Phosphorylation dynamics of LHCSR3 were investigated in Chlamydomonas reinhardtii by quantitative proteomics and genetic engineering. LHCSR3 protein expression and phosphorylation were induced in high light. Our data revealed synergistic and dynamic N-terminal LHCSR3 phosphorylation. Phosphorylated and non-phosphorylated LHCSR3 associated with PSII-LHCII supercomplexes. The phosphorylation status of LHCB4 was closely linked to the phosphorylation of multiple sites at the N-terminus of LHCSR3, indicating that LHCSR3 phosphorylation may operate as a molecular switch modulating LHCB4 phosphorylation, which in turn is important for PSII-LHCII disassembly. Notably, LHCSR3 phosphorylation diminished under prolonged high light, which coincided with onset of CEF. Hierarchical clustering of significantly altered proteins revealed similar expression profiles of LHCSR3, CRX and FNR. This indicates the existence of a functional link between LHCSR3 protein abundance and phosphorylation, photosynthetic electron flow and the oxidative stress response.

Inclusion lists compiled with Skyline were used for scheduled fragmentation of target peptides. Peptide quantifications were performed using non-enriched samples only. See Table S3 for details on LC and MS parameters. LHCSR3 phosphosite mutants: Raw PRM spectra files were imported into Skyline for the extraction of ion chromatograms and integration of fragment ion peak areas. The total peak areas (area under curve, AUC) of a minimum of 3 fragment ions per peptide were determined with manual adjustment of peak borders. Data points were discarded if the correlation score (dotp) between the measured product ion peak areas and the fragment ion intensities in the spectral library was lower than 0.7. Then protein ratios (mutant/rescue vs. wild type) were calculated using the mean 14N/15N (label swap: 15N/14N) ratios of at least three distinct proteotypic peptides per protein after combining Skyline results from the lysC and trypsin treated samples. In case a peptide was quantified in lysC and trypsin digested samples, the mean ratio was used for further calculations. The median of all 14N/15N peptide ratios of a sample was used for normalization to account for small inaccuracies that occurred during mixing of labelled and unnlabeled protein prior to digestion. Standard deviations were calculated using ratios of all quantified peptides of a protein. High light kinetics (200 µmol photons m-2 s-1 versus 500 µmol photons m-2 s-1): Raw MS data was imported as described above. The integrated areas (AUC) of at least 3 fragment ions were summed to determine peptide abundances. Data points were discarded if the correlation score (dotp) between the measured product ion peak areas and the fragment ion intensities in the spectral library was lower than 0.7. Total protein abundances were expressed as the sums of the AUCs of a minimum of two peptides. Data was normalized to the total AUC of three peptides from chloroplast GAP-DH (GAP3, Cre01.g010900). Standard deviations were calculated over all replicates (200 µmol photons m-2 s-1: n=4, 500 µmol photons m-2 s-1: n=3).

LHCSR3 phosphosite mutants: Each unlabeled mutant or the rescue strain was mixed with the 15N-labelled WT strain based on equal amounts of chlorophyll. A label swap experiment was performed with labelled mutant/rescue strains and unlabeled WT. Cells were lysed immediately upon mixing in 4% SDS in 100 mM Tris-HCl (pH 8) in the presence of protease and phosphatase inhibitors (1 mM benzamidine, 1 mM PMSF, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate and 10 mM β-glycerophosphate). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. Samples were split into two aliquots, each containing 75 µg of protein, and digested using trypsin and lysC (enzymes to protein ratio 1:50, both enzymes obtained from Promega), respectively, in 0.5 mL ultracentrifugation devices (Amicon Ultra 0.5, 30 kDa cutoff) according to the FASP protocol (Wiśniewski et al., 2009). After overnight digestion at 37°C, a fraction of each peptide sample corresponding to 50 µg of protein was submitted to phosphopeptide enrichment using titanium dioxide tips (NT3TIO, Glygen) according to the manufacturer’s instructions. Residual peptide solutions were dried by vacuum centrifugation and stored at -80°C (‘non-enriched samples’). LC-MS/MS analyses of phosphopeptide enriched samples were performed exclusively by data-dependent acquisition (DDA), since they were required only for the construction of spectral libraries. All LC-MS/MS-based quantifications were carried out using non-enriched peptide samples. High light kinetics (200 µmol photons m-2 s-1 versus 500 µmol photons m-2 s-1): For protein quantitation, a label-free approach was chosen. Cells were harvested by centrifugation for 5 min at 1,500 g. Supernatants were removed and cell pellets were immediately frozen in liquid nitrogen. Cell lysis, protein isolation and determination of protein concentrations were performed as described above. Proteins (2 mg per sample) were on-filter digested (FASP) in 15 mL ultracentrifugation devices (Amicon Ultra 15, 30 kDa cutoff) using trypsin. Peptides were desalted with Sep-PAK tC18 cartridges (Waters) and eluted in 2 mL 50 % acetonitrile, 0.1 % trifluoroacetic acid. A sample volume corresponding to 25 µg of peptides was dried by vacuum centrifugation and stored at -80°C (‘non-enriched samples’).