Analysis of the Phosphoproteome of Chlamydomonas reinhardtii Provides New Insights into Various Cellular Pathways

Abstract

The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases.

The Phosphoproteome of aChlamydomonas reinhardtiiEyespot Fraction Includes Key Proteins of the Light Signaling Pathway

“…Phosphorylation of the C. reinhardtii SOUL/HBP at two sites of its N terminus (Thr-42 and Ser-44) might play a role for its heme-binding capacity, since His and Cys possibly involved in the necessary complexation of Fe 21 in the heme are also situated at the N terminus (T. Schulze and M. Mittag, unpublished data). A second Chlamydomonas SOUL/HBP (ID 154433; Vs2), which is not present in the eyespot proteome, is also phosphorylated at the N terminus (Ser-2 and Ser-3; Wagner et al, 2006). Also a predicted unusual protein kinase (ID 153985) could be involved in signaling events.…”

“…Seventeen of the phosphoproteins were identified by a single phosphopeptide and 16 were covered by more than one, with up to nine phosphopeptides (Table I), where slightly different peptides with overlapping sequences or identical peptides with different phosphorylation sites are also counted. From the identified 33 phosphoproteins in this study, only six were previously described in phosphoproteomic approaches to wholecell extracts and thylakoids of C. reinhardtii (Table II; Turkina et al, 2006b;Wagner et al, 2006) and an additional three were found when comparing the phosphoproteins from Supplemental Table S2. Thus, most phosphorylation sites described here are novel.…”

“…They correspond to 27 gene models of Vs2 and 29 models of Vs3 of the C. reinhardtii genome. For three models present in Vs2, but missing or reduced in Vs3, ESTs as well as proteomic support for the Vs2 model exist (Table I; Schmidt et al, 2006;Wagner et al, 2006;this article). Seventeen of the phosphoproteins were identified by a single phosphopeptide and 16 were covered by more than one, with up to nine phosphopeptides (Table I), where slightly different peptides with overlapping sequences or identical peptides with different phosphorylation sites are also counted.…”

“…Precipitated proteins were solubilized with 6 M guanidine hydrochloride. We then applied the protocol of Wagner et al (2006), which has successfully been used for phosphoproteins of crude extracts from C. reinhardtii. In this protocol, proteins are digested with trypsin and afterward phosphopeptides are enriched with Ga 31 -based IMAC.…”

“…One of them, immobilized metal-ion affinity chromatography (IMAC), is based on the presence of the negatively charged phosphate groups and enriches for phosphorylated Ser, Thr, and Tyr. This method has already been applied for phosphoproteome analyses in different systems like lymphoma cells (Shu et al, 2004), higher plants (Nü hse et al, 2004), and C. reinhardtii (Turkina et al, 2006a(Turkina et al, , 2006bWagner et al, 2006). It relies on the direct identification of phosphopeptides in MS in contrast to other methods that chemically substitute phosphate residues.…”