TPPP Orthologs Are Ciliary Proteins

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FEBS Letters 582 (2008) 3757–3764

Hypothesis TPPP orthologs are ciliary proteins

Ferenc Orosz*, Judit Ova´di Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1113 Budapest, Hungary

Received 1 August 2008; revised 1 October 2008; accepted 2 October 2008

Available online 16 October 2008

Edited by Michael R. Bubb

compartment separate from the cytoplasm via a process Abstract Tubulin polymerization promoting protein, (TPPP/ p25), was identified as a brain-specific protein. The potential called intraflagellar transport (IFT), mediated by the IFT function of this protein resembled that of MAPs. It is mainly ex- multiprotein complex (IFT particle) and IFT motors [8]. pressed in oligodendrocytes; however, immunopositivity was also However, Plasmodium cilia and Drosophila sperm flagella detected in glial and neuronal inclusions in synucleinopathies. are assembled in the cytoplasm by a process that does not ap- Here, we show that TPPP gene(s) are conserved in the genomes pear to require IFT [4,5]. The molecular makeup of motile of ciliated organisms, but are lacking from the nonciliated ones. and non-motile cilia is likely to overlap substantially, This recognition is based upon homologous gene sequence anal- although each type will boast components required accommo- ysis, in silico comparative genomic studies, bioinformatic search dating their specific functions. For example, non-motile cilia and experimental evidence. Cilia (flagella) are microtubule-based lack motility-associated proteins (e.g. outer and inner dynein cellular extensions of sensory and/or motile function. TPPP arm and radial spoke proteins, microtubule central pair-asso- orthologs are among the only 16 genes that can be found in all ciliated organisms, suggesting that TPPP orthologs may be ciated proteins) but can be enriched for proteins related to associated with a basic function of cilia. sensory perception [9]. Ó 2008 Federation of European Biochemical Societies. Recently, we isolated a partially unfolded protein from bo- Published by Elsevier B.V. All rights reserved. vine brain [10] and named it tubulin polymerization promoting protein/p25, (TPPP/p25), which has no well-defined Keywords: TPPP/p25; TPPP ortholog; Cilia; Flagella; structure [11,12]. It was first identified by co-purification to- Microtubule; Synucleinopathy gether with the tau protein kinase, Cdk5, in the bovine brain [13], which probably causes its phosphorylation, together with other kinases, ERK2 and PKA [14]. We have shown that it promotes tubulin polymerization into normal and double 1. Introduction walled tubules and polymorph aggregates; its binding to pac- litaxel-stabilized microtubules induces their bundling [14,15]. The research on eukaryotic cilium/flagellum has come to a It exhibits microtubule associated proteins-like functions sta- renaissance of interest, which was triggered by the recognition bilizing the microtubular network both in vitro and in trans- that defects of this organelle are at the centre of several hu- fected HeLa and rat kidney cells [14,16]. The physiological man disorders, including most importantly polycystic kidney role of TPPP/p25 is unknown, although our data strongly disease, the most common inherited disease in the US [1,2]. suggest that TPPP/p25 may take part in the stabilization of The cilium evolved very early in the development of eukary- the microtubular network. Under pathological conditions, otes. This organelle, virtually in its present form, had been TPPP/p25 is enriched in filamentous a-synuclein bearing present in the last common ancestor of eukaryotes [3,4]. Lewy bodies of ParkinsonÕs and diffuse Lewy body diseases, However, it has been lost secondarily in multiple lineages as as well as in glial inclusions of multiple system atrophy, as red algae, cellular slime molds and in the majority of land demonstrated by immunohistochemistry and confocal micros- plants or fungi [5,6]. However, gametes and sperms of several copy [11,12,17,18]. TPPP/p25 promotes the fibrillization of a- fungi and ancient plants such as mosses, ferns or Ginkgo pos- synuclein in vitro [19]. sess flagella/cilia [6,7]. There are two main types of cilia, motile and non-motile or sensory. Their axonemes consist of nine doublet microtubules; motile cilia (named also flagella) 2. Phyletic distribution of TPPP proteins contain an additional central pair of microtubules. The axonemal microtubules of all cilia nucleate and extend from a ba- TPPP/p25 is the first member of a new protein family, the sal body, a centriolar structure. Most cilia are assembled in a primary sequence of which differs from that of other known proteins, but shows homology with TPPP-like hypothetical proteins [15] sought via BLAST. There are two homologous *Corresponding author. Fax: +36 1 4665465. gene sequences in the human genome encoding two shorter E-mail address: [email protected] (F. Orosz). proteins, TPPP2/p18 and TPPP3/p20. Only the latter one was isolated from bovine brain which displays similar micro- Abbreviations: TPPP, tubulin polymerization promoting protein; IFT, intraflagellar transport; PSC, photoreceptor sensory cilium; CCV, tubule bundling activity as TPPP/p25 [20]. The paralogous clathrin-coated vesicle; SV, synaptic vesicle genes can be found only in vertebrates but not in other

0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.10.011 3758 F. Orosz, J. Ova´di / FEBS Letters 582 (2008) 3757–3764

organisms. BLASTP or TBLASTN analysis [21] was per- formed on complete genome sequences and EST collections

FAP265 available at the NCBI website using the sequences of human R R K R R 151 128 176 164 172 TPPP proteins. Fig. 1 shows the sequence homology of sev- S D R E KF SC SA SE TE TD GQ Homo sapiens eral TPPP proteins from unicellular organism to vertebrates. K K N K K K V V V V V LD Similarity can be seen in all parts of the proteins except the G G P P A A N-terminal tail of TPPP/p25s, which is missing in other K K K K K K

I V L V V V homologs. A standard and simple method for determining

EEA EES EAA QAK KNA orthology is the reciprocal best hit approach. It helped to re-

L L L L L veal 1:1 orthology also in cases when the BLAST E score was I SSPT KTGGA VKVGG DS GGV TQAEA QKVQGGK219 AKVKKQKVSKKTH

K K RTE R K higher than 1eÀ10. Our search has shown that such proteins/ A A TSKSGG A A A D D D D Chlamydomonas reinhardtii S S ST S T M genes can be found throughout the animal kingdom from K K K K Q N Y Y Y Y F F F F F F

T T T T T S protists to vertebrates but not in prokaryotes, land plants T T T T EL SA DMAG DKE DDQ DA T V V I I I T V V V A and fungi (Table 1). Thus they cannot be found in the com- TPPP/p25 (NP_008961); Hs3, G G G G I I I I L I

G G G G G pleted genomes of Arabidopsis thaliana, Oryza sativa or Sac-

D D D charomyces cerevisiae etc. No evidence for TPPP orthologs HA NA GD GE F VD TD was found either in bacteria or archaea. The species in Table K K K K K DV D D DA EV 1 are only representative examples. (The only possible excep- IIS ANV VMV AT S QS S Y Y Y Y T T SVD T T T MAAST MG MS MS Homo sapiens (XP_001743131); Cr, P P P P P P tion was the moss, Physcomitrella patens, which has a best-re- V G N S T S ciprocal-hit protein, although the similarity is very low, and SG SA GN GN RGV T T T T T T

V V V V IPAV probably it cannot be considered as a TPPP ortholog. The

Y Y Y Y H very recent establishment of the draft genome sequence of

G G G G G this model species and the comparison of its features with RNV KS V KE S QKF KS L KVI those of ﬂowering plants and the unicellular green algae re- LI EGKA LVAGKE VI NGGG I CKGKG VTRAGG VEAAS ES DS AS vealed genomic changes concomitant with the evolutionary Monosiga brevicollis R Q K K K K movement from aquatic environment to land, including loss

KAAQ RSPAD of some but not all ﬂagellar components for gametic motility VI DG VADG VLGP LYD LI S ILD

D D D D D as proteins of the outer dynein arms of ﬂagella [22]. However, we guess that it would be premature to make a ﬁnal conclu- CQ CK CK LH SG sion concerning the occurrence of TPPP gene/protein in land D D D D ETK D

LHA plants with ciliated sperm and we should wait until more gen- K K K R K K omes will be available.) TPPP is also absent in the slime . The alignment was reﬁned manually. Hs1, RVDLV RQDI L RVDKV RVDRD EDS RAKLAGHL GT

(XP_001628751); Mb, mold, Dictyostelium discoideum. Interestingly, it occurs in LC LC CL MF LC CI G G G G G G [51] the biﬂagellated green alga, Chlamydomonas reinhardtii but K K K K K K not in the red alga, Cyanidioschyzon merolae, which does KA IA LD IE LA not have cilia/ﬂagella. A detailed investigation of protists re- WS WA FA FG FT FS G G G G G vealed that a TPPP ortholog occurs in many species, however N N N K K N K K K K K QAAG it is absent from some of them. It can be found in Alveolates G G G G H including Apicomplexa (e.g. Plasmodium falciparum) and Cili- K K K K K ophora (e.g. Tetrahymena thermophila), in Euglenozoa (e.g. HGK NGK TGK DNA DNK Nematostella vectensis G G G G G RFKDKSSEEAVREVH RF QGKS KEEAF DAIKKI C NKKF GS KDRGVS VDS EDVF N QVGKLT E TGQDVS AVEA Trypanosoma brucei) and in Diplomonadida (Giardia lamblia) M M M M LDSA M K K E K K K but not in Entamoeba histolytica (Entamoebidae) and Acan- E K T A E K A thamoeba castellanii (Acanthamoebidae). DP S DES GAD DKS DS E A A A A A A The data listed in Table 1 show a tight parallel between the F F F F F phyletic distribution of cilia and the TPPP proteins, which sug- R R R R R EL EL KV EV HF gests that the TPPP gene is conserved in the genomes of cili- E E E E E NGGP TNI DK (BW358083); Nv, E E E E E GLC ated organisms but is absent from those that are non- K K K K K K RAT GRE KA S GQE KA TKG A QP L QMMKQD K L L L L L L ciliated. TPPP genes can be found in all organisms possessing H H H H H Y A A A A A these organelles, independently of the type of the cilium: (i) in S S S S S V organisms with motile cilia (most of the ciliated organisms); (ii) G G G G G G DA DP NS AGA KG CT QE KK KVG KV RS

T T T T T T in organisms with non-motile cilia only (e.g. C. elegans); (iii) in G G G G G F F F F F F

F Y Y Y Y Y organisms with compartmentalized (but not cytosol-assem- Ciona intestinalis Q E Q Q E Q bled) cilia (most of the ciliated organisms); (iv) and in organ- E E K N A A

TK SR SQ KC SQ isms with cytosol-assembled cilia (e.g. P. falciparum). F Y F F F F AVH AI H MVM CAF ASY T T T T T KNL There are only two unicellular green algae, Ostreococcus F F F F F T N D N G N D D D D D D I I I I I I lucimarinus and Ostreococcus tauri, whose genomes contain T T T T T K T T V K genes that can be considered as TPPP orthologs but these R R L L M L M R R KT R

FRR FRK YKK FES FI A species do not have cilia (cf. Table 1). Ostreococcus belonging to the Prasinophyceae, an early-diverging class within the sMAKKAANTPSGPKRARSEEAEAAASP ADKAKPAKAANRTPPKSPGDPSKDRAAKRLSLESEGAGEGA Hs1M Cr H D Hs1 Hs3 Ci Nv Mb Hs1 Hs3 Ci Nv Mb Hs3 Ci Nv Mb Cr Cr Hs3 Ci Nv Mb Cr Hs1 green plant lineage, are the smallest known eukaryotes. They have a very simple cellular organization, with no cell wall or Fig. 1. Multiple sequence alignment of several members of the TPPP family by ClustalW TPPP3/p20 (NP_057048); Ci, (XP_001695016). Residues identical and similarsequences) in involves at the least major all part but of one species the are proteins indicated except by the black N-terminal and gray tail backgrounds,flagella, specific respectively. for The TPPP/p25s. so-called Pfam05517 domain (2–4 lines of the amino acid and with a single chloroplast and mitochondrion. In .Ooz .Ova J. Orosz, F. ´ Table 1 3757–3764 (2008) 582 Letters FEBS / di Phyletic distribution of TPPP proteins. Species Clade Group GenBank gi (Hypothetical) Protein *EST E-value Reciproc-al E-value Identitiy (%) Cilia/flagella Mus musculus Opisthokonts Metazoa 33469051 NP_878259 2eÀ96 7eÀ101 91 Yes Monodelphis domestica Opisthokonts Metazoa 126320804 XP_001363210 8eÀ97 4eÀ98 87 Yes Ornithorhynchus anatinus Opisthokonts Metazoa 149443502 XP_001505656 3eÀ92 1eÀ99 82 Yes Gallus gallus Opisthokonts Metazoa 118086293 XP_001231864 2eÀ90 5eÀ99 80 Yes Xenopus tropicalis Opisthokonts Metazoa 156717851 NP_001096466 4eÀ76 1eÀ70 77 Yes Danio rerio Opisthokonts Metazoa 41152061 NP_958492 3eÀ69 3eÀ64 70 Yes Ciona intestinalis Opisthokonts Metazoa 47769884 BW358083* (Ci0100135725) 2eÀ35 9eÀ35 47 Yes Branchiostoma floridae Opisthokonts Metazoa 66297093 BW710519* 2eÀ55 3eÀ55 63 Yes Strongylocentrotus purpuratus Opisthokonts Metazoa 115634898 XP_782492 1eÀ33 5eÀ29 48 Yes Drosophila melanogaster Opisthokonts Metazoa 21357209 NP_648881 (CG4893) 5eÀ27 1eÀ17 38 Yes Crassostrea virginica Opisthokonts Metazoa 31903501 AUF_101_G19_TJ* 2eÀ19 1eÀ18 32 Yes Caenorhabditis elegans Opisthokonts Metazoa 17505795 NP_491219 (C32E8.3) 6eÀ28 1eÀ23 39 Yes Schistosoma japonicum Opisthokonts Metazoa 29841410 AAP06442 4eÀ19 2eÀ17 36 Yes Nematostella vectensis Opisthokonts Metazoa 156371398 XP_001628751 4eÀ28 3eÀ32 48 Yes Saccharomyces cerevisiae Opisthokonts Fungi No reciprocal best hit 3.5 – – No Monosiga brevicollis Opisthokonts Choanoflagellida 163778230 XP_001743131 3eÀ23 3eÀ25 45 Yes Tetrahymena thermophila Alveolata Ciliophora 146181921 XP_001023601 2eÀ11 6eÀ2 23 Yes Cryptosporidium hominis Alveolata Apicomplexa 126649214 XP_001388280 3eÀ61eÀ3 16 Yes Plasmodium falciparum Alveolata Apicomplexa 124806570 XP_001350760 2eÀ71eÀ6 20 Yes Theileria parva Alveolata Apicomplexa 71028521 XP_763904 8eÀ82eÀ5 13 Yes Trypanosoma brucei Euglenozoa Kinetoplastida 72387999 XP_844424 4eÀ11 1eÀ11 28 Yes Giardia lamblia Diplomonadida Diplomonadida 159110571 XP_001705540 2eÀ84eÀ6 25 Yes Chlamydomonas reinhardtii Viridiplantae Chlorophyta 159473789 XP_001695016 (FAP265) 3eÀ15 2eÀ11 33 Yes Ostreococcus lucimarinus Viridiplantae Chlorophyta 145353793 XP_001421186 2eÀ6 0.028 17 No Ostreococcus tauri Viridiplantae Chlorophyta 116056988 CAL51415 (Unnamed protein) 6eÀ6 0.021 15 No Physcomitrella patens Viridiplantae Streptophyta 168035642 XP_001770318a 0.41 2.2 7 Yes (in sperm only) Arabidopsis thaliana Viridiplantae Streptophyta No reciprocal best hit 9.6 – – No Cyanidioschyzon merolae Rhodophyta Bangiophyceae No hit –– – No Dictyostelium discoideum Mycetozoa Dictyosteliida No reciprocal best hit 2.6 – – No Acanthamoeba castellani Acanthamoebidae Acanthamoeba No hit –– – No Entamoeba histolytica Entamoebidae Entamoeba No reciprocal best hit 1.8 – – No Escherichia coli Proteobacteria Gammaproteobacteria No hit –– – Flagellin based Salmonella typhimurium Proteobacteria Gammaproteobacteria No reciprocal best hit 1.1 – – Flagellin based Methanococcus aeolicus Euryarchaeota Methanococci No reciprocal best hit 0.46 – – Flagellin based The presence of orthologous TPPP proteins in different species was established using BLAST searches [21] and the reciprocal best hit approach. Protein and nucleotide sequences were searched using BLASTP and TBLASTN, respectively, at http://www.ncbi.nlm.nih.gov/BLAST/, using the sequences of human TPPP proteins. Sequence identities were determined using CLUSTALW [51]. Asterisks stand for ESTs. aThis protein cannot be considered as a TPPP ortholog since no best-reciprocal-hit was found except between Homo sapiens and Physcomitrella patens. However, the Chlamydomonas reinhardtii ortholog has a different reciprocal best hit in Physcomitrella patens (XP_001761224) with higher similarity than the human (E-values are 0.14 and 0.37). 3759 3760 F. Orosz, J. Ova´di / FEBS Letters 582 (2008) 3757–3764 the evolution of its small size, Ostreococcus have lost a num- that the expression of C. elegans genes required for cilium bio- ber of genes involved in flagellum biosynthesis that are found genesis is controlled by the regulatory factor X (RFX)-type in Chlamydomonas, the flagellar unicellular green alga [23]. transcription factor, DAF-19, which binds to a conserved pro- The TPPP orthologs we found in Ostreococcus are the most moter element termed the ‘‘X-box’’. This indicated that C. ele- divergent members of the family, with the far highest E-val- gans X-box-containing genes, expressed exclusively in ciliated ues. Even the Chlamydomonas reinhardtii ortholog, FAP265 cells, are likely to encode proteins with ciliary functions. Nota- protein, are more similar to the human TPPPs than to the bly, human RFX3 and at least one of the two Drosophila RFX Ostreococcus homologs. FAP265 is identical in 31–33% with transcription factors also regulate proper cilium assembly and the three human TPPPs but only in 21–23% with the Ostreo- function. These findings prompted genome-wide searches for coccus orthologs. Taking into account that the phylogenetic such genes in C. elegans. Because the conserved C. elegans distance is much smaller between the two green alga families X-boxes are typically 100-bp upstream of start codons [26], than between mammals and algae, it can be hypothesized that X-boxes within 250-bp from start codon were searched using although the Ostreococcus ‘‘TPPPs’’ have not disappeared different algorithms and X-box consensus sequences [27,28]. from their genomes but they might lose their original func- X-boxes positioned in this region were found in 164 [28] or tions. 293 [27] of these gene promoters, including the X-box of TPPP ortholog (C32e8.3, 87-bp from start codon) in the latter study [27]. This finding was corroborated in [25] as well. These genes 3. In silico studies suggest that TPPPs are ciliary proteins can be considered as potential ciliogenic or ciliary genes, although the two complementary data sets only overlap par- The availability of numerous sequenced genomes from cili- tially. ated and non-ciliated eukaryotes has recently set the stage for powerful comparative genomic studies. In silico subtrac- tion of homologous genes found in non-ciliated organisms from a ciliated genomic data set should enrich for genes that 4. Experimental support have unique, cilium-specific functions. Li et al. [24] applied such an approach by using a panel of genomes from different Amputation of the flagella in Chlamydomonas strongly in- organisms. The non-ciliated Arabidopsis genome was sub- duces expression of flagellar genes. A 3- to 12-fold upregula- tracted from the genomic intersection between the ciliated hu- tion of the mRNAs for structural components of the flagella, man and Chlamydomonas genomes. The result was defined as the IFT particle, and its molecular motors was observed the so-called flagellar apparatus-basal body (FABB) proteome 30 min after deflagellation, whereas genes encoding non-flagel- containing 688 proteins, including TPPPs. The same ‘‘calcula- lar proteins were not induced [29]. Proteins that function in tion’’ was carried out using other ciliated organism genomes both the flagellum and the cell body usually were only weakly (M. musculus, C. intestinalis, C. elegans and D. melanogaster) induced. Therefore, several groups used the deflagellation of C. instead of the human genome, which resulted in data sets that reinhardtii to identify further flagellar genes. DNA oligonu- overlapped to a large degree with the FABB proteome. A cleotide microarray [30] or real-time PCR [30,31] were used smaller amount of overlap was found in the case of C. elegans, to measure changes in transcript level during flagellar regener- probably due to the lack of motile cilia in the nematode. ation. Indeed, a substantial fraction of the genes was upregu- A similar study by Avidor-Reiss et al. [25] involved six cili- lated. The TPPP ortholog was also induced, more than two- ated organisms (D. melanogaster, H. sapiens, C. elegans, C. fold [31]. It should be noted that in addition to revealing genes reinhardtii, P. falciparum and T. brucei) and three non-ciliated that encode flagellar/ciliary components, this approach also organisms (A. thaliana, S. cerevisiae and D. discoideum). Fol- identifies genes whose products are involved in regulating fla- lowing genome subtractions, 187 ‘‘ciliary’’ genes were identi- gellar assembly or function but are themselves not components fied in total, which were divided into four classes, based on of cilia or flagella. However, proteomic analysis after isolation the occurrence of the different type of cilia in the species. of flagella/cilia and basal body can differentiate among these Namely, genes conserved in organisms: (i) with motile cilia possibilities. (all ciliated organisms except C. elegans; 18 genes); (ii) with The very few proteomic studies have been carried out mostly compartmentalized cilia (all ciliated organisms except P. falci- on unicellular organisms, and, except a pioneering one in 2002 parum; 103 genes); (iii) with compartmentalized and motile ci- [32], have been published in the last two years. In these works lia (i.e. Homo sapiens, Chlamydomonas, Trypanosoma and the cilia were isolated, and peptides were generated by proteo- Drosophila but not C. elegans or Plasmodium; 50 genes); and lytic digestion either directly or following 1D and/or 2D gel (iv) those are present in all ciliated organisms (16 genes). electrophoresis separations of the proteins, which was followed These comparative genomic studies can identify cell body or by liquid chromatography (LC)–mass spectrometric (MS) basal body proteins as well that support cilia function but are analyses of the peptides. Generally, proteins represented by not specifically localized to the organelle (e.g. cannot be de- at least two (but preferentially more) peptides are considered tected in proteomic analysis of isolated cilia). In other words, as potential ciliary ones. they identify genes/proteins related specifically to cilia func- The most detailed investigations involved the quasi model tion. Importantly, TPPP homologs were found among the only organism of ciliary/flagellar species, the biflagellated green 16 genes, which can be found in all ciliated organisms. This alga, Chlamydomonas reinhardtii. Not only the flagellum but finding suggests that they are connected to not a specific but also its sub-organellar parts (axoneme, membrane, matrix) some kind of basic function of cilia. [31] and the basal bodies [33] were isolated and studied sepa- Bioinformatic search supported further the possible ciliary rately. Six hundred fifty-two proteins were represented by at function of TPPPs. The study by Svoboda et al. [26] revealed least two peptides in the flagella preparation and 45 proteins F. Orosz, J. Ova´di / FEBS Letters 582 (2008) 3757–3764 3761 were validated as basal body candidate proteins. The TPPP identified, with significant amount of contaminating material. ortholog (XP_001695016 or FAP265) was found exclusively TPPP/p25 was not found in this preparation. It is in accordance in the flagella, in the matrix preparation. The matrix contains with the data of [31] where the TPPP ortholog was found in the components that are not tightly associated with either the axo- matrix but not in the membrane fraction. neme or the membrane such as IFT proteins. However, it is The mammalian proteomic reports identified TPPP/p25 in questionable whether FAP265 protein itself is an IFT protein. the modified sensory cilia of photoreceptor cells [36] and It was not found in the isolated IFT particle of C. reinhardtii TPPP3/p20 in the motile cilia of bronchial epithelial tissue cells [8] and the TPPP orthologous gene can be found in each Plas- [32]. Further studies will enlighten whether the two paralogs modium species, the cilia of which are assembled in the cyto- really share in the roles: are TPPP/p25 and TPPP3/p20 present plasm by a process that does not require IFT. in non-motile and motile cilia, respectively? Recently, the ciliary proteome of the uniflagellated parasite, Tripanosoma brucei [34], and the ciliated Tetrahymena thermophila [35] was also established. Although both protists are known to possess a TPPP orthologous gene (thus probably 5. Potential role of TPPPs in ciliary function the protein as well), the TPPP protein has not been found in these studies. In the latter case [35] the reason may have been The combination of the results of the different complemen- the loss of extremely basic proteins, such as TPPP during 2D tary studies suggests that TPPP orthologs are ciliary proteins electrophoresis. In general, biochemical fractionation might re- and they are integral part of cilia/flagella. The TPPP orthologs move ciliary components therefore proteins of low abundance were found among the only 16 genes, which can be found in all might not be identified by at least two peptides. For example, ciliated organisms. This finding suggests that they are con- although all the so-called Bardet–Biedl syndrome (a ciliary dis- nected to a basic function of cilia. The most general feature of order) proteins studied in C. elegans have been shown to be eukaryotic cilium is that its axoneme consisting of nine dou- components of the IFT machinery, none of the homologous blets of microtubules serves as its ‘‘skeleton’’. Beside microtu- proteins from humans, Chlamydomonas or Tetrahymena were bules, the axoneme contains many other proteins and protein identified in the proteomic studies [9]. complexes necessary for its function. TPPP proteins can stabi- The first attempt to identify a mammalian ciliary axoneme lize microtubules via their high bundling activity. Experiments proteome, using cilia released from human bronchial epithelial with isolated TPPP proteins and with human cell lines have tissue culture cells as starting material, was described by shown that TPPP/p25 and TPPP3/p20, in addition to their Ostrowski et al. [32]. A total of 214 proteins were identifiable tubulin polymerization promoting activities, cross-link micro- on the basis of one or more peptide match(es). Sixty-five of tubules making them resistant against the effects of anti-micro- them were identified as known or likely ciliary components, tubular agents such as vinblastine [16,20]. Such stabilizing effect including TPPP3/p20. (TPPP3/p20 was identified on the basis of TPPP proteins might manifest itself at the ultrastructural of a single peptide only. The results of more recent proteomic organization of both the axonemal and the axonal microtu- studies usually involve only proteins identified on more bules. matches. In addition, the peptides, which the identifications Another, almost universally cilia-associated characteristic is were based on, were not given, in contrast to recent studies. that their assembly relies on IFT except Plasmodium cilia and Thus data provided in this early paper showing that TPPP3/ Drosophila sperm flagella, which are assembled in the cyto- p20 is a ciliary protein would not be satisfactory according plasm by a process that does not appear to require IFT. IFT to the present-day standards of proteomic works). is characterized by a bidirectional movement of protein parti- All the studies mentioned above analyzed motile cilia/fla- cles in cilia between the microtubular axoneme and the outer gella. The first proteomic study of non-motile (sensory) cilia membrane [38]. The movement of IFT particles is microtubule is a very recent one by Liu et al. [36]. Specialized versions of dependent and driven by motor proteins kinesin and dynein. non-motile cilia are involved in many aspects of sensation. The IFT particle can be isolated as a multiprotein complex The single photoreceptor sensory cilium (PSC) or outer seg- [8]. Electron microscopic studies indicate that the IFT complex ment elaborated by each rod and cone photoreceptor cell of is linked to the ciliary membrane but the mechanism of associ- the retina is a classic example. ation is unknown. As suggested by Je´kely and Arendt [4], the The PSC complex comprises the outer segment and its cyto- IFT probably evolved as a special form of clathrin-coated ves- skeleton, including the axoneme, basal body and ciliary rootlet icle (CCV) transport. CCVs are postulated to be responsible extending into the inner segment of photoreceptor cells. A de- for the recycling of synaptic vesicles (SVs) during neurotrans- tailed analysis of the mouse PSC complex proteome revealed mission (For review see Ref. [39]). After exocytosis, SVs are re- mammalian cilia to be several times more complex than the ci- trieved by clathrin-dependent endocytosis to restore the lia of unicellular organisms. The PSC complex contains 1968 primed vesicle pool and are locally recycled to regenerate exo- proteins represented by three or more unique peptides, includ- cytosis-competent vesicles. CCVs retrieve SV membranes from ing 1500 proteins not detected in cilia from lower organisms. the plasma membrane after SVs collapse, concomitant with 1185 of them are derived from the outer segment (i.e. cilium neurotransmitter release. Very recently, it has been suggested and basal body), including TPPP/p25, represented by six pep- [39] on the basis of proteomic analysis of SVs and CCVs that tides. TPPP/p25, as a protein transiently associated with the SV/CCV This year proteomic analysis of a membrane preparation from membrane during the SV life cycle, may have a role in the rat olfactory sensory cilia was also published; however, this organelle transport and interaction of the vesicles with the study has not intended to identify the entire set of ciliary proteins nerve terminal cytoskeleton. TPPPs are not integral part of but only that of the ciliary membrane [37]. Two hundred sixty- either SVs/CCVs or IFT particle but TPPP/p25 was identified eight proteins, the half of which was membrane proteins, were as a vesicle membrane-associated protein that attaches and 3762 F. Orosz, J. Ova´di / FEBS Letters 582 (2008) 3757–3764 detaches during the vesicle cycle in a time-dependent manner. lation (roughly two-fold) of the TPPP ortholog gene in One can hypothesize a similar role for TPPPs in IFT as well. deflagellated C. reinhardtii [31] also suggests this dual localiza- As it was suggested in [4] the eukaryotic cilium/flagellum tion. Their functions can be similar in both cellular environ- probably is an autogenous innovation of eukaryotes. This is ments: stabilization of microtubules and/or participation in likely true for the IFT machinery as well, and the hypothesis cytoskeleton based transport processes. These functions can is also supported by the evolutionary history of ciliary axo- be only analogous and manifest themselves depending on the neme proteins, including first of all tubulin. The fact that an- cell type. However, we are intrigued by the possibility of a more other ciliary protein, TPPP, has no homologs in prokaryotes intimate relationship between TPPP/p25, cilia and cell cycle possessing flagellin based flagellum fits to this view. However, progression: the genesis and disassembly of the cilia is con- it does not exclude the other explanations, as the endosymbi- trolled by cell cycle, and vice versa, which is affected by otic [40] or the viral invasion [41] hypothesis. TPPP/p25 via modulation of the reorganization of microtubule Until very recently [42] TPPP/p25 has been considered as a ultrastructures. brain-specific protein since it was identified in oligodendrocytes and in the neuropil [11,43], and was studied in brain tis- Acknowledgements: The authors are grateful to La´szlo´ Patthy for his sues by immunohistochemistry up to now. A study of the comments on the manuscript. This work was supported by FP6– 2003-LIFESCIHEALTH-I: BioSim to J.O.; and by the Hungarian spatial and temporal distribution of TPPP/p25 in the develop- National Scientific Research Fund Grants Nos. OTKA T-046071 ing rat brain showed that the protein appeared only in differ- and T-67963 to J.O. and T-049247 to F.O., respectively. entiating oligodendrocytes and co-localized with bIV-tubulin when the cells reached the myelinating state [44]. (This tubulin isoform appears in myelinating oligodendrocytes but not in References oligodendrocyte progenitor cells). Our recent data [45] ob- tained with oligodendrocyte progenitor cells from rat brain [1] Pan, J., Wang, Q. and Snell, W. (2005) Cilium-generated signaling also revealed that TPPP/p25 is virtually not present in progen- and cilia-related disorders. Lab. Invest. 85, 452–463. [2] Fliegauf, M., Benzing, T. and Omran, H. 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