Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12-15 core and 4 -9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photooxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.

of the reaction center (RC) subunits, PSAA/PSAB, as an RC, the redox components involved in the primary photochemical reactions, such as the primary electron donor, P700, and the intermediate electron acceptors, A 0 , A 1 , and F X , as well as core antenna pigments and several peripheral subunits. One of the peripheral subunits, PSAC, binds the secondary electron acceptors, F A and F B , and, together with PSAD and PSAE subunits, forms a stromal ridge providing a ferredoxin docking site. PSAF and PSAN subunits are involved in plastocyanin (or cytochrome c) docking. In vascular plants, three hydrophobic subunits, PSAI, PSAL, and PSAH, are located on the opposite side of PSAF with respect to the PSI RC. The PSAO subunit, which has two putative transmembrane helices, also is located close to PSAL subunit and, together with PSAI, PSAL, and PSAH subunits, is proposed to be involved in the interaction with light-harvesting chlorophyll a/b complex II (LHCII; Jensen et al., 2004). In addition, the vascular plant PSI core binds the outer antenna, light-harvesting chlorophyll a/b complex I (LHCI), to form a PSI-LHCI supercomplex (Boekema et al., 2001; Ben-Shem et al., 2003). The association of LHCIs significantly increases light-harvesting capacity. The

The chloroplast-encoded Ycf4 plays an essential role in PSI complex assembly in the green alga Chlamydomonas reinhardtii. To gain insight into how Ycf4 functions, we generated several mutants in which residues R120, E179 and/or E181, which are conserved among oxygenic photosynthetic organisms, were changed to A or Q. Although the single mutants R120A and R120Q accumulated 80% less Ycf4 than the wild type, they assembled a functional PSI complex and grew photosynthetically like the wild type. Thus we inferred that under laboratory growth conditions, wild-type cells accumulate a superfluous amount of Ycf4. Single mutants E179A, E179Q and E181Q assembled a functional PSI complex like the wild type, whereas the single mutant E181A and double mutant E179/181A accumulated a functional PSI complex to significantly reduced levels. Double mutant E179/181Q, in contrast, accumulated Ycf4 at the wild-type level but did not assemble any mature PSI complex, suggesting that the two glutamic acid residues play crucial roles in the functionality of Ycf4. Interestingly, sucrose density gradient centrifugation of the thylakoid extracts separated a small amount of PSI subcomplex. The apparent size of the subcomplex (150-170 kDa), its composition and pulse-chase protein labeling indicate that it was an unstable subcomplex consisting of a PsaA-PsaB heterodimer. We inferred that the subcomplex was a PSI complex assembly intermediate that was detected because subsequent assembly steps were blocked by the E179/181Q mutation. We concluded that Ycf4 is involved in early processes of PSI complex assembly.

ObjectiveCardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca2+ indicator GmNL(Ca2+), and its application in a customized microscope for high-throughput drug screening.ResultsGmNL(Ca2+) gives a 140% signal change with Ca2+, and can image drug-induced changes of Ca2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca2+) with this adaptation, we could image spontaneous Ca2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3421-7) contains supplementary material, which is available to authorized users.