Amyloid ␤ protein (A␤), a pathogenic molecule associated with Alzheimer's disease, is produced by ␥-secretase, which cleaves the ␤-carboxyl terminal fragment (␤CTF) of ␤-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that ␤CTF is cleaved first at the membranecytoplasm boundary, producing two long A␤s, A␤ 48 and A␤ 49 , which are processed further by releasing three residues at each step to produce A␤ 42 and A␤ 40 , respectively. To test this hypothesis, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the specific tripeptides that are postulated to be released. Using CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonate)-reconstituted ␥-secretase system, we confirmed that A␤ 49 is converted to A␤ 43/40 by successively releasing two or three tripeptides and that A␤ 48 is converted to A␤ 42/38 by successively releasing two tripeptides or these plus an additional tetrapeptide. Most unexpectedly, LC-MS/MS quantification revealed an induction period, 3-4 min, in the generation of peptides. When extrapolated, each time line for each tripeptide appears to intercept the same point on the x-axis. According to numerical simulation based on the successive reaction kinetics, the induction period exists. These results strongly suggest that A␤ is generated through the stepwise processing of ␤CTF by ␥-secretase.

PsbP, an extrinsic subunit of photosystem II (PSII), is a nuclear-encoded protein that optimizes the water-splitting reaction in vivo. In addition to PsbP, higher plants have two nuclear-encoded genes for PsbP homologs (PsbP-like proteins [PPLs]) that show significant sequence similarity to a cyanobacterial PsbP homolog (cyanoP); however, the function of PPLs in higher plants has not yet been elucidated. In this study, we characterized Arabidopsis (Arabidopsis thaliana) mutants lacking either of two PPLs, PPL1 and PPL2. Phylogenetic analysis suggests that PPL1 would be an ortholog of cyanoP, and PPL2 and PsbP may have a paralogous relationship with PPL1. Analysis on mRNA expression profiles showed that PPL1 expressed under stress conditions and PPL2 coexpressed with the subunits of chloroplast NAD(P)H dehydrogenase (NDH) complex. Consistent with these suggestions, PSII activity in a ppl1 mutant was more sensitive to high-intensity light than wild type, and the recovery of photoinhibited PSII activity was delayed in ppl1 plants. Therefore, PPL1 is required for efficient repair of photodamaged PSII. Furthermore, the stoichiometric level and activity of the chloroplast NDH complex in thylakoids were severely decreased in a ppl2 mutant, demonstrating that PPL2 is a novel thylakoid lumenal factor required for accumulation of the chloroplast NDH complex. These results suggest that during endosymbiosis and subsequent gene transfer to the host nucleus, cyanoP from ancient cyanobacteria evolved into PPL1, PPL2, and PsbP, and each of them has a distinct role in photosynthetic electron transfer in Arabidopsis.

PsbP and PsbQ proteins are extrinsic subunits of photosystem II (PSII) and participate in the normal function of photosynthetic water oxidation. Both proteins exist in a broad range of the oxygenic photosynthetic organisms; however, their physiological roles in vivo have not been well defined in higher plants. In this study, we established and analyzed transgenic tobacco (Nicotiana tabacum) plants in which the levels of PsbP or PsbQ were severely down-regulated by the RNA interference technique. A plant that lacked PsbQ showed no specific phenotype compared to a wild-type plant. This suggests that PsbQ in higher plants is dispensable under the normal growth condition. On the other hand, a plant that lacked PsbP showed prominent phenotypes: drastic retardation of growth, pale-green-colored leaves, and a marked decrease in the quantum yield of PSII evaluated by chlorophyll fluorescence. In PsbP-deficient plant, most PSII core subunits were accumulated in thylakoids, whereas PsbQ, which requires PsbP to bind PSII in vitro, was dramatically decreased. PSII without PsbP was hypersensitive to light and rapidly inactivated when the repair process of the damaged PSII was inhibited by chloramphenicol. Furthermore, thermoluminescence studies showed that the catalytic manganese cluster in PsbP-deficient leaves was markedly unstable and readily disassembled in the dark. The present results demonstrated that PsbP, but not PsbQ, is indispensable for the normal PSII function in higher plants in vivo.

Arabidopsis has three PsbQ-like (PQL) proteins in addition to the PsbQ subunit of the oxygen-evolving complex of PSII. Recent bioinformatic and proteomic studies suggested that the two PQL proteins, PQL1 (At1g14150) and PQL2 (At3g01440), might function in the chloroplast NAD(P)H dehydrogenase (NDH) complex; however, their molecular function has not been characterized. In this study, we examined the function of the chloroplast NDH in the Arabidopsis pql1 and pql2 mutants. Post-illumination increases in Chl fluorescence, which are caused by an NDH-dependent cyclic electron flow, were absent in both mutants, indicating that PQL1 and PQL2 are required for NDH activity. In the thylakoid membranes of wild-type plants, PQL1 and PQL2 were tightly associated with the NDH-PSI supercomplex and protected from protease treatments, while unassembled PQLs were not stably accumulated in mutants lacking known NDH subunits. Subunit stability of the NDH complex was affected differently in the thylakoid membranes of the pql1 and pql2 mutants. These data indicate that PQL1 and PQL2 are novel NDH subunits and differ in their functional roles and in their binding sites in the NDH complex. Furthermore, functional analysis on PQL3 (At2g01918) using the pql3 mutant suggests that PQL3 is also required for NDH activity. Proteins homologous to each PQL protein are found in various plant species, but not in cyanobacteria, algae, mosses or ferns. These results suggest that seed plants that have NDH activity in chloroplasts specifically developed three PQL proteins for the function of the chloroplast NDH complex.