Evidence is given for the existence of an electron transport pathway to oxygen in the thylakoid membranes of chloroplasts (chlororespiration). Plastoquinone is shown to be a redox carrier common to both photosynthetic and chlororespiratory pathways. It is shown that, in dark-adapted chloroplasts, an electrochemical gradient is built up across the thylakoid membrane by transfer of electrons through the chlororespiratory chain as well as by reverse functioning of the chloroplast ATPases. It is proposed that these mechanisms ensure recycling of the ATP and NAD(P)H generated by the glycolytic pathway converting starch into triose phosphates. Chlororespiration is thus an 02-uptake process distinct from photorespiration and the Mehler reaction. The evolutionary significance of chlororespiration is discussed.Respiration is a process common to living cells in which reduced organic substrates are oxidized at the expense of molecular oxygen. The free energy recovered in this process is partly converted into heat and partly recovered by the coupled phosphorylation of ADP into ATP. The hydrolysis of ATP releases free energy available for cell functions. Conversely, photosynthesis generates strong reductants at the expense of the energy of light. This formation of strong reductants corresponds to the main energy-conversion process of photosynthesis. A minor part of the light energy is however used to phosphorylate ADP into ATP. The photosynthetic and respiratory apparatuses show strong similarities: (i) both implicate membrane-bound electron transfer chains, (ii) several membrane components are present in both systems (cytochromes, iron-sulfur proteins, quinones, ATPases), and (iii) both membrane systems form closed vesicles and generate ATP.Photosynthetic eukaryotic cells possess distinct organelles in which photosynthesis and respiration proceed-namely, chloroplasts and mitochondria. These organelles might be symbiotic prokaryotes that have transferred some of their genetic information to the host cell. In prokaryotic photosynthetic organisms, the photosynthetic and respiratory chains are located in the same membranes and share both electron transport components and coupling factors (1-3). One could thus question whether respiration occurs in the chloroplast as well. I show in this paper that, indeed, chlororespiration exists and is distinct from mitorespiration. MATERIALS AND METHODSChlamydomwnas reinhardtii wild-type 137C and mutant strains were grown in TAP medium (4) under an illumination of 200 lux. The mutants used in this work have been described (5-7). Chlorella pyrenoidosa was grown phototrophically as described (8).Open cell preparations were obtained as follows: 200 ml of cell suspension (2 X 106 cells/ml) was collected and suspended in 20 ml of 20 mM N-tris(hydroxymethyl)methylglycine(Tricine)/KOH, pH 7.8/10 mM NaCl/10 mM MgCl2/1 mM K2-HPO4/0.1 M sucrose/5% Ficoll. The cell suspension was passed through a Yeda press operated at 90 kg/cm2, diluted with 200 ml of Ficoll-lacking buffer, and centrifuged, a...

Unstacked thylakoid membrane vesicles were obtained from a homogenate of Chlamydomonas reinhardtii by flotation in a 1.8 M sucrose layer containing 5 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid)-10 mM EDTA (pH 7.5). Sodium dodecyl sulfate-gradient gel electrophoresis showed that the wildtype membranes have a total of at least 33 polypeptides ranging in molecular weights from 68,000 to less than 10,000. The wild-type and three non-photosynthetic mutant strains were studied with respect to their photosynthetic electron transport properties, their fluorescence rise kinetics, and their membrane polypeptide compositions. The results showed a strong correlation between the presence of a membrane polypeptide (molecular weight = 47,000) and the activity of the photosystem II reaction center. This polypeptide is missing from F34 (a Mendelian mutant lacking Q, the primary electron acceptor of photosystem II), but is partially restored in a suppressed strain of F34 in which there is an incomplete recovery of photosystem II activity. In a thermosensitive mutant, T4, the same polypeptide is present in reduced amount only in cells grown at 350 but not in those grown at 250. Although the mechanisms of the photosynthetic electron transport reactions have been intensively studied, relatively little is known about the molecular architecture of the thylakoid membranes on which these reactions are localized (compare ref. 1). Chemical analysis revealed that the thylakoid membranes are made up of approximately 50% lipids and 50% proteins (2). There is evidence that there are at least 10 to 20 polypeptides of different molecular weights in these membranes (3-11).Several approaches are available for the identification of the functions of the thylakoid membrane polypeptides. One approach is to fractionate the membranes by either detergents (5, 6, 9) or passage through a French pressure cell (9, 11) into small fragments enriched in either photosystem I (PS I) or photosystem II (PS II) activities. The polypeptide components of these subchloroplastic fragments or pigmentprotein complexes can then be identified by sodium dodecyl sulfate-gel electrophoresis. Another approach is to analyze the membrane polypeptides of mutant strains which are either pigment-deficient (12-16) or have specific lesions in the electron transport pathway (6,17,18). The missing or altered Abbreviations: WT, wild-type; PS I, photosystem I; PS II, photosystem II; DCMU, 3,4-dichlorophenyl dimethylurea; PBQ, p-benzoquinone; DPIP,2,6-dichlorophenol indophenol; MV, methyl viologen; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. 2175de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, polypeptides can then be correlated with the deleted functions in the mutant.In this paper, we have adopted the mutant approach and compared the polypeptide profile of thylakoid membrane of wild-type Chlamydomonas reinhardtii with those of mutant strains lacking or deficient in PS II activity. Our results suggest that a membrane polypeptide of mol...

The psbC gene of Chlamydomonas reinhardtii encodes P6, the 43 kd photosystem II core polypeptide. The sequence of P6 is highly homologous to the corresponding protein in higher plants with the exception of the N‐terminal region where the first 12 amino acids are missing. Translation of P6 is initiated at GUG in C. reinhardtii. The chloroplast mutant MA16 produces a highly unstable P6 protein. The mutation in this strain maps near the middle of the psbC gene and consists of a 6 bp duplication that creates a Ser‐Leu repeat at the end of one transmembrane domain. Two nuclear mutants, F34 and F64, and one chloroplast mutant, FuD34, are unable to synthesize P6. All of these mutants accumulate wild‐type levels of psbC mRNA. The FuD34 mutation has been localized near the middle of the 550 bp 5′ untranslated region of psbC where the RNA can be folded into a stem‐loop structure. A chloroplast suppressor of F34 has been isolated that partially restores synthesis of the 43 kd protein. The mutation of this suppressor is near that of FuD34, in the same stem‐loop region. These chloroplast mutations appear to define the target site of a nuclear factor that is involved in P6 translation.

In Chlamydomonas reinhardtii the oxygen evolving enhancer protein 1 (OEE1), which is part of the oxygen evolving complex of photosystem H (PS I), is coded for by a single nuclear gene (psbl). The nuclear mutant FuD44 specifically lacks the OEE1 polypeptide and is completely deficient in photosynthetic oxygen evolution. In this mutant a 5 kb DNA insertion into the 5' region of the psbl gene results in the complete absence of OEE1 mRNA and protein. A revertant, FuD44-R 2, which is capable of 30% of the photosynthetic oxygen evolution of wild-type cells, has lost 4 kb of the 5 kb DNA insert, and accumulates both OEE1 mRNA and protein, although at levels somewhat less than those of wild-type cells. Absence of the OEE1 protein in the FuD44 mutant does not affect the accumulation of other nuclear encoded PS II peripheral polypeptides. OEE1 absence does, however, result in a more rapid turnover of the chloroplast encoded PS II core polypeptides, thus resulting in a substantial deficiency of PS II core polypeptides in FuD44 cells. These PS II core proteins again accumulate in revertant FuD44-R2 cells.