PSA3, a Protein on the Stromal Face of the Thylakoid Membrane, Promotes Photosystem I Accumulation in Cooperation with the Assembly Factor PYG7

Abstract

PSI is a large protein-pigment complex located in the thylakoid membrane in cyanobacteria, plants, and algae. Although the structure and components of PSI are well characterized, mechanisms that orchestrate its assembly are poorly understood. In this study, we discovered a novel nucleus-encoded protein, Photosystem I Assembly3 (PSA3), that is required for PSI accumulation. PSA3 is conserved among green photosynthetic eukaryotes but is lacking in cyanobacteria. Mutations in the psa3 gene cause the specific loss of PSI in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Ribosome profiling and pulse-labeling analyses showed that chloroplast-encoded PSI subunits are synthesized at normal rates in psa3 mutants, indicating that PSA3 is involved in the biogenesis of PSI at a posttranslational step. PSA3 resides on the stromal face of the thylakoid membrane, where it is found in a complex that is slightly smaller than PSI. Structural predictions suggest that PSA3 binds a basic peptide in a manner that is sensitive to the oxidation state of Cys pairs flanking the predicted peptide binding groove. PSA3 and the previously described PSI biogenesis factor PYG7 interact in yeast two-hybrid and bimolecular fluorescence complementation assays, and they are found in thylakoid membrane complexes of similar size. These and other results indicate that PSA3 cooperates with PYG7 to promote the stable assembly of PSI, and that the PsaC subunit is likely to be the primary target of their action.

Exploring the proteome associated with the mRNA encoding the D1 reaction center protein of Photosystem II in plant chloroplasts

“…Equal aliquots of the membrane fraction were treated with thermolysin or proteinase K, or washed with sodium carbonate or NaBr and centrifuged to recover pellet (P) and supernatant (S) fractions. These samples are the same as those validated previously by analysis of marker proteins (Shen et al , ). Equivalent portions of each fraction were analyzed by immunoblotting.…”

“…Stromal and thylakoid fractions were prepared from purified maize chloroplasts as described previously (Shen et al , ). Thylakoid membranes (162 µg of chlorophyll per immunoprecipitation) were solubilized with 1% n‐dodecyl ß D‐maltoside (Sigma D‐4641) dissolved in 95 µl Co‐IP buffer (20 m m Tris–HCl pH 7.5, 150 m m NaCl, 1 m m EDTA pH 8, 5 m m MgCl 2 , 5 µg/ml aprotinin) by incubation for 15 min at 4°C with gentle mixing.…”

The Arabidopsis pentatricopeptide repeat protein LPE1 and its maize ortholog are required for translation of the chloroplast psbJ RNA

“…In past work, we observed a small decrease in psbA ribosome occupancy in ribosome profiling analyses of various nonphotosynthetic mutants (e.g. Zoschke et al, 2013Zoschke et al, , 2016Shen et al, 2017). Therefore, we considered the possibility that the small reduction in psbA translation in lpe1 mutants is a secondary effect of their PSII defect.…”

Tetratricopeptide repeat protein Pyg7 is essential for photosystem I assembly by interacting with PsaC in Arabidopsis

“…In Arabidopsis , mutation of Pyg7 leads to the abolition of photoautotrophic growth and an almost complete loss of the PSI complex (Stöckel et al ., ). Very recently, PSA3 has been shown to promote PSI accumulation in cooperation with Pyg7 (Shen et al ., ). However, the role of Pyg7 in the PSI complex accumulation remains elusive.…”