A Nuclear Mutant of Chlamydomonas reinhardtii Defective in Photosynthetic Photophosphorylation

Abstract

Use of the Flagyl selection procedure [Schmidt et al. (1977) Proc. Nut1 Acad. Sci. USA, 74, 610-6141 led to the isolation of a nuclear mutant of Chlamydomonas reinhardtiidesignated thm-24. This mutant displays normal electron transport rates in vitro, possesses high latent ATPase activity bound to the thylakoid membrane, but is incapable of photophosphorylation. Decay of the transmembrane potential, as indicated by the kinetics of the 520-nm absorption change after illumination, is unusually slow and markedly biphasic. Sodium dodecylsulfate/ polyacrylamide gel electrophoresis of purified thylakoid membranes shows mutant thm-24 to be lacking a number of polypeptides including those previously designated 4.1, 4.2 and 8.1.Treatment of purified thylakoid membranes of wild-type and mutant algae, using the chloroform-release procedure of Beechey et al. [(1975) Biochem. J . 148, 533-5371 resulted in the removal of ATPase activity from each strain. In wild-type cells, the ATPase activity was of heterogeneous enzymatic origin ; fractionation of the chloroform-release extracts by non-denaturing polyacrylamide gel electrophoresis yielded three distinct bands displaying ATPase activity, designated ATPases I, I1 and 111. In contrast, extracts from membranes of mutant thm-24 yielded only one ATPase-containing fraction, co-migrating with ATPase I from wild-type. Use of electrophoretic, immunological and enzymatic methods established a correspondence of the polypeptide subunits of ATPases I1 and I11 and those of spinach coupling factor, CF1. ATPase I from either algal strain was shown to be structurally distinct from higher plant CFI and to C. reinhardtii ATPases I1 and 111.Recent publications have established the identity of a number of polypeptide bands resolved upon dodecyl sulfate/ polyacrylamide gel electrophoresis of purified thylakoid membranes of Chlamydomonas reinhardtii [l -31. Identification of these polypeptides has been accomplished principally by two methods : isolation of enzymes and pigment-protein complexes in a nearly-native state, followed by comparison of the mobilities of the polypeptide components of these entities with the mobilities of polypeptides in the unfractionated thylakoid membrane; and electrophoretic analysis of thylakoid membranes from mutants defective in particular aspects of photosynthetic function and deficient in the synthesis or incorporation of specific thylakoid membrane polypeptides. Monospecific antibodies raised against purified polypeptides from the thylakoid membrane have been used to confirm identifications based on electrophoretic mobility and to establish structural relationships between different components of the thylakoid membrane as well as between analogous coinponents in different plant species [4].In this paper we continue our study of the complete set of thylakoid membrane polypeptides in C. reinhardtii, focussing on the ATP-synthesizing complex. Using a combination of the approaches listed above, we have identified four thylakoid membrane polypeptides as subunits a, p...

The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features

“…Denaturing electrophoresis (SDS /PAGE) was carried out in 12–18% acrylamide gradient gels containing 8 M urea (45). Proteins were stained with Coomassie blue R-250, or electroblotted onto nitrocellulose membrane (Hybond-ECL, GE Healthcare).…”

Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo‐oxidative stress in response to iron availability in Chlamydomonas reinhardtii

“…Proteins from solubilized cells were prepared from 30 ml cultures (Piccioni et al, 1981) and resuspended in 20 mM HEPES, pH 7.5, 0.1 M DTT, 20 mM EDTA buffer. Whole cells were resuspended in H6 (5 mM HEPES, pH 7.5, 10 mM EDTA) and stored at )80°C.…”

Multistep Processing of an Insertion Sequence in an Essential Subunit of the Chloroplast ClpP Complex

“…Biochemical Analysis-SDS-PAGE was performed on 12-18% acrylamide gels containing 8 M urea (22); cell samples were loaded on an equal chlorophyll basis. For purified complex, protein concentration was determined using a BCA assay kit (Sigma).…”