The power of functional proteomics

Abstract

One of the key modifications of proteins that can affect protein functions, activities, stabilities, localizations and interactions, represents phosphorylation. For functional phosphoproteomics, phosphopeptides are enriched from isolated sub-cellular fractions of interest and analyzed by liquid chromatography-electrospray ionization-mass spectrometry. Such an approach was recently applied to the eyespot apparatus of the green flagellate alga Chlamydomonas reinhardtii, which represents a primordial visual system. Thereby, 32 phosphoproteins of known eyespot proteins along with 52 precise in vivo phosphorylation sites were identified. They include enzymes of carotenoid and fatty acid metabolism, (putative) light signaling components and proteins with unknown function. Strikingly, the two unique green algal photoreceptors, channelrhodopsin-1 and -2 were found to be phosphorylated in the cytoplasmic loop next to their seven transmembrane regions in a similar distance as observed in vertebrate rhodopsins.

The green alga Chlamydomonas reinhardtii serves already for many years as a model to study diverse processes such as photosynthesis, the composition, function and development of the chloroplast, the flagella and basal apparatus along with their relevance for human diseases as well as stress and light or circadian gated pathways.¹ In the last years research on C. reinhardtii has entered a new era based on the availability of its complete genome (nucleus, mitochondria, chloroplast) and EST sequences.^2–4 Since this green alga can be grown relatively easy in a short time-range, sufficient biological material is available to efficiently establish biochemical purification procedures of specific sub-cellular fractions. Combined with the available genome sequences, this has allowed identifying the components of such fractions by conducting large-scale proteome analysis. This led, for example, to the identification of the majority of proteins that are present within the flagella, the basal apparatus, also named centriole, and the eyespot apparatus.^5–7

The eyespot apparatus of flagellate green algae is a primitive visual system, which can detect both, light direction and intensity and is thus important for the phototactic orientation of these algae.⁸ In C. reinhardtii, it is a singular structure usually composed of two layers of highly ordered carotenoid-rich lipid globuli that are situated at the periphery of the chloroplast. Thylakoid membranes subtend these globule layers. Moreover, the outermost globule layer is attached to specialized areas of the chloroplast envelope membranes and the adjacent plasma membrane, in which the photoreceptors are generally considered to be localized. Until 2005, only six components of this early visual system were known, including EYE2 and MIN1, two proteins that are relevant for eyespot assembly, two splicing variants of the retinal binding protein COP (Chlamydomonas opsin), and two unique seven-transmembrane domain (TMD) photoreceptors, COP3 and 4, which are better known as channelrhodopsins ChR-1 and ChR-2.^9,10 Recently, purification of a fraction enriched in the eyespot lipid globuli and the associated parts of chloroplast and plasma membranes paved the way for its proteomic analysis by tandem mass spectrometry (MS/MS). Thereby, 202 proteins of the eyespot apparatus that were covered by at least two peptides per protein were identified.⁷ These proteins included the already known six proteins of the eyespot as well as a variety of functional groups ranging from calcium-sensing and binding proteins, channels, membrane associated/structural proteins such as proteins with PAP-fibrillin domains to proteins involved in retinal, carotenoid, chlorophyll biosynthesis and lipid metabolism. Notably, known proteins from thylakoids such as the alpha, beta and gamma subunits of the chloroplast ATP synthase seem to have a specialized localization and possibly function within the eyespot.¹¹ Moreover, a limited number of kinases and phosphatases were found among the proteins of the eyespot, suggesting that reversible protein phosphorylation might play a role in the light-signaling cascade.

To identify the targets of the kinases and phosphatases, functional phosphoproteomics can be applied. For this purpose, it is necessary to enrich a given fraction, such as the eyespot apparatus, for phosphopeptides, because the phosphorylation status of a given protein can be very low and phosphoproteins involved in signaling are often anyhow low abundant. Immobilized metal affinity chromatography is frequently used for phosphopeptide enrichment. In C. reinhardtii, phosphopeptides from proteins of the cellular and thylakoid phosphoproteomes were treated by this way and resulted in the identification of numerous in vivo phosphorylation sites.^12–14 Since the proteins of the eyespot have a rather hydrophobic character, a specialized protocol involving digestion with the endopeptidase LysC prior to Trypsin was used for generating the phosphopeptides from the eyespot.¹⁵ Therefore, its proteins were dissolved in 4 M urea, in which LysC still has an activity of 86%. In total, 68 different phosphopeptides corresponding to 32 known proteins of the eyespot along with 52 precise in vivo phosphorylation sites were identified by MS/MS and neutral loss triggered MS/MS/MS analysis. The identified phosphoproteins belong mainly to the following functional categories: carotenoid and fatty acid metabolism, (putative) light signaling pathway(s) and retina-related proteins as well as thylakoid and chloroplast envelope-related proteins. But there are also several proteins of yet unknown functions. The most prominent phosphoproteins clearly involved in the light signaling pathway(s) represent the two photoreceptors ChR-1 and ChR-2. They contain three and one phosphorylation sites, respectively (Fig. 1A and B). These sites are localized in a cytoplasmatic loop with close proximity to the seven TMD channel-forming regions. It is striking that the relative position of the functional sites of phosphorylation is highly conserved within the green algal and vertebrate rhodopsins, implying functional relevance for the regulation of these unique directly light-gated channels. It will be of special interest to find out (i) the physiological role of photoreceptor phosphorylation in C. reinhardtii and (ii) the kinase(s) involved in the phosphorylation of the two ChRs, because it (they) might have a critical role in guiding phototransduction. The amino acids surrounding the phosphorylated Ser residue in ChR-1 and ChR-2 and the closely related ChR-1 of Volvox carteri are highly conserved.¹⁵ No additional putative ortholog sequences of this motif were found by NCBI protein BLAST searches against the Chlamydomonas genome using the 60-amino acid long sequence motif surrounding the phosphorylated Ser-358 in ChR-1. Because specificity of the active sites of kinases toward their substrates is primarily based on the linear sequence surrounding the phosphorylation site, the green algal ChRs might be targets of a specialized kinase.^15,16

Figure 1.

Protein architecture of exemplary phosphoproteins from the eyespot along with the location of their phosphorylation sites. P with a gray background and arrowheads indicate phosphorylation sites within the proteins and the hydrophobicity plots, respectively. Protein architecture was adapted from the NCBI CD Web site (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). Numbering above the schematic protein models indicate the number of amino acids (AA), whereas the numbers at the beginning of the models are the protein ID's (genome Vs2). Gray bars within the models indicate TMDs, and specialized domains are shown below the schematic protein model, if present. (A and B) ChR- 1 (A) and 2 (B), respectively, along with their conserved rhodopsin domains. TMDs were set accordingly to Kateriya et al.¹⁰ (C) SOUL3 heme-binding protein along with the SOUL domain.¹⁹ (D) EFh: EF hand calcium binding motif. TMDs were predicted by TMHMM as outlined in Wagner et al.¹⁵ (E) Protein with no significant hit in NCBI BLASTp and no functional domain along with its corresponding hydrophobicity plot using TMpred as outlined in Wagner et al.¹⁵ TMDs were predicted by TMHMM. This protein was not found in former eyespot analysis, but may have been easily missed due to the lack of adequate sites for Trypsin.⁷,¹⁵ (F) Protein with no significant hit in NCBI BLASTp and no functional domain along with its corresponding hydrophobicity plot using TMpred.

Analysis of the phosphorylation sites from the phosphoproteins of the eyespot revealed a bias for surrounding amino acids with regard to basic, acidic, aromatic amino acids and Gly as well as a tendency for clustering outside known functional domains. In the majority of the cases (23 proteins), the phosphorylation sites were found outside the predicted domains. Two examples of such phosphoproteins are depicted in Figure 1C and D. The EF hand containing Ca²⁺-binding protein and the SOUL3 heme-binding protein represent additional potential members of the light-signaling pathway(s) within the eyespot. It is known that extra-cellular calcium-fluxes are intricately involved in the behavioral responses of C. reinhardtii to light and that both ChRs can conduct Ca²⁺.^17,18 A SOUL heme-binding protein was found in a screen for chicken mRNAs specifically expressed in the retina and pineal gland, indicating that certain proteins seem to be indeed conserved from primitive visual algal systems to the highly sophisticated visual system of animals.¹⁹ Because Descartes considered the pineal gland as the “soul”, Zylka and Reppert named the protein accordingly.¹⁹

Phosphorylation sites were frequently located in regions with a more hydrophilic character. A typical example (protein of unknown function; Vs2, ID 170226), where the phosphorylation sites were found in the most hydrophilic region, is shown in Figure 1E. The protein with the highest number (nine) of phosphorylation sites (Vs2, ID 167609) is a rather hydrophilic protein of yet unknown function (Fig. 1F). There, the phosphorylation sites tend to cluster within a 230 amino acid area of the 763-amino acid long protein. Such candidates of yet unknown function are also of special interest for future functional characterization, because these proteins could be specific for green algal eyespots.

Our recent proteomic approaches to the eyespot of Chlamydomonas have just generated an inventory of the diverse (phospho)proteins of this light sensing organelle. Nonetheless, they form the basis to study many intriguing questions regarding the light signaling pathway(s) initiated by excitation of the ChRs as well as its structural maintenance and positioning. Considering the fact that until 2005 only six proteins of the eyespot of C. reinhardtii were known at the molecular level, these studies are yet another good example of the intriguing power of large-scale proteomic approaches to sub-cellular fractions rather subtle in isolation. Moreover, the functional phosphoproteome approach highlights eyespot proteins that are regulated at the posttranslational level and suggests relevant members of the light signaling pathway(s). Clearly the arduous task of functional analysis of the diverse proteins by RNA interference technology, mutant analyses and methods for identifying their (potential) protein interaction partners must be tackled now to gain deeper insights into the functions of this fascinating green algal cell organelle.

Abbreviations

ChR

channelrhodopsin

COP

retinal binding protein

MS

mass spectrometry

TMD

transmembrane domain