We report here a comprehensive proteomics analysis of the mouse PSC complex. The PSC complex proteome determined in this study contains 1968 proteins detected by three or more unique peptides, making it one of the largest organelle proteomes reported to date (44
). Our PSC complex proteome contains many novel cilia and photoreceptor proteins. This dataset has the potential to open new avenues for research directed at fundamental questions about cilia biology, such as how these highly polarized structures are built and maintained and how these processes are disrupted in human disease.
The PSC complex proteome contains many cilia proteins not identified previously in photoreceptors. This includes 13 proteins produced by genes that harbor mutations that cause cilia disease (). The detection of these proteins in photoreceptors is consistent with the hypothesis that defects in these proteins cause disease by disrupting cilia structure or function and focuses future investigation of disease pathogenesis (2
). Our PSC complex proteome also contains seven IFT proteins not described previously in mammalian cilia or photoreceptors (). These data confirm the importance of IFT in mammalian cilia and the high degree of conservation of this axonemal transport system. These transport processes are especially important in photoreceptor cilia, where large numbers of proteins are required to maintain the outer segment, which are completely renewed every 10 days (45
The PSC complex proteome also contains ~1500 proteins not identified previously in cilia. This includes novel candidate disease genes. The identification of 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders will help prioritize genes in these linkage intervals for mutation screening (Supplemental Table S6
). This is especially true for the 34 genes in the list that produce proteins in the PSC-OS group. For example, the gene for transmembrane protein 27 maps within the critical region for retinitis pigmentosa 23 (RP23) (46
). Expression of the protein produced by this gene, which is also called collectrin, has been reported previously only in the kidney and pancreas, both organs in which primary cilia play an important role (47
). Furthermore it is likely that other PSC complex proteins are encoded by genes located in unidentified disease loci as evidenced by the recent detection of two novel disease proteins in the PSC complex proteome.3
The novel cilia proteins detected in the PSC complex proteome may also be informative about photoreceptor biology. For example, 27 components of the UPS were detected in the PSC complex but are not shared with other cilia (). The UPS plays an important role in several cellular pathways via the coordinated degradation of their protein components (48
). Although proteasomes are typically concentrated at the centrosome, the role of the UPS in cilia and photoreceptor function has not been evaluated in detail (49
). It has been suggested that the UPS may play a role in setting the levels of phototransduction proteins, which are tightly regulated (50
). The UPS also has an important role in removing damaged proteins from the cell (51
). Dysfunction of this process is associated with several neurodegenerative disorders, including Parkinson disease (48
). PSC complexes are subjected to high levels of oxidative stress resulting from obligatory exposure of the retina to light (52
). The finding of UPS components in the PSC complex suggests that local recognition and clearance of oxidatively modified proteins within the PSC complex by the UPS may be one of the mechanisms by which photoreceptor cells cope with chronic light stress. Consistent with these hypotheses, ubiquitin is abundant in the PSC complex and was detected at multiple molecular weights, indicating conjugation to PSC complex proteins.
The results of the mass spectrometric analyses show that the PSC complex proteome is relatively comprehensive and has only limited contamination by non-PSC proteins. For example, the 98% agreement between the analyses of the rootletin KO and wild-type PSC complex preparations shows excellent reproducibility of the LC-MS/MS analyses (Figs. and ). In addition, the agreement between the two datasets demonstrates good depth of analysis provided by separating the protein preparations into 30 fractions by electrophoresis and using long LC gradients with a linear ion trap mass spectrometer. Furthermore 36 of 38 proteins produced by retinal degeneration disease genes that are known to be located in the PSC complex were detected (). The two proteins that were not detected in our analyses are harmonin and the β
3 subunit of the cone cyclic nucleotide-gated channel (Cngb3). Evidence suggests that harmonin is a soluble protein (53
); thus it is possible it was lost during the isolation of the PSC complexes. Membrane proteins such as the cone cyclic nucleotide-gated channel β
3 are difficult to detect using mass spectrometry in part because their membrane-spanning regions are very hydrophobic and thus lack the arginine and lysine residues required for cleavage by trypsin. Most tryptic peptides derived from channel proteins are thus outside the size and hydrophobicity ranges detected by the mass spectrometric methods used (54
). Because only 3% of photoreceptors in the mouse retina are cones, it is likely that the combination of having six membrane-spanning regions and being present only in cones prevented detection of Cngb3.
As another assessment of the specificity of the PSC proteome, we performed immunofluorescence analyses of mouse retina using antibodies to 51 proteome members that were not described previously in PSC complexes. All but six of these antibodies confirmed the mass spectrometric data (Supplemental Table S5
). The other six antibodies stained cells in the inner retina, such as Müller cells, suggesting a low level of contamination of the PSC complex preparations with components of other cells, such as Müller cell processes. Further analysis of the proteins to which these antibodies are directed is warranted as they may also be present at low abundance in PSCs. There are several examples of PSC complex proteins that require immunoelectron microscopy for detection in retinal sections, such as myosin VIIa (55
). Myosin VIIa was readily detected in the PSC complex proteome (Supplemental Table S1; eight peptides
), illustrating the power of proteomics analysis to reveal low abundance proteins. Proteins from mitochondria and ribosomes, which can segregate with PSC complexes due to their high abundance in photoreceptor inner segments, accounted for less than 15% of the total proteins detected; this is comparable to the levels of mitochondrial and ribosomal proteins detected in other cilia and basal body proteomics analyses (9
). In sum, these analyses support the conclusion that the majority of the proteins in the proteome are truly present in the PSC complex.
Acceleration of the Pace of Discovery
Analysis of the PSC complex proteome has the potential to accelerate the pace of discovery regarding cilia biology. For example, an insight derived from our analyses is that the PSC complex as a functional organelle includes components in both the inner and outer segments of photoreceptor cells ( and Supplemental Table S1
). The detection of ~650 proteins predicted to be functionally part of the cilium structure that extends into the inner segment of photoreceptor cells emphasizes the essential connection between the inner and outer segment portions of the PSC complex. Further study of the PSC-IS proteins detected in our analyses may help identify additional proteins required for PSC complex function. For instance, almost all of the components of the exocyst complex were detected in the PSC-IS group of proteins. The exocyst complex has been shown to be located in primary cilia in renal tubule epithelial cells and plays a central role in the development of renal tubules and cysts (56
). There is recent evidence that the exocyst also plays an important role in vesicle-mediated transport of proteins to the Drosophila
rhabdomere, but the role of the exocyst in mammalian photoreceptors has not yet been investigated (57
The link between the inner and outer segment components of the PSC complex is also highlighted by the recent recognition that the movement of signal transduction proteins between the inner and outer segments of photoreceptor cells is an important mechanism by which photoreceptor cells adapt to changes in light intensity (30
). Stimulus-dependent redistribution of signaling proteins into and out of cilia also mediates transduction of external signals by primary cilia, including Hedgehog signaling and sensation of urine flow, both of which ultimately lead to modulation of transcription of target genes (3
). Protein movement may thus be a general mechanism by which cilia transduce information about the external environments of their cells. The PSC-IS group proteins detected in the PSC complex are candidates to participate in the movement of proteins in response to light or other stimuli in the PSC and other cilia.
Conservation of the Mammalian PSC
Based on the analysis of PSC complexes from rootletin KO mice, our data show that the PSC complex proteome contains 1185 proteins confirmed to be part of the PSC-OS (). Comparison of the PSC-OS proteome with other cilia datasets shows that the outer segment contains 2–5 times more proteins than reported for cilia from unicellular organisms, whose proteomes range from 223 in Tetrahymena to 652 in Chlamydomonas ().
Although a portion of the additional proteins in the PSC complex are associated with visual function, including 44 proteins associated with phototransduction, detection of these additional proteins likely reflects a general increase in complexity of mammalian cilia. This conclusion is supported by the observation that many protein classes in cilia from unicellular organisms are augmented in the PSC complex proteome. For example, of the 11 kinesin components and 14 microtubule-associated proteins found in the PSC, only five and three are present in flagella of Chlamydomonas
, respectively (10
). The increased number of proteins in the PSC complex reflects, at least in part, the increased complexity of the mouse genome as the percentage of genes devoted to encoding cilia proteins is similar in the mouse, Chlamydomonas
, and Trypanosoma
genomes; this is not true of the Tetrahymena
genome (). The increased complexity of mammalian cilia also suggests that additional members of other protein classes will be found in mammalian cilia.
Cilia proteomes and genome sizes
It is also evident from the comparisons that there is notable conservation of cilia proteins. At least 344 proteins are shared between the non-redundant cilia-related proteins in the Cilia-Proteome (16
) and the PSC complex proteome and represent a set of common cilia proteins, emphasizing the preservation of the basic components of cilia structure throughout evolution. For example, the PSC complex shares 137 proteins with flagella from Chlamydomonas
, a unicellular green alga estimated to have emerged as a species 700 million years ago (58
). Furthermore the relative proportions of the major enriched functional protein groups are similar in the common cilia proteins and the PSC complex proteome ().
Implications of Quantification of the PSC Complex Proteome Components
Precise regulation of protein levels is a major theme in cell biology. In photoreceptor cells over- or underexpression of specific proteins can lead to cell dysfunction and death with alterations in rhodopsin levels being the classic example (59
). It is difficult, however, to measure the levels of individual proteins on a large scale especially of proteins for which activity assays or quantitative immunoassays are not available. We found that total spectral counts normalized to total expected peptides (S/PP) within the analyzed mass range correlated well with the known amounts of 15 outer segment proteins (). Specifically 14 of the benchmark points lie within a factor of 3 of the best fitting line. This is consistent with prior reports that the number of spectra detected for a given protein in MS/MS analyses can be used to estimate the relative abundance of that protein (26
The value S/PP is an estimate of protein abundance and is not corrected for inherent variations in peptide “detectability.” For example, as indicated above peptides from membrane proteins are less efficiently detected in LC-MS/MS experiments due to their hydrophobicity (54
). Rhodopsin, which has seven transmembrane segments, is a relevant example. Because the majority of tryptic peptides from rhodopsin are too large to be detected using the LC-MS/MS parameters applied in our analysis, we used the hydrophilic peptide from the C terminus of rhodopsin for the analysis in . Even this peptide appears to be less “detectable” than the peptides in the majority of the other benchmark outer segment proteins as the point for rhodopsin (most abundant protein) falls below the regression line (). That said, it is evident from that S/PP provides a good estimate of relative protein abundance. This may in part be because the S/PP calculation is an average for all peptides detected from a given protein and thus “averages over” some of the inherent variation in peptide detectability. The S/PP calculation can be refined in the future as the reasons for the variation in peptide detectability are better defined (61
The estimated copy number of each protein in our PSC-OS proteome ( and Supplemental Table S8
) provides a basis for predicting the stoichiometries of protein complexes in outer segments and could be useful for functional studies of these proteins and their interactions and roles in the pathogenesis of diseases caused by cilia dysfunction. Knowledge of the amounts of specific proteins in photoreceptor outer segments may also be important for directing the development of accurate gene augmentation therapies for inherited retinal degenerations and other cilia disorders, some of which are nearing clinical application (62
). For example, mutations in CEP290
have recently been reported to cause the cilia disorders Joubert syndrome and Leber congenital amaurosis (LCA), a form of congenital blindness (63
). The proteome data indicate that ~9000 copies of Cep290 protein are present in each mouse outer segment. In contrast, ~580,000 copies of Rds, whose mutation causes several forms of retinal degeneration, are present (Supplemental Table S8
). These examples reveal that different delivery and expression strategies will be needed to effect successful therapy for different types of inherited retinal degenerations and other cilia disorders.