Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: wild-type and mutant strains deficient in photosystem II reaction center.

Abstract

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...

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“…10 ~tl of 0.2 M-iodoacetamide were added to each sample 2 h prior to electrophoresis. Electrophoresis was performed according to the procedure of Cr~UA and BENNOUN (8). Native l~C-labelled hordeins were co-electrophoresed as markers.…”

In vitro synthesis and transport of barley endosperm proteins: Reconstitution of functional rough microsomes from polyribosomes and stripped microsomes

“…After removal of insoluble proteins by centrifugation, the isopropanol extracts were dried down and prepared for electrophoresis as described in section 2.7. Electrophoresis was performed on 12.5% polyacrylamide gels employing the buffer system of CHUA and BENNOUN (9), with native ~4C-labelled hordeins as markers.…”

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