The phosphoproteome of a Chlamydomonas reinhardtii eyespot fraction includes key proteins of the light signaling pathway

Abstract

Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca(2+)-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.

Figure 1.

Western-blot analysis of proteins from the eyespot fraction of C. reinhardtii with phospho-amino acid-specific antibodies. A, Proteins (4 μg) were separated by 11% SDS-PAGE, transferred to a PVDF membrane, and either analyzed with a polyclonal anti-phospho-Thr antibody (Anti-pThr; 1:1,000) or stained with Coomassie Brilliant Blue R250 (Coomassie). B, The membrane, to which proteins (4 μg) had been blotted, was treated with 1 m KOH according to Kamps and Sefton (1989) prior to incubation with the antiserum to reduce the amount of phosphorylated Ser and Thr residues. The developing time was identical to that in A (3 min). C, Proteins (4 μg) from two independent eyespot isolations were separated by SDS-PAGE (11%), blotted, and probed with a monoclonal anti-phospho-Tyr antibody (Anti-pTyr; 1:1,000, developing time 15 min). Arrowheads indicate the two major labeled protein bands. Positions of molecular mass markers are indicated on the left (kD).

Figure 2.

Schematic overview of phosphopeptide enrichment from the eyespot fraction of C. reinhardtii for LC-ESI-MS analysis. Details are described in “Materials and Methods.” Nano-LC-ESI-MS (MS² and MS³) analysis was carried out in a mass spectrometer with a linear ion trap that permits the acquisition of data-dependent neutral loss (Finnigan LTQ; Thermo Electron).

Figure 3.

Identification of a phosphopeptide from ChR-1 (ID 166415; Vs2) by nano-LC-ESI-MS² and by neutral-loss-triggered MS³. A, Identification of the phosphopeptide ASpLDGDPNGDAEANAAAGGK by the MS² fragmentation pattern of peptide ion 942.02; “p” indicates the phosphorylation site on Ser. B, Identification of the peptide ADhaLDGDPNGDAEANAAAGGK by the MS³ fragmentation pattern of the detected neutral-loss fragment 892.6; “Dha” indicates the site of the neutral loss of phosphoric acid from phospho-Ser. Only prominent y- and b-fragment ions have been labeled.

Figure 4.

Frequency of selected amino acids in single positions relative to their total frequency in positions −6 to +6 surrounding the Ser/Thr phosphorylation sites. Only those phosphorylation sites, which were unambiguously assigned, were included in the analysis. For the analyses, sequences of Vs2 were used when models were present in both genome versions.

Figure 5.

Identified phosphorylation sites in the two photoreceptors ChR-1 and ChR-2. A, Partial sequences of ChR-1 and ChR-2 are depicted. In both cases, parts of the C-terminal sequences are missing. For ChR-1, only the first 420 and for ChR-2 only the first 328 amino acids are shown, respectively. Peptides identified by MS analyses have been labeled in bold and phosphorylation sites are marked by a “p” with gray background. All TMDs are underlined. B, Localization of the phosphorylation sites in relation to the TMD arrangement of ChR-1 and ChR-2 (modified from Kateriya et al., 2004). Phosphorylation sites are marked by an asterisk. C, ClustalW alignment of the experimentally determined first phosphorylation site of the two ChRs from C. reinhardtii (CrChr-1 and CrChR-2) and the orthologous sequence of the ChR of Volvox carterii (VcChR-1). *, Perfect matches; :, high similarity; ·, low similarity. Phosphorylated Ser is highlighted by a gray background and charged amino acid residues (+/−) at identical positions are indicated above the sequence.