U. Turku Plant Biology - CURT1B

U. Turku Plant Biology - CURT1B
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Light-dependent dynamics of phosphorylation of the thylakoid structural protein CURT1B
ProteomeXchange: PXD014562
  • Organism: Arabidopsis thaliana
  • Instrument: TSQ Vantage
  • SpikeIn: No
  • Keywords: Arabidopsis thaliana, Thylakoids, grana margins, LHCII, CURT1,
  • Lab head: Andrea Trotta Submitter: Andrea Trotta
Abstract
The regulation of the photosynthetic apparatus in higher plants is highly connected to the organization of the thylakoid membrane into appressed and non-appressed regions. The two photosystems (I and II) are the spatially strongest segregated protein complexes by this thylakoid heterogenous organization, with PSII predominantly present in the appressed membranes (grana), while PSI is confined to the stroma-exposed non-appressed thylakoid membranes (lamellae). Stacking of thylakoid membranes in grana is highly dynamic and influenced by the levels of PSII and LHCII protein phosphorylation which, in turn, are dependent on changes in light intensity and quality and thus, ultimately, on the redox state of the electron transfer chain. Dynamics of this lateral heterogeneity controls the spillover of excitation energy from PSII to PSI and optimizes the photochemistry in constantly fluctuating light conditions. Recently, a family of membrane-integral thylakoid proteins named CURVATURE THYLAKOID 1 (CURT1) have been proposed as new key components in shaping both the width and the number of layers per granum in response to changes in light intensity. CURT1B, in particular, has been known for a long time as a relatively abundant phosphorylated and acetylated protein, but so far the role and dynamics of these post-translational modifications (PTMs) have not been revealed. To this end, we have quantified by means of targeted proteomics the amount of CURT1 proteins and the level of CURT1B PTMs after short-term fluctuating light treatments in wild type Arabidopsis thaliana and the knock-out mutants of the kinases STN7 and STN8 and of the phosphatase TAP38. The CURT1B protein was first localized to a specific curvature domain largely depleted of the chlorophyll-protein complexes. Moreover, we have found that CURT1B is either phosphorylated or acetylated in the N-terminus, but never both. While the level of acetylation is never affected by the light treatments, the phosphorylation increases in light conditions that lead to sudden increase in PSII core protein phosphorylation, and these dynamics are largely abolished in stn8 plants but not in stn7 or tap38. Intriguingly, the phosphorylation dynamics as well as the level of the other CURT1 proteins, are instead highly affected in psb33 plants, which lack LHCII phosphorylation dynamics, and in the psal plants, which show constitutively phosphorylated LHCII. These results provide important information for assessing the relationships between PSII-LHCII phosphorylation, CURT1B phosphorylation and the dynamics of the appressed thylakoids, which in turn allow optimal photosynthesis and provide photoprotection under fluctuations in light intensity.
Experiment Description
Isolated thylakoids corresponding to 5 µg of chlorophyll were denatured in 6M urea 6% acrylamide SDS-PAGE and in-gel digested with trypsin (1:10 µg of trypsin to µg of chlorophyll). The peptide mixture was first analyzed in DDA with a Q-exactive (Thermo Fisher Scientific) and the MS/MS acquired utilized to generate the SRM transitions. The sum of the intensities (integrated peak area) of the three most intense transition for every peptide have been utilized to calculate the ratio between protein complexes and targeted proteins. Three unique proteotypic peptides from subunits of PSI (PsaA, PsaB, PSAH1and 2, PSAL, PSAK), of LHCI (1 to 6) and the CURT1 proteins (A to D) were used to calculate protein abundance. The PTMs of the N-terminus of CURT1A and B were also monitored to calculate the percentage of phosphorylation.
Sample Description
Thylakoids membranes were isolated from 32 days-old Arabidopsis plants, grow under a light intensity of 120 µE, at the end of 16 hours dark period (D1), after subsequent treatment for 2 hours at light intensity substantially lower than the growth light (LL- 20 µE) and further for 2 hours in substantially higher light intensity than the growth light (HL- 1000 µE). Thereafter thylakoids were digested with trypsin and the obtained peptides identified in DDA using a Q-exactive (Thermo Fisher Scientific). The proteotypic peptides from all four CURT1 proteins were identified, including the previously described CURT1A and CURT1B N-terminal peptides acetylated on Ala-63 (63Ac-ASSEETSSIDTNELITDLK81) and Ala-64 (64Ac-ATTEVGEAPATTTEAETTELPEIVK88), respectively, as well as the CURT1B N-terminal single phosphorylated peptide (64ApTTEVGEAPATTTEAETTELPEIVK88). The amount of CURT1 proteins and the level of acetylation and phosphorylation of CURT1A and CURT1B were quantified by SRM from plants illuminated under changing white light intensities. For standardization between the measurements of thylakoid replicates, we also measured the level of the PSI core subunits PsaA and PsaB in parallel with CURT1 proteins. Moreover, since CURT1B and CURT1C have been reported to interact with small subunits of PSI, the levels of PSAL and PSAH that form the LHCII interacting domain of the PSI complex and that of PSAK as a control peripheral subunit, were likewise quantified. As a control, the level of the six LHCI subunits were monitored in all the measurements. Thylakoids for assessing the dynamics of the levels of above mentioned proteins and of CURT1A and CURT1B PTMs were isolated at six time points from 32 days-old Arabidopsis plants, grow under a light intensity of 120 µE, treated with a fluctuating white light cycle in the end of the 16 h dark period (D1), after subsequent 2 h at low light (LL1), and then after 2 h of high light (HL), again after 2 h of low light (LL2), and finally after transfer for 1 h to darkness (1h D), followed by 15 h of darkness (D2). The same analysis was conducted in parallel in the knock-out mutants (KO) of the protein kinase STN7 and of the protein phosphatase TAP38, responsible for LHCII phosphorylation and dephosphorylation, respectively, in KO of the kinase STN8, responsible for Photosystem II phosphorylation, in KO of the protein PSB33, which lack LHCII phosphorylation dynamics, and in KO of the PSI subunit PSAL, which show constitutively phosphorylated LHCII.
Created on 7/10/19, 9:43 AM

This data is available under the CC BY 4.0 license.