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Comparative Phosphoproteome Profiling Reveals a Function

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Comparative Phosphoproteome Profiling Reveals a Function Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF) Sonja Reilanda,b, Giovanni Finazzic,d,e,f, Anne Endlera,b, Adrian Willigg,h, Katja Baerenfallera,b, Jonas Grossmanna,b, Bertran Gerritsa,b, Dorothea Rutishausera,b, Wilhelm Gruissema,b, Jean-David Rochaixg,h, and Sacha Baginskya,b,1,2 aDepartment of Biology, Eidgenössische Technische Hochschule (ETH) Zurich, 8092 Zurich, Switzerland; bFunctional Genomics Center Zurich, 8057 Zurich, Switzerland; cCentre National Recherche Scientifique, Unité Mixte Recherche 5168, Laboratoire Physiologie Cellulaire et Végétale, F-38054 Grenoble, France; dCommissariat à l’Energie Atomique et Energies Alternatives, l’Institut de Recherches en Technologies et Sciences pour le Vivant, F-38054 Grenoble, France; eUniversité Grenoble 1, F-38041, France; fInstitut National Recherche Agronomique, UMR1200, F-38054 Grenoble, France; and Departments of gMolecular Biology and hPlant Biology, University of Geneva, 1211 Geneva, Switzerland Edited* by Bob B. Buchanan, University of California, Berkeley, CA, and approved June 24, 2011 (received for review March 24, 2011) Important aspects of photosynthetic electron transport efficiency in a defect in state transitions (11). This process balances the chloroplasts are controlled by protein phosphorylation. Two thyla- absorbed light excitation energy between the two photosystems. koid-associated kinases, STN7 and STN8, have distinct roles in short- State transitions are regulated by light quality and intensity and and long-term photosynthetic acclimation to changes in light qual- mediated by phosphorylation of photosystem II (PSII) light-har- ity and quantity. Although some substrates of STN7 and STN8 are vesting complex (LHCII) proteins (4, 12). It is now well estab- known, the complexity of this regulatory kinase system implies that lished that STN7 activity is required for state transitions, although currently unknown substrates connect photosynthetic perfor- it is currently unclear whether STN7 directly phosphorylates mance with the regulation of metabolic and regulatory functions. LHCII proteins or triggers their phosphorylation through a cas- We performed an unbiased phosphoproteome-wide screen with cade. STN8 is a paralog of STN7 and is also associated with the Arabidopsis WT and stn8 mutant plants to identify unique STN8 thylakoid membrane system. Analyses with phosphothreonine- targets. The phosphorylation status of STN7 was not affected in specific antibodies identified the D1 (PsbA) and D2 (PsbD) stn8, indicating that kinases other than STN8 phosphorylate STN7 proteins of PSII, PsbH, CP43, and a Ca2+-sensitive thylakoid under standard growth conditions. Among several putative STN8 phosphoprotein, calcium-sensing receptor (CaS), as STN8 sub- substrates, PGRL1-A is of particular importance because of its pos- strates (13–15). However, loss of STN8 function not only affects sible role in the modulation of cyclic electron transfer. The STN8 the phosphorylation of thylakoid membrane proteins but also the phosphorylation site on PGRL1-A is absent in both monocotyledon- expression of nucleus- and plastid-encoded genes for photosyn- ous plants and algae. In dicots, spectroscopic measurements with thetic proteins (13). Arabidopsis stn7 stn8, stn7 stn8 WT, , and / double-mutant plants These data suggest multiple functional interactions of STN8 indicate a STN8-mediated slowing down of the transition from cy- fl fi within the chloroplast phosphoprotein network that extend clic to linear electron ow at the onset of illumination. This nding beyond our current mechanistic knowledge. For example, light- suggests a possible link between protein phosphorylation by STN8 – fi fl quality dependent changes of photosystem core protein phos- and ne-tuning of cyclic electron ow during this critical step of phorylation mediated by STN8 no longer occur in the stn7 back- photosynthesis, when the carbon assimilation is not commensurate ground in Arabidopsis, suggesting functional crosstalk between to the electron flow capacity of the chloroplast. STN7 and STN8 (16, 17). The Chlamydomonas ortholog of STN8, called Stl1, is a phosphoprotein in vivo whose phosphorylation phosphoproteomics | Arabidopsis thaliana depends on Stt7 (18). It is conceivable that a similar crosstalk PLANT BIOLOGY exists between the corresponding Arabidopsis orthologs STN8 and fi n the eld of chloroplast biogenesis, interest in protein phos- STN7. However, although STN7 is an abundant phosphoprotein, Iphorylation historically focused on photosynthesis-related pro- comprehensive phosphoproteome analyses failed to identify any teins, with the initial discovery of thylakoid membrane protein STN8 phosphorylation in Arabidopsis chloroplasts under different – phosphorylation dating back to the late 1970s (1 4). Almost conditions (9, 10, 19). Interestingly, the sequence of the C-ter- a decade later, AtpB, RNA-binding proteins, and transcription minal region of STN7 containing the four mapped phosphory- factors were recognized as phosphoproteins in thylakoid mem- lated sites diverges from the corresponding region in Stt7, branes and stroma fractions (5–7). Because of recent large-scale suggesting a function of STN7 phosphorylation in adaptation functional genomics and phosphoproteomics approaches, ∼200 processes that are specific to higher plants (10). Although it is chloroplast phosphoproteins are known today, and several kinases have been identified that are most likely involved in their phos- phorylation (8). However, the exact kinase/substrate relationships Author contributions: S.R., G.F., J.-D.R., and S.B. designed research; S.R., G.F., A.E., and are not known for most of the proteins, and efforts are underway A.W. performed research; S.R., G.F., K.B., J.G., B.G., D.R., W.G., J.-D.R., and S.B. analyzed to identify in vivo substrates of known kinases. Phosphoproteo- data; and G.F. and S.B. wrote the paper. mics data suggest that chloroplast functions are regulated by The authors declare no conflict of interest. a highly complex phosphoprotein network in which one kinase *This Direct Submission article had a prearranged editor. phosphorylates several substrates and one substrate is probably Data deposition: The MS data and tandem MS spectra have been deposited in the phosphorylated by several kinases at different sites (9, 10). PRoteomics IDEntifications (PRIDE) database, http://www.ebi.ac.uk/pride/ (accession nos. 13754–13761). Although we currently do not understand all nodes in the 1Present address: Institute of Biochemistry and Biotechnology, Martin-Luther-University chloroplast phosphoprotein network, candidate proteins and ex- Halle-Wittenberg, 06120 Halle (Saale), Germany. perimental tools are available to address the above questions. 2To whom correspondence should be addressed. E-mail: sacha.baginsky@biochemtech. Two of the best-characterized chloroplast kinases are STN7 and uni-halle.de. STN8. STN7 is the ortholog of the Stt7 kinase from Chlamydo- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. monas reinhardtii that was identified in screens for strains with 1073/pnas.1104734108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1104734108 PNAS | August 2, 2011 | vol. 108 | no. 31 | 12955–12960 Downloaded by guest on September 26, 2021 currently unknown which kinase phosphorylates STN7, analysis of Quantitative phosphopeptide analysis was performed in three the phosphorylation motifs has suggested that one of the phos- biological replicates by spectral counting and extracted ion phorylation sites might be used by casein kinase II (10). chromatogram quantification (Fig. 1A). To distinguish between Here we report STN8 substrates that we identified in a com- changes in protein abundance and changes in phosphorylation parative proteome-wide analysis of protein phosphorylation in state, we quantified the unphosphorylated proteins from the flow- WT and in STN8-deficient (stn8) plants. We quantified unphos- through fraction of the IMAC. The protein quantification from phorylated proteins in plastids of both genotypes by normalized the flow-through enables a quantitative comparison of the plastid spectral counting to distinguish changes in phosphorylation states proteomes in the two different genetic backgrounds (Fig. 1B and from changes in protein abundance. Our data show that other SI Appendix, Table S3). The average Spearman rank correlation kinase(s) besides STN8 are involved in the phosphorylation of coefficient, ρ, for the spectral count data from 756 identified STN7 and establish PGRL1-A as a STN8 substrate. chloroplast proteins is 0.871, suggesting minor quantitative adaptations of the chloroplast proteome to a loss of STN8 (Fig. Results and Discussion 1B). The high similarity between the plastid proteomes of WT and Phosphoproteome Profiling from stn8 and WT Arabidopsis Leaf stn8 plants allowed a valid quantitative comparison of protein Tissue. We analyzed the leaf phosphoproteome of WT and stn8 phosphorylation in the plastids of the two genotypes. plants in three biological replicates by using a combined immo- bilized metal-ion affinity chromatography/titanium dioxide affin- Quantitative Comparison of Phosphopeptide Detection in WT and STN8-Deficient Plants. We searched for phosphoproteome differ- ity chromatography (IMAC/TiO2) phosphopeptide enrichment strategy followed by LTQ-Orbitrap mass spectrometry (MS). In ences between WT and stn8 plants by comparing the spectral total, 15,492 spectra were assigned to 3,589 phosphopeptides
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