharacterized proteins 87 fractions were highly pure, at some level the dataset is likely to Predicted membrane proteins 39 contain proteins that are contaminants. The number of peptides found by electrospray MS of complex mixtures roughly corre- aOnly proteins identified by two or more peptides are included here. Proteins may be included in more than one category. lates 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 ex- spanned exon–exon junctions. From these four fractions, 8,345 ceptions include several outer dynein arm subunits, which unique peptides were identified and grouped by the proteins would not have been present in flagella of the outer dynein arm- from which they were derived. 360 proteins were identified by less mutant used for all but the Tergitol-insoluble membrane five or more unique peptides, 292 by two to four unique pep- axonemes fraction; Tctex1 (one peptide), which is very small; tides (Table S1) and another 482 by a single peptide (Table S2). and EB1 (two peptides), which is located only at the tip of the All protein and peptide sequences, as well as the Mascot scores flagellum and therefore is of relatively low abundance. Obvious for the peptides, are available at http://labs.umassmed.edu/ contaminants in the dataset (tRNA synthetases, ribosomal pro- chlamyfp/index.php. teins, and histones) were usually identified by one or two pep- Downloaded from The list of flagellar proteins identified by two or more tides, even though these proteins are highly abundant in the cell peptides is rich in motor proteins, signal transduction proteins, body. Thus, the 360 proteins identified by five or more peptides proteins with predicted coiled-coil domains, and predicted are highly likely to be true flagellar proteins, with possible membrane proteins (Table I), and contains a number of pro- exceptions including methionine synthase, elongation factors, teins whose homologues are associated with disease in humans arginyl-tRNA synthetase, carbonic anhydrase, and two cell wall www.jcb.org and model vertebrates. Nearly 90 proteins are highly conserved proteins. However, even some of the latter proteins may func- in humans (BLAST E score 1e-10) but have not been previ- tion in the flagellum: methionine synthase gene expression is in- ously characterized in any organism. Individual proteins of in- duced by flagellar adhesion (Kurvari et al., 1995); elongation terest are described in the Discussion. factor 1a binds calmodulin and localizes to Tetrahymena cilia on July 8, 2008 (Ueno et al., 2003); and arginyl-tRNA synthetase is reported to The dataset contains nearly all known modify protein substrates to allow degradation via the ubiquitin flagellar proteins pathway (Ferber and Ciechanover, 1987), several other compo- A measure of the completeness of the dataset can be obtained by nents of which were found in the flagellum. Several glycolytic determining what percentage of known proteins is present. At enzymes also are present in this data subset and are likely to this time, 101 C. reinhardtii flagellar proteins have been identified function in the flagellum (see Discussion). by biochemical, genetic, and bioinformatic methods (Table S3). The 292 proteins identified by two to four peptides should 97 of these are present in our dataset. This includes all known be considered candidate flagellar proteins as this group contains outer dynein arm subunits and associated proteins except one, both known flagellar proteins and likely contaminants. Within all known and predicted inner dynein arm subunits except one, this group, the genes of 19 out of 51 (37%) randomly chosen all known IFT components, and all known radial spoke proteins proteins that were not previously reported to be in the flagellum (Table S3). The gene (ODA5) encoding the outer dynein arm– were found to be induced at least 1 by deflagellation (i.e., their associated protein that is missing is not present in the genome transcript levels doubled; see Induction of genes by deflagella- assembly and so could not have been found by our analysis. The tion section). Flagellar proteins would include these plus known gene (DHC3) encoding the putative inner dynein arm subunit flagellar proteins plus some whose genes are not induced by de- that is missing was identified by PCR analysis of genomic DNA flagellation, so that the percent of true flagellar proteins in this but the protein has not been shown biochemically to be in fla- subset is likely to be substantially higher than 37%. The group gella. This gene was much less strongly expressed than other of proteins identified by single peptides was not analyzed in dynein heavy chain genes as measured by Northern blot or re- detail but likely contains low abundance and small flagellar verse-transcriptase PCR analysis (Porter et al., 1996). The gene proteins along with contaminants. encoding LF4 is mostly missing from the genome assembly. The only other missing protein is p80 katanin, which was shown Distribution of proteins in flagellar genetically to be involved in central pair assembly, but the fractions abundance of the native protein is unknown (Dymek et al., In the subset of proteins identified by five or more peptides, the 2004). Although fractions from flagella lacking outer dynein peptides of 3/4 of the proteins were present predominantly in arms were used for all but the initial analysis, all known outer the KCl extract and extracted axoneme fractions and thus
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Figure 3. Distribution of proteins in flagellar fractions. (A and B) Distribution of proteins identified by five or more peptides (A) and by two to four pep- Figure 4. Deflagellation-induced change in transcript levels for 176 pre- tides (B) in the extracted axoneme (Ex Ax), KCl extract (KCl Ex), and dicted flagellar proteins as measured by real-time PCR. (Yellow) Proteins