Christer Jansson

Reconstitution of photosynthetic water splitting in inside-out thylakoid vesicles and identification of a participating polypeptide☆

Inside-out spinach thylakoid vesicles have been used for inhibition and reconstitution of photosynthetic water splitting. Washing inside-out vesicles with a buffer containing 250 mM NaCl inhibited 75% of the Photosystem II reduction of phenyl-p-benzoquinone. In contrast, the same treatment of right-side-out vesicles gave quite a small inhibition. The site of inhibition was shown by fluorescence induction to be located at the water-splitting side of P-680. The salt-induced inhibition was accompanied by the release of two polypeptides (23 and 16 kDa) from the inner thylakoid surface. Readdition of the salt-washed supernatant or a crude chloroplast extract to the washed inside-out vesicles at low ionic strength gave a 2.7-fold stimulation of the water-splitting activity, thereby restoring about 60% of the activity lost by salt washing. This stimulation was abolished by proteolysis of the reconstituting fractions. Restoration of the water-splitting reaction was accompanied by rebinding of the 23 and 16 kDa polypeptides to the inside-out vesicles. After polypeptide purification by ion-exchange chromatography it was shown that the 23kDa polypeptide was responsible for the restorative effect. These observations provide strong evidence that the 23 kDa polypeptide, electrostatically bound to the inner thylakoid surface, is involved in the photosynthetic water-splitting reaction.

Localization of a 34 000 and a 23 000 Mr polypeptide to the lumenal side of the thylakoid membrane

Several attempts have been made to localize different components across the thylakoid membrane. The methods used include treatments with membrane-impermeable agents such as antibodies [ 11, chemical modifiers [ 2,3] and proteolytic enzymes [3]. This has given valuable information about the outer surface of the thylakoid membrane and has revealed that components like ferredoxin, ferredoxin-NADP-reductase, the coupling factor and the light-harvesting complex ‘are all accessible from the outside. Information about the inner thylakoid surface is poor however and the localization of components on this side has often been deduced from indirect measurements. A direct study of the inner side has become possible after the isolation of insideout thylakoids [4-91. In [IO] trypsination of insideout thylakoid vesicles was used to demonstrate that at least a part of the water-splitting system is exposed to the lumen. Here, inside-out thylakoid vesicles have been treated with trypsin or alkaline-Tris. The subsequent changes in the polypeptide pattern were followed by SDS-PAGE.

Differential expression of the psbA genes in the cyanobacterium Synechocystis 6803.

The 5′ region and transcription initiation sites of the psbA-2 and psbA-3 genes of Synechocystis 6803 were determined. The otherwise highly homologous genes were shown to diverge significantly in the 5′ noncoding regions. The transcription start site for the psbA-2 gene was mapped to position — 49 upstream of the coding region and for the psbA-3 gene to position — 88, i.e. 38 by upstream of the psbA-2 transcription start point. Both genes exhibit promoter elements, which conform in sequence and position to Escherichia coli consensus motifs. The two genes share identical — 35 sequences but differ in their — 10 sequences. Primer extension analysis demonstrated that the psbA-2 and psbA-3 genes are differentially expressed, with > 90 % of the total psbA transcripts being produced by the psbA-2 gene and the rest by the psbA-3 gene. Inactivation of the psbA-2 gene resulted in an eightfold up-regulation of the psbA-3 gene. The strikingly higher stability of the psbA transcripts in darkness compared to light, and the accumulation of a specific decay intermediate under dark conditions was reported previously. We show here that this dark-stability applies to both the psbA-2 and psbA-3 transcripts. The psbA-3 transcript did not appear to produce the processed intermediate, arguing for the involvement of the 5′ non-coding region as a determinant in psbA transcript degradation.

Modification of the chloride requirement for photosynthetic O2 evolution: The role of the 23 kDa polypeptide

In thylakoid preparations from spinach and the halophyte Avicennia marina a correlation is observed between functional O2 evolution at low chloride concentrations and the presence of the 23 kDa protein. Addition of spinach 23 kDa protein to polypeptide‐depleted halophyte inside‐out thylakoid vesicles lowers their chloride requirement for optimal O2 evolution activity from 250 to 5 mM. It is suggested that the specific role for the 23 kDa protein is to increase the affinity of the water oxidation site for chloride.

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

SummarySpinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector λgt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CFo-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A+-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.

Immunological studies on the organization of proteins in photosynthetic oxygen evolution

Abstract Studies on inside-out thylakoid vesicles and several Photosystem-II particles have suggested the involvement of three proteins of 33, 23 and 16 kDa in photosynthetic oxygen evolution. In this study, monospecific antibodies were raised against the purified 33, 23 and 16 kDa proteins. The antibodies were used to investigate the organization and function of these proteins in the oxygen-evolving complex. Quantification of the 33, 23 and 16 kDa proteins by rocket immunoelectrophoresis revealed one copy of each polypeptide per some 200 chlorophylls in unfractionated thylakoids. Isolated inside-out thylakoids, derived from the grana partitions, showed 6–8 times more of the 33, 23 and 16 kDa proteins, on a chlorophyll basis, compared to the stroma lamellae vesicles. Agglutination studies revealed that the proteins are exposed on the lumenal side of the thylakoid membrane. An obligatory role for the 23 kDa protein in the photosynthetic water oxidation is suggested from the close correlation obtained between the inhibition of oxygen evolution and the release of this protein as caused by washing the inside-out thylakoids with increasing concentrations of NaCl. For the 16 kDa protein no such correlation was obtained. Rebinding experiments, using both salt-washed and alkaline Tris-washed inside-out thylakoids revealed that the 33 kDa protein was required for the binding of the 23 kDa protein, which in turn enhanced the binding of the 16 kDa protein. It is concluded that the three proteins are closely organized as a complex at the inner thylakoid surface in association with membrane spanning proteins of Photosystem II.

Enzymic degradation of polymers: II. Viscometric determination of cellulase activity in absolute terms

Abstract The method of determining the activity of polymer degrading enzymes formulated in a previous paper has been applied to a purified fungal cellulase with a CM-cellulose sample as a substrate. It has been possible to determine cellulase activity in absolute terms, i.e. the number of β-1,4-glucosidic bonds broken per unit time. The molecular activity of the cellulase has also been calculated.

Intracellular coding sites of polypeptides associated with photosynthetic oxygen evolution of photosystem II

SummaryThree hydrophilic polypeptides of approximately 34, 23, and 16 kd located on the inner thylakoid surface are associated with the water-splitting activity of photosystem II. Stable transcripts for the three proteins were found only in cytosolic (polyadenylated) RNA, suggesting that they are encoded in nuclear genes. The immunologically reacting products synthesized in a rabbit reticulocyte cell-free translation system are larger in size than the authentic mature proteins by about 6–10 kd. These larger precursors are imported post-translationally into isolated, intact chloroplasts, and are processed to their mature forms during or after translocation. The imported proteins can be extracted from thylakoids by procedures used to isolate the three native proteins of the water-splitting complex, suggesting that they have assembled properly into their final destination, the inner thylakoid surface.

Ultrastructural and biochemical characterization of a Synechocystis 6803 mutant with inactivated psbA genes

A constructed Synechocystis 6803 mutant with a deletion of the three psbA genes was subjected to ultrastructural and biochemical characterization. This D1-depleted mutant also lacks the D2 protein and the chlorophyll a-binding protein CP-47. A general ultrastructural comparison between the wild type and the mutant did not reveal any major changes in cell appearance. We found by freeze-fracture analysis that approximately 60% of the endoplasmic face particles found in the wild-type thylakoids were missing in the mutant. A corresponding increase in protoplasmic face particles in the mutant thylakoids may represent a subcomplex of those photosystem II (PS II) polypeptides which accumulate in the absence of the D1 protein. Correlation of the PS I:PS II ratio with freeze-fracture data indicates that there is only one reaction center in each PS II freeze-fracture particle. Fluorescence measurements show that the CP-43 polypeptide in the mutant binds chlorophyll and that it may be connected to the phycobilisomes. Excitation energy can be transferred from the phycobilisomes to photosystem I in the absence of the photosystem II reaction center heterodimer and CP-47. This suggests that exciton transfer to photosystem I is mediated either directly by a terminal phycobilisome transmitter or via CP-43.

Reconstitution of oxygen evolution in high salt washed photosystem II particles

Photosystem II thylakoid particles possessing high rates of oxygen evolution, were shown to have a very simple polypeptide composition. Upon washing of these particles with 250 mM NaCl the oxygen evolution was inhibited up to 80% concomitant with a release of two polypeptides of 23 and 16 kDa. Readdition of the pure 23 kDa protein to the depleted thylakoids under low ionic strength reconstituted more than half of the lost activity. No stimulation was obtained with the 16 kDa protein alone or in combination with glycerol. The results give further strong evidence that the 23 kDa protein is an essential component in the oxygen evolving complex. The possible involvement of other proteins in this complex is discussed in light of the demonstrated simple polypeptide pattern of the photosystem II particles.