Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstructed lateral migration of the photosystem II reaction center protein D1 and of processing protease FtsH between the stacked and unstacked membrane domains, suppressing turnover of damaged D1 in the leaves exposed to high light. These findings show that the high level of photosystem II phosphorylation in plants is required for adjustment of macroscopic folding of large photosynthetic membranes modulating lateral mobility of membrane proteins and sustained photosynthetic activity.The use of captured sunlight energy to split water and drive oxygenic photosynthesis by photosystem II (PSII) (Barber, 2006) inevitably generates reactive oxygen species and causes oxidative damage to the PSII protein pigment complex. The lightinduced damage to PSII, in particular to the D1 reaction center protein, requires PSII repair to sustain its photosynthetic function (Takahashi and Murata, 2008). Impairment and degradation of D1 increase with rising light intensities, and this protein has the fastest turnover rate among the photosynthetic proteins of plants, algae, and cyanobacteria (Yokthongwattana and Melis, 2006). However, in plants, the PSII is segregated in highly stacked membrane layers of very large thylakoid membranes (Andersson and Anderson, 1980;Kirchhoff et al., 2008), which are densely folded to fit inside chloroplasts (Mullineaux, 2005;Shimoni et al., 2005). As a consequence, the PSII repair cycle in plants is slower than in cyanobacteria (Yokthongwattana and Melis, 2006), and it includes migration of the PSII complex from the stacked membrane domains (grana) to the unstacked membranes (stroma lamellae), where proteolysis and insertion of a newly synthesized D1 protein occurs (Baena-Gonzalez and Aro, 2002;Yokthongwattana and Melis, 2006). High light also causes quantitative phosphorylation of the membrane surface-exposed regions of D1, D2, CP43, and PsbH proteins of PSII in plants (Rintamä ki et al., 1997;Vener et al., 2001), but the function of this phosphorylation is largely unknown and reports on its importance for the D1 protein turnover ...

State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b 6 f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light–induced reduction of this bond may occur through a transthylakoid thiol–reducing pathway driven by the ferredoxin-thioredoxin system which is also required for cytochrome b 6 f assembly and heme biogenesis.

Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in shortand long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8, indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL1-A is absent in both monocotyledonous plants and algae. In dicots, spectroscopic measurements with Arabidopsis WT, stn7, stn8, and stn7/stn8 double-mutant plants indicate a STN8-mediated slowing down of the transition from cyclic to linear electron flow at the onset of illumination. This finding suggests a possible link between protein phosphorylation by STN8 and fine-tuning of cyclic electron flow during this critical step of photosynthesis, when the carbon assimilation is not commensurate to the electron flow capacity of the chloroplast.phosphoproteomics | Arabidopsis thaliana I n the field of chloroplast biogenesis, interest in protein phosphorylation historically focused on photosynthesis-related proteins, with the initial discovery of thylakoid membrane protein phosphorylation dating back to the late 1970s (1-4). Almost a decade later, AtpB, RNA-binding proteins, and transcription factors were recognized as phosphoproteins in thylakoid membranes and stroma fractions (5-7). Because of recent large-scale functional genomics and phosphoproteomics approaches, ∼200 chloroplast phosphoproteins are known today, and several kinases have been identified that are most likely involved in their phosphorylation (8). However, the exact kinase/substrate relationships are not known for most of the proteins, and efforts are underway to identify in vivo substrates of known kinases. Phosphoproteomics data suggest that chloroplast functions are regulated by a highly complex phosphoprotein network in which one kinase phosphorylates several substrates and one substrate is probably phosphorylated by several kinases at different sites (9, 10).Although we currently do not understand all nodes in the chloroplast phosphoprotein network, candidate proteins and experimental tools are available to address the above questions. Two of the best-characterized chloroplast kinases are STN7 and STN8. STN7 is the ortholog of the Stt7 kinase from Chlamydomonas reinhardtii that was identified in screens for strains with a defect in state transitions (11). This process ba...

Photosynthetic organisms are subjected to frequent changes in light quality and quantity and need to respond accordingly. These acclimatory processes are mediated to a large extent through thylakoid protein phosphorylation. Recently, two major thylakoid protein kinases have been identified and characterized. The Stt7/STN7 kinase is mainly involved in the phosphorylation of the LHCII antenna proteins and is required for state transitions. It is firmly associated with the cytochrome b 6 f complex, and its activity is regulated by the redox state of the plastoquinone pool. The other kinase, Stl1/STN8, is responsible for the phosphorylation of the PSII core proteins. Using a reverse genetics approach, we have recently identified the chloroplast PPH1/TAP38 and PBPC protein phosphatases, which counteract the activity of STN7 and STN8 kinases, respectively. They belong to the PP2C-type phosphatase family and are conserved in land plants and algae. The picture that emerges from these studies is that of a complex regulatory network of chloroplast protein kinases and phosphatases that is involved in light acclimation, in maintenance of the plastoquinone redox poise under fluctuating light and in the adjustment to metabolic needs.

The chloroplast serine-threonine protein kinase STN7 of Arabidopsis (Arabidopsis thaliana) is required for the phosphorylation of the light-harvesting system of photosystem II and for state transitions, a process that allows the photosynthetic machinery to balance the light excitation energy between photosystem II and photosystem I and thereby to optimize the photosynthetic yield. Because the STN7 protein kinase of Arabidopsis is known to be phosphorylated at four serine-threonine residues, we have changed these residues by site-directed mutagenesis to alanine (STN7-4A) or aspartic acid (STN7-4D) to assess the role of these phosphorylation events. The corresponding mutants were still able to phosphorylate the light-harvesting system of photosystem II and to perform state transitions. Moreover, we noticed a marked decrease in the level of the STN7 kinase in the wild-type strain under prolonged state 1 conditions that no longer occurs in the STN7-4D mutant. The results suggest a possible role of phosphorylation of the STN7 kinase in regulating its turnover.

Microsatellite instability (MSI) is a molecular signature of mismatch repair deficiency (dMMR), a predictive marker of immune checkpoint inhibitor therapy response. Despite its recognized pan-cancer value, most methods only support detection of this signature in colorectal cancer. In addition to the tissue-specific differences that impact the sensitivity of MSI detection in other tissues, the performance of most methods is also affected by patient ethnicity, tumor content, and other sample-specific properties. These limitations are particularly important when only tumor samples are available and restrict the performance and adoption of MSI testing. Here we introduce MSIdetect, a novel solution for NGS-based MSI detection. MSIdetect models the impact of indel burden and tumor content on read coverage at a set of homopolymer regions that we found are minimally impacted by sample-specific factors. We validated MSIdetect in 139 Formalin-Fixed Paraffin-Embedded (FFPE) clinical samples from colorectal and endometrial cancer as well as other more challenging tumor types, such as glioma or sebaceous adenoma or carcinoma. Based on analysis of these samples, MSIdetect displays 100% specificity and 96.3% sensitivity. Limit of detection analysis supports that MSIdetect is sensitive even in samples with relatively low tumor content and limited microsatellite instability. Finally, the results obtained using MSIdetect in tumor-only data correlate well (R=0.988) with what is obtained using tumor-normal matched pairs, demonstrating that the solution addresses the challenges posed by MSI detection from tumor-only data. The accuracy of MSI detection by MSIdetect in different cancer types coupled with the flexibility afforded by NGS-based testing will support the adoption of MSI testing in the clinical setting and increase the number of patients identified that are likely to benefit from immune checkpoint inhibitor therapy.

State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b 6 f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light-induced reduction of this bond may occur through a transthylakoid thiol-reducing pathway driven by the ferredoxinthioredoxin system which is also required for cytochrome b 6 f assembly and heme biogenesis.