Yasuhiro Kashino

Proteomic analysis of a highly active photosystem II preparation from the cyanobacterium Synechocystis sp. PCC 6803 reveals the presence of novel polypeptides.

A highly active oxygen-evolving photosystem II (PSII) complex was purified from the HT-3 strain of the widely used cyanobacterium Synechocystis sp. PCC 6803, in which the CP47 polypeptide has been genetically engineered to contain a polyhistidine tag at its carboxyl terminus [Bricker, T. M., Morvant, J., Masri, N., Sutton, H. M., and Frankel, L. K. (1998) Biochim. Biophys. Acta 1409, 50-57]. These purified PSII centers had four manganese atoms, one calcium atom, and two cytochrome b(559) hemes each. Optical absorption and fluorescence emission spectroscopy as well as western immunoblot analysis demonstrated that the purified PSII preparation was devoid of any contamination with photosystem I and phycobiliproteins. A comprehensive proteomic analysis using a system designed to enhance resolution of low-molecular-weight polypeptides, followed by MALDI mass spectrometry and N-terminal amino acid sequencing, identified 31 distinct polypeptides in this PSII preparation. We propose a new nomenclature for the polypeptide components of PSII identified after PsbZ, which proceeds sequentially from Psb27. During this study, the polypeptides PsbJ, PsbM, PsbX, PsbY, PsbZ, Psb27, and Psb28 proteins were detected for the first time in a purified PSII complex from Synechocystis 6803. Five novel polypeptides were also identified in this preparation. They included the Sll1638 protein, which shares significant sequence similarity to PsbQ, a peripheral protein of PSII that was previously thought to be present only in chloroplasts. This work describes newly identified proteins in a highly purified cyanobacterial PSII preparation that is being widely used to investigate the structure, function, and biogenesis of this photosystem.

Increases in the fluorescence Fo level and reversible inhibition of Photosystem II reaction center by high-temperature treatments in higher plants

Effects of high temperatures on the fluorescence Fm (maximum fluorescence) and Fo (dark level fluorescence) levels were studied and compared with those of the photochemical reactions of PS II. These comparisons were performed during and after the high temperature treatments. The following results were obtained; (1) increases in the Fo level at high temperatures were partly reversible, (2) the Fm level in the presence of dithionite in spinach chloroplasts decreased at high temperatures and also showed a partial reversibility, (3) photoreductions of pheophytin a and Qa were reversibly inhibited at high temperatures parallel to the decrease in the difference between the Fm and Fo levels, and (4) the decrease in the fluorescence Fm level seemed to be related to denaturation of chlorophyll-proteins. All the data suggested that, as well as the separation of light-harvesting chlorophyll a/t b protein complexes of PS II from the PS II core complexes, partly reversible inactivation of the PS II reaction center at high temperatures is the cause of the increase in the Fo level.

Effects of high temperatures on the photosynthetic systems in spinach: Oxygen-evolving activities, fluorescence characteristics and the denaturation process

Activities of oxygen evolution, fluorescence Fv (a variable part of chlorophyll fluorescence) values, and amounts of the 33 kDa protein remaining bound to the thylakoids in intact spinach chloroplasts were measured during and after high-temperature treatment. The following results were obtained. (1) Both the Fv value and the flash-induced oxygen evolution measured by an oxygen electrode were decreased at high temperatures, but they showed partial recovery when the samples were cooled down and incubated at 25°C for 5 min after high-temperature treatment. (2) Oxygen evolution was more sensitive to high temperatures than the Fv value, and the decrease in the Fv/Fm ratio at high temperatures rather corresponded to that in the oxygen evolution measured at 25°C after high-temperature treatment. (3) Photoinactivation of PS II was very rapid at high temperatures, and this seems to be a cause of the difference between the Fv values and the oxygen-evolving activities at high temperatures. (4) At around 40°C, the manganese-stabilizing 33 kDa protein of PS II was supposed to be released from the PS II core complexes during heat treatment and to rebind to the complexes when the samples were cooled down to 25°C. (5) At higher temperatures, the charge separation reaction of PS II was inactivated, and the PS II complexes became less fluorescent, which was recovered partially at 25°C. (6) Increases in the Fv value due to a large decrease in the electron flow from QA to QB became prominent after high-temperature treatment at around 50°C. This was the main cause of the discrepancy between the Fv values and the oxygen-evolving activities measured at 25°C. Relationship between the process of heat inactivation of PS II reaction center complexes and the fluorescence levels is discussed.

Reduction of QA in the dark: Another cause of fluorescence Fo increases by high temperatures in higher plants

Increases in the chlorophyll fluorescence Fo (dark level fluorescence) during heat treatments were studied in various higher plants. Besides the dissociation of light-harvesting chlorophyll a/b protein complexes from the reaction center complex of PS II and inactivation of PS II, dark reduction of QA via plastoquinone (PQ) seemed to be related to the Fo increase at high temperatures. In potato leaves or green tobacco cultured cells, a part of the Fo increase was quenched by light, reflecting light-induced oxidation of QA- which had been reduced in the dark at high temperatures. Appearance of the Fo increase due to QA reduction depended on the plant species, and the mechanisms for this are proposed. The reductants seemed to be already present and formed by very brief illumination of the leaves at high temperatures. A ndhB-less mutant of tobacco showed that complex I type NAD(P)H dehydrogenase is not involved in the heat-induced reduction of QA. Quite strong inhibition of the QA reduction by diphenyleneiodonium suggests that a flavoenzyme is one of the electron mediator to PQ from the reductant in the stroma. Reversibility of the heat-induced QA reduction suggests that an enzyme(s) involved is activated at high temperatures and mostly returns to an inactive form at room temperature (25 °C).

An improved sodium dodecyl sulfate-polyacrylamide gel electrophoresis system for the analysis of membrane protein complexes

Membrane protein complexes such as the reaction center complexes of oxygenic photosynthesis or the complex I of mitochondira are composed of many subunit polypeptides. To analyze their polypeptide compositions by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), a wide range of molecular sizes has to be resolved, especially in the low molecular mass range. We have improved the traditional Tris/HCl buffer systems adopting a Tris/2‐(N‐morpholino)ethanesulfonic acid (MES) buffer system containing 6 M urea. This gel system was used with an 18–24% acrylamide gradient for the separation of polypeptides with molecular masses from below 5 kDa to over 100 kDa. This buffer system can also be applied to the usual uniform concentration of acrylamide gel and also to minislab gels.

The PsbQ protein defines cyanobacterial Photosystem II complexes with highest activity and stability

Light-induced conversion of water to molecular oxygen by Photosystem II (PSII) is one of the most important enzymatic reactions in the biosphere. PSII is a multisubunit membrane protein complex with numerous associated cofactors, but it continually undergoes assembly and disassembly due to frequent light-mediated damage as a result of its normal function. Thus, at any instant, there is heterogeneity in the subunit compositions of PSII complexes within the cell. In particular, cyanobacterial PSII complexes have five associated extrinsic proteins, PsbO, PsbP, PsbQ, PsbU, and PsbV. However, little is known about the interactions of the more recently identified PsbQ protein with other components in cyanobacterial PSII. Here we show that PSII complexes can be isolated from the cyanobacterium Synechocystis sp. PCC 6803 on the basis of the presence of a polyhistidine-tagged PsbQ protein. Purification of PSII complexes using a tagged extrinsic protein has not been previously described, and this work conclusively demonstrates that PsbQ is present in combination with the PsbO, PsbU, and PsbV proteins in cyanobacterial PSII. Moreover, PsbQ-associated PSII complexes have higher activity and stability relative to those isolated using histidine-tagged CP47, an integral membrane protein. Therefore, we conclude that the presence of PsbQ defines the fully assembled and optimally active form of the enzyme.

Separation methods in the analysis of protein membrane complexes.

The separation of membrane protein complexes can be divided into two categories. One category, which is operated on a relatively large scale, aims to purify the membrane protein complex from membrane fractions while retaining its native form, mainly to characterize its nature. The other category aims to analyze the constituents of the membrane protein complex, usually on a small scale. Both of these face the difficulty of isolating the membrane protein complex without interference originating from the hydrophobic nature of membrane proteins or from the close association with membrane lipids. To overcome this difficulty, many methods have been employed. Crystallized membrane protein complexes are the most successful example of the former category. In these purification methods, special efforts are made in the steps prior to the column chromatography to enrich the target membrane protein complexes. Although there are specific aspects for each complex, the most popular method for isolating these membrane protein complexes is anion-exchange column chromatography, especially using weak anion-exchange columns. Another remarkable trend is metal affinity column chromatography, which purifies the membrane protein complex as an intact complex in one step. Such protein complexes contain subunit proteins which are genetically engineered so as to include multiple-histidine tags at carboxyl- or amino-termini. The key to these successes for multi-subunit complex isolation is the idea of keeping the expression at its physiological level, rather than overexpression. On the other hand, affinity purification using the Fv fragment, in which a Strep tag is genetically introduced, is ideal because this method does not introduce any change to the target protein. These purification methods supported by affinity interaction can be applied to minor membrane protein complexes in the membrane system. Isoelectric focusing (IEF) and blue native (BN) electrophoresis have also been employed to prepare membrane protein complexes. Generally, a combination of two or more chromatographic and/or electrophoretic methods is conducted to separate membrane protein complexes. IEF or BN electrophoresis followed by 2nd dimension electrophoresis serve as useful tools for analytical demand. However, some problems still exist in the 2D electrophoresis using IEF. To resolve such problems, many attempts have been made, e.g. introduction of new chaotropes, surfactants, reductants or supporting matrices. This review will focus in particular on two topics: the preparative methods that achieved purification of membrane protein complexes in the native (intact) form, and the analytical methods oriented to resolve the membrane proteins. The characteristics of these purification and analytical methods will be discussed along with plausible future developments taking into account the nature of membrane protein complexes.

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis

Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP-PS I super complex (approximately 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A1 instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.

Highly Efficient Transformation of the Diatom Phaeodactylum tricornutum by Multi-Pulse Electroporation

A highly efficient nuclear transformation method was established for the pennate diatom Phaeodactylum tricornutum using an electroporation system that drives multi-sequence pulses to introduce foreign DNAs into the cells. By optimizing pulse conditions, the diatom cells can be transformed without removing rigid silica-based cell walls, and high transformation efficiency (about 4,500 per 10(8) cells) is achieved.

Responses to desiccation stress in lichens are different from those in their photobionts.

In order to clarify the role of symbiotic association in desiccation tolerance of photosynthetic partners in lichens, responses to air-drying and hypertonic treatments in a green-algal lichen (a chlorolichen, Ramalina yasudae Räsänen) and its green algal photobiont (freshly released and cultured Trebouxia sp.) were studied. Responses to dehydration in the isolated Trebouxia sp. were different from those in the lichen, R. yasudae, i.e. (i) the PSII reaction was totally inhibited in R. yasudae when photosynthesis was completely inhibited by desiccation, but it remained partially active in isolated Trebouxia sp; (ii) dehydration-induced quenching of PSII fluorescence was less in the isolated Trebouxia sp. compared with that in R. yasudae, suggesting that a substance(s) or a mechanism(s) to dissipate absorbed light energy to heat was lost by the isolation of the photobiont; and (iii) the air-dried isolated Trebouxia sp. showed a higher sensitivity to photoinhibition than R. yasudae. These results support the idea that association of the photobionts with the mycobionts increases tolerance to photoinhibition under drying conditions.