We identified 195 proteins as potential primary cilia proteins by using the calcium shock isolation method, MudPIT correlation profiling, and subtractive proteomic analysis. This study is the first proteomic analysis of generic mammalian primary cilia. Previous proteomic analyses of non-motile mammalian cilia focused on specialized sensory cilia, namely the connecting cilium of photoreceptors and olfactory cilia [3
], which differ in appearance from “generic” primary cilia but share the same fundamental structure of a microtubule axoneme surrounded by a membrane.
Our primary cilia candidate list includes almost all known IFT proteins except Ift43 (Table S5
). We also identified kinesin and dynein IFT motor proteins. Notably absent were any BBS proteins, which are the causative gene products of Bardet-Biedl syndrome and are localized to primary cilia [24
]. We do not understand why most of the IFT proteins, but none of the BBS proteins, were detected in our analysis. Nevertheless, the fact that we recovered almost every known IFT proteins, including IFT proteins not used to construct the reference protein profile, confirms that our calcium shock method and protein correlation profiling were effective for identifying bona fide ciliary proteins. Our candidate list also includes proteins that potentially regulate protein import into the cilium. We identified the septins (Sept2, Sept7 and Sept9) in our ciliary proteome. Septins have been reported to form a diffusion barrier that controls protein entry into cilia and regulates ciliogenesis [25
]. We also detected the small GTPase Ran as well as importin, which regulate both nuclear and ciliary entry of proteins [27
]. However, we failed to detect many known ciliary membrane proteins, such as polycystin-1 and polycystin-2. This may reflect the reduced solubility of such proteins under extraction conditions used for protein separation in MudPIT.
Around 75% of our candidate primary cilia proteins are shared components with motile or specialized sensory cilia (Table S2
). Especially, most of IFT proteins and Nme7 were also identified in comparative genomics and X-box promoter analysis (Table S2
). We speculate that Nme7 is a part of IFT complex or related with IFT. However, Nme7 may regulate protein transport or signal transduction rather than assembly of cilia [28
]. This indicates these proteins are necessary for all types of cilia and flagella. On the other hand, prior to our analysis we had expected to recover two major classes of axonemal proteins in our candidate list: tektins and pf-ribbon proteins [29
]. As these proteins are known to be stable and abundant components of the axoneme, we assumed that they would be among the most abundant hits in our list, just as they have been reported to be common in proteomic analysis of motile cilia/flagella and basal bodies [4
]. Structural studies have suggested that tektins and pf-ribbon proteins form filamentous structures tightly associated with the outer doublets of the axoneme, which are conserved among all animal cilia, and may therefore be critical determinants of outer doublet architecture in all cilia [31
]. Surprisingly, neither tektin nor pf-ribbon proteins were found in the candidate list from our primary cilia proteome. Tektin and pf-ribbon proteins have also not been identified in the proteomes of specialized sensory cilia [3
]. Moreover, knock out or knock down of tektin induces defects in ciliary motility but not in ciliogenesis per se [32
]. Therefore, it is possible that tektin and pf-ribbon proteins are only components of basal bodies and motile cilia, but not primary cilia.
The remaining 25% of our candidate proteins are possible primary cilia specific proteins because these proteins have not been identified in proteomic studies of motile or specialized sensory cilia. However, some primary cilia specific proteins might be also localized to sensory or motile cilia because Inpp5e was also identified in inner segment of photoreceptor [34
]. Some of the primary cilia specific proteins are apparently involved in signal transduction, such as, Evc2, Inpp5e, Inversin, Broad-Minded (Bromi) and Nphp3. Evc2 and Bromi function in hedgehog signal transduction [20
], while Inpp5e is involved with phosphatidylinositol and PDGF signaling [34
Because ciliary defects are linked to many genetic disorders, we were particularly interested to see if any ciliary disease loci would turn up in our candidate list. In fact, multiple candidate proteins were found to correspond to known causative gene products of genetic disorders (Table S6
). We identified several known ciliopathy gene products, including Inpp5e, Evc2, Nphp3, Nek8 and Lca5 (see referenes in Supplemental Information
). A recent study reported that TSGA14 is a plausible causative gene of autism spectrum disorders [37
]. Moreover, WDR11 has been also recently reported as a causative gene of idiopathic hypogonadotropic hypogonadism and Kallmann syndrome [38
]. In light of our results that Tsga14 and Wdr11 are present within primary cilia (), we hypothesize that the autism and abnormal genital development in such patients might illustrate new symptoms of ciliopathy.
In this study, we isolated primary cilia from cultured mammalian cells and utilized this material to generate the first reported proteome of mammalian primary cilia. Our analysis revealed that the majority of primary cilia components are shared with motile and specialized sensory cilia. However, around 25% of the candidate proteins were only identified in this analysis, including Evc2, Inpp5e, Inversin, Nphp3 and Broad-Minded. These novel candidates, a number of which have already been implicated in ciliopathies and signal transduction, are potentially specific to primary cilia. Tsga14 and Wdr11 are new candidates of causative genes of ciliopathy. The results of this study should serve as a starting point toward a greater understanding of primary cilia functions and mechanistic insights into ciliary diseases.