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Seven genes seem to encode for putative ClC chloride channels (AtClC-a to AtClC-g) in Arabidopsis thaliana. Their function and localization is still largely unknown. AtClC-f shares considerable sequence similarity with putative ClC channel proteins from Synechocystis, considered to represent the precursor of chloroplasts. We show by biochemical and mass spectrometry analysis that ClC-f is located in the outer envelope membrane of spinach chloroplasts. Consistent with the plastidial localization of ClC-f, p-chlorophenoxy-acetic acid (CPA) reduces photosynthetic activity and the protein is expressed in etioplasts and chloroplasts but not in root tissue. These findings may represent a step toward the molecular identification of ion channel activities in chloroplast membranes.

Fractionation of photosynthetic membranes from the cyanobacterium Synechocystis 6803 by polyacrylamide gel electrophoresis in the presence of Deriphat-160 allowed the isolation of a number of pigmented bands. Two of them, with molecular masses of 240+/-20 and 110+/-15 kDa respectively, showed peroxidase activity and, by means of polypeptide composition, immunoblotting and N-terminal sequencing, were identified as dimeric and monomeric cytochrome b6/f complexes, containing 1.3+/-0.35 chlorophyll molecules per cytochrome f. Further fractionation of monomeric complexes by mild gel electrophoresis in the presence of sodium dodecyl sulfate indicated that it is the cytochrome b6 polypeptide which provides the actual binding site for the chlorophyll molecule observed in the complex.

Exposure of isolated reaction centres of photosystem II to UV-B radiation generates specific breakdown products of the D2 protein. When the quinone, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone is present a 22 kDa fragment containing the N-terminus of the mature protein is generated. Concomitant with the appearance of the N-terminal fragment, two fragments containing the C-terminus of the D2 protein having apparent molecular masses around 10-12 kDa are observed. It is concluded that the primary cleavage occurs in the hydrophilic loop linking putative transmembrane segments IV and V. No such cleavage was observed when silicomolybdate was used as an electron acceptor, suggesting that this UV-B damage is dependent on binding of the added quinone to the QA site.

The nearest neighbour relationships within the D1/D2/cyt b559 complex (PSIIRC) and the CP47/D1/D2/cyt b559 complex (RC-CP47) were investigated by using different length bifunctional crosslinking agents. The crosslinking products were identified by immunoblotting with polyclonal antibodies and by two-dimensional gel electrophoresis. Seven products (CP47/D2, D1/D2/alpha, D1/D2, D2/alpha, D1/alpha, alpha/alpha, alpha/beta) have been revealed in both complexes. The crosslinking of both complexes does not increase their photostability. The photocrosslinking products (D1/alpha and D2/alpha) appeared under illumination of complexes with light of high intensity.

When isolated photosystem II reaction centers from spinach are exposed to photoinhibitory light in the presence of an electron acceptor, breakdown products of the D2 protein at 28, 25, 23, 18, 9, 5 and 4.5 kDa are detected by immunoblotting with a monospecific anti-D2 polyclonal antibody. In a time-course experiment the 23 and 4.5 kDa fragments show a transient appearance, whilst the others are photoaccumulated. The regions of the D2 protein containing the cleavage sites for the 28 and 18 kDa photoinduced fragments have been identified. Significant degradation of D2 takes place only in the presence of an electron acceptor, and breakdown of the protein is partially prevented by serine-type protease inhibitors.

When isolated reaction centres of photosystem II are subjected to photoinhibitory illumination, a 41 kDa SDS-PAGE band is observed under all experimental conditions. The same band is also found, together with lower molecular weight fragments of the D1 protein, in whole thylakoids and in all PSII sub-particles investigated up to now. In the case of isolated reaction centres the 41 kDa band is represented by a heterodimer of the D1 polypeptide and the alpha-subunit of cytochrome b559. The cross-linkage between D1 and alpha-cyt b559 involves a region on D1 between the N-terminal residue and Arg-225, and is an early event in photo-induced damage to the D1 protein.

Photoinduced degradation of the photosystem II (PSII) reaction center D1 protein was studied in isolated thylakoids and different PSII subparticles. A 16 kDa fragment corresponding to the C-terminus of the protein is detected in thylakoids when they are inactivated at the donor side before illumination. The same D1 fragment is found in different types of PSII preparations at different integration levels characterized by different polypeptide compositions so long as they have an inactivated donor side and an active electron acceptor for the reduced pheophytin. However, when the PSII particle is equal to or smaller than the 43-less PSII core complex, other fragments are observed which are not found in more integrated systems.

When isolated reaction centres of photosystem 2 from pea or wheat are exposed to photoinhibitory illumination in the presence of an electron acceptor, breakdown products of the D1-protein are observed having molecular masses ranging from about 24 to 10 kDa. By using antibodies raised to the C-terminal or N-terminal portions of D1 it was shown that the major breakdown fragment of 24 kDa was derived from the C-terminus. This conclusion was supported by phosphorylation studies and from the digestion pattern obtained by lysine specific endoprotease-induced proteolysis. The complementary N-terminal breakdown fragment was found to have an apparent molecular mass of 10 kDa. The implications of these data are discussed in terms of the possible relationship between the 24 kDa C-terminal fragment and the 23.5 kDa breakdown fragment detected in vivo by Greenberg et al. [1987, EMBO J. 6, 2865-2869] and it is suggested, based on limited proteolysis using papain, that the latter may not be derived from the N-terminus as previously thought but also originates from the C-terminus.