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O Vallon

Publications and source records attributed to O Vallon.

6 recordsLinked to original sources
PubMed1992

Purification and membrane topology of PSI-D and PSI-E, two subunits of the photosystem I reaction center.

Structural studies have been conducted on polypeptides PSI-D and PSI-E, which are extrinsic but firmly bound to the photosystem I reaction center. These subunits are predicted to be involved in the correct interaction with soluble electron acceptor(s), like ferredoxin. We designed an original method to extract both polypeptides directly from thylakoid membranes and to purify them: a stepwise extraction with NaSCN followed by size fractionation and reverse-phase HPLC. Investigation of the in situ topology of PSI-D and PSI-E was undertaken using monoclonal antibody binding, controlled proteolysis, peptide sequencing and electron microscopy. The precise identification of numerous proteolytic sites indicates that the entire N-terminal regions of PSI-E (up to Glu15) and PSI-D (up to Lys15) are exposed to the medium. Partial mapping of the exposed epitopes was possible using purified fragments of each polypeptide. In the case of PSI-E, this mapping confirmed the accessibility of the N-terminal part, and suggested the need for another exposed sequence, probably located after Met39 in the second half of the protein. For PSI-D, this mapping revealed that the sequence between Met74 and Met140, including the most basic amino acid clusters, is also partly accessible. These experiments provide the first detailed informations, although still partial, on the topology of these polypeptides. They give a preliminary basis for hypotheses concerning the sites of interaction with the soluble counterparts.

Amino Acid Sequence↗
PubMed1991

Lateral redistribution of cytochrome b6/f complexes along thylakoid membranes upon state transitions.

The cytochrome b6/f complex operates in photosynthetic electron transfer either in linear electron flow from photosystem II to photosystem I or in cyclic flow around photosystem I. Using membrane fractionation and immunocytochemistry, we show a change in lateral distribution of cytochrome b6/f complexes along the thylakoid membranes during state transitions. This change is seen in maize as well as in the green algae Chlamydomonas reinhardtii. When either of the two organisms were adapted to state II in vivo, the proportion of cytochrome b6/f complexes found in the photosystem I-enriched stroma lamellae regions was significantly larger than after adaptation to state I. A similar observation was made upon state I to state II transitions done in vitro by illuminating, in the presence of ATP, broken maize chloroplasts prepared from dark-adapted leaves. This reorganization of the electron-transfer chain is concurrent with the change in light-energy distribution between the two photosystems, which requires lateral displacement of light-harvesting complex II. That the changes in lateral distribution of both cytochrome b6/f and light-harvesting II complexes seen upon state transition in vitro similarly required addition of exogenous ATP, suggests that the change in cytochrome b6/f organization also depends on kinase activity. The increased concentration of cytochrome b6/f complexes in the vicinity of photosystem I in state II is discussed in terms of an increase in cyclic electron flow, thus favoring ATP production. Because transition to state II can be triggered in vivo by ATP depletion, we conclude that state transitions should be regarded not only as a light-adaptation mechanism but also as a rerouting of photosynthetic electron flow, enabling photosynthetic organisms to adapt to changes in the cell demand for ATP.

Chlamydomonas↗
PubMed1989

Visualization of antibody binding to the photosynthetic membrane: the transmembrane orientation of cytochrome b-559.

We have used immuno-gold labeling and electron microscopy to study the topography of thylakoid membrane polypeptides. Thylakoid vesicles formed by passage through a French press were adsorbed onto a plastic film supported by an electron microscope grid and processed for single or double immuno-gold labeling. After shadowing with platinum, the inside-out and right-side-out vesicles were identified by their distinctive morphologies. Right-side-out vesicles were labeled by a monoclonal antibody recognizing an epitope located in the trypsin-cleaved, N-terminal portion of the LHC II apoprotein, and by an antibody to CF1. A monoclonal antibody to the alpha-subunit of cytochrome b-559 reacted with a synthetic tridecapeptide corresponding to the C-terminal portion of the polypeptide. Both this antibody and a polyclonal antibody to the synthetic peptide labeled inside-out vesicles exclusively, indicating that the polypeptide C-terminus was exposed on the lumenal (exoplasmic) surface of the membrane.

Amino Acid Sequence↗
PubMed1988

Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product.

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

MP26 in the bovine lens: a post-embedding immunocytochemical study.

Gold immunolabeling of bovine lens tissue embedded in Lowicryl K4M, using a polyclonal antibody specific for a major component of lens fiber plasma membrane of 26 K molecular weight, shows that this constituent is absent from the epithelial cell plasma membrane and associated only with the junctional and non-junctional domains of the lens fiber plasma membrane.

Animals↗