The chloroplast ClpP complex in Chlamydomonas reinhardtii contains an unusual high molecular mass subunit with a large apical domain

The composition of the chloroplast-localized protease complex, ClpP, from the green alga Chlamydomonas reinhardtii was characterized by nondenaturing electrophoresis, immunoblotting and MS. The detected ClpP complex has a native mass of approximately 540 kDa, which is approximately 200 kDa higher than ClpP complexes in higher plant chloroplasts, mitochondria or bacteria. The 540-kDa ClpP complex contains two nuclear-encoded ClpP proteins (ClpP3 and P5) and five ClpR (R1, R2, R3, R4 and R6) proteins, as well two proteins, ClpP1L and ClpP1H, both probably derived from the plastid clpP1 gene. ClpP1H is 59 kDa and contains a approximately 30-kDa insertion sequence (IS1) not found in other ClpP proteins, responsible for the high MW of the complex. Based on comparison with other sequences, IS1 protrudes as an additional domain on the apical surface of the ClpP/R complex, probably preventing interaction with the HSP100 chaperone. ClpP1L is a 25-kDa protein similar in size to other ClpP proteins and could arise by post-translational processing of ClpP1H. Chloramphenicol-chase experiments show that ClpP1L and ClpP1H have a similar half-life, indicating that both are stable components of the complex. The structure of the ClpP complex is further discussed in conjunction with a phylogenetic analysis of the ClpP/R genes. A model is proposed for the evolution of the algal and plant complex from its cyanobacterial ancestor.     

Expression by Chlamydomonas reinhardtii of a chloroplast ATP synthase with polyhistidine-tagged beta subunits

The green alga Chlamydomonas reinhardtii is a model organism for the study of photosynthesis. The chloroplast ATP synthase is responsible for the synthesis of ATP during photosynthesis. Using genetic engineering and biolistic transformation, a string of eight histidine residues has been inserted into the amino-terminal end of the beta subunit of this enzyme in C. reinhardtii. The incorporation of these amino acids did not impact the function of the ATP synthase either in vivo or in vitro and the resulting strain of C. reinhardtii showed normal growth. The addition of these amino acids can be seen through altered gel mobility of the beta subunit and the binding of a polyhistidine-specific dye to the subunit. The purified his-tagged CF1 has normal Mg(2+)-ATPase activity, which can be stimulated by alcohol and detergents and the enzyme remains active while bound to a nickel-coated surface. Potential uses for this tagged enzyme as a biochemical tool are discussed.     

The chloroplast ATP synthase in Chlamydomonas reinhardtii. I. Characterization of its nine constitutive subunits

We have characterized the subunit composition of the chloroplast ATP synthase from Chlamydomonas reinhardtii by means of a comparison of the polypeptide deficiencies in a mutant defective in photophosphorylation, with the polypeptide content in purified coupling factor (CF)1 and CF1.CF0 complexes. We could distinguish nine subunits in the enzyme, four of which were CF0 subunits. Further characterization of these subunits was undertaken by immunoblotting experiments, [14C]dicyclohexylcarbodiimide binding and analysis of their site of translation. In particular, we were able to show the presence of an as yet unidentified delta subunit in CF1 from C. reinhardtii. We have identified a 70-kDa peripheral membrane protein in the thylakoid membranes of C. reinhardtii, which is immunologically related to the beta subunit of CF1. We discuss its conceivable ATPase function with respect to the Ca2+-dependent ATPase activity previously reported in the thylakoid membranes from C. reinhardtii.     

Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii.

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.     

Chlorophyll synthesis modulates retention of apoproteins of light-harvesting complex II by the chloroplast in Chlamydomonas reinhardtii

Localization of apoproteins of the major light-harvesting complex (LHCII) in Chl b -less cells of Chlamydomonas reinhardtii cbn 1–113 was determined by immunoelectron microscopy. In dark-grown cells, a low amount of apoproteins was detected in cytoplasmic vacuoles. The amount in vacuoles, and in the cytosol, increased dramatically when the rate of protein synthesis was enhanced in the dark by raising the temperature to 38°C. After exposure of cells to light, the apoproteins accumulated also in the chloroplast. Mature-sized apoproteins were recovered in an alkali-soluble fraction of cellular proteins commensurate with accumulation in the cytoplasm. At 25°C, content of apoproteins in the chloroplast of pale-green cells grown in medium lacking acetate was one-half of the amount in cells grown with acetate, yet the total amount remained similar. Cytoplasmic vacuoles, which were nearly filled with immunoreactive, electron-opaque material, were more abundant in cells grown without acetate as compared with cells grown with acetate. Accumulation of apoproteins outside of the chloroplast suggested that translocation into the organelle of a portion of the apoproteins, apparently synthesized in excess of the amount accommodated by Chl synthesis, was aborted after processing of precursors. These results suggested that assembly of LHCII was required for retention of apoproteins by the chloroplast.     

Sedimentation behavior of chloroplast ribosomes from Chlamydomonas reinhardtii.

The identity of peaks generated by chloroplast ribosomes of Chlamydomonas reinhardtii were determined by zone velocity sedimentation on sucrose density gradients, and analysis of distribution of ribosomal RNAs in the gradients. The sedimentagion coefficient of the principal peak was 66-70 S (usually 69 S), in good agreement with previously reported values for chloroplast ribosomes of C. reinhardtii, and other organisms. The fast sedimenting side of the 69 S peak contained an excess of chloroplast large subunit. When ribosome dissociation was prevented by sedimentation at low velocity, by aldehyde fixation, or by the presence of nascent polypeptide chains, the principal peak had a sedimentation coefficient of about 75 S. Thus the 69 S peak was an artifact caused by dissociation during centrifugation. Peaks that contained chloroplast ribosomal RNAs were also observed at '60 S' and '45 S' when chloroplast ribosomes were centrifuged unfixed at high velocity. The amounts of '60 S' and '45 S' components were decreased by centrifugation at low speed, or fixation, but sedimentation coefficients remained unchanged. The '60 S', and '45 S' components were identified as large, and small subunits of chloroplast ribosomes, respectively. The artifacts produced by centrifugation of chloroplast ribosomes, are similar to the artifacts produced by centrifuging ribosomes of Escherichia coli. Similar explanations appear to apply to both. We concluded that the 69 S chloroplast ribosome peak occurs because of dissociation of 'tight' couples, and incomplete separation of subunits. Subunit peaks (60 S and 45 S) arise from free subunits, and/or from dissociation of 'loose' couples.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.     

Conserved gene clusters in the highly rearranged chloroplast genomes of Chlamydomonas moewusii and Chlamydomonas reinhardtii

We have extended to about 75 the number of genes mapped on the Chlamydomonas moewusii and Chlamydomonas reinhardtii chloroplast DNAs (cpDNAs) by partial sequencing of the very closely related C. eugametos and C. moewusii cpDNAs and by hybridizations with Chlamydomonas chloroplast gene-specific sequences. Only four of these genes (tscA and three reading frames) have not been identified in any other algal cpDNAs and thus may be specific to Chlamydomonas. Although the C. moewusii and C. reinhardtii cpDNAs differ by complex sequence rearrangements, 38 genes scattered throughout the genome define 12 conserved clusters of closely linked loci. Aside from the rRNA operon, four of these gene clusters share similarity to evolutionarily primitive operons found in other cpDNAs, representing in fact remnants of these operons. Our results thus indicate that most of the ancestral bacterial operons that characterize the chloroplast genome organization of land plants and early-diverging photosynthetic eukaryotes have been disrupted before the emergence of the polyphyletic genus Chlamydomonas. All gene rearrangements between the C. moewusii and C. reinhardtii cpDNAs, with the exception of those accounting for the relocations of atpA, psbI and rbcL, occurred within corresponding regions of the genome. One of these rearrangements seems to have led to disruption of the ancestral region containing rpl23, rpl2, rps19, rpl16, rpl14, rpl5, rps8 and the psaA exon 1. This gene cluster, which bears striking similarity to the Escherichia coli S10 and spc operons, spans a continuous DNA segment in C. reinhardtii, while it maps to two separate fragments in C. moewusii.     

Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.