Arminio Boschetti

A chlorophyll b-less mutant of Chlamydomonas reinhardii lacking in the light-harvesting chlorophyll ab-protein complex but not in its apoproteins

Abstract The mutant pg 113, derived from Chlamydomonas reinhardii, arg2− mt+ (parent strain), completely lacks chlorophyll (Chl) b but is still able to grow under autotrophic conditions. The light-harvesting Chl a b- protein complex (LHCP) is absent. This is shown (a) by the lack of the corresponding signal in the CD spectrum of thylakoids and (b) by the absence of the band of the LHCP after electrophoresis of partially solubilized thylakoid membranes on lithium dodecyl sulfate polyacrylamide gels. All the other chlorophyll-protein complexes are present. In spite of the absence of the LHCP, all the polypeptide components of this complex are present in the mutant in the same ratios as in the parent strain, although in slightly reduced amounts. The LHC apoproteins are synthesized, processed and transported into the thylakoid membrane of the mutant. Moreover, the phosphorylation of thylakoid membrane polypeptides, which is related to the regulation of the energy distribution between Photosystem I and II, is the same in the mutant and in the parent strain, indicating that phosphorylation is not dependent on the presence of Chl b. Electron micrographs of thin sections of whole cells show that there are stacked regions of thylakoids in both the mutant and the parent strain chloroplasts. However, in the mutant, stacks are located near the chloroplast envelope, while long stretches or sometimes circles of unstacked membranes are found in the interior, mostly around the pyrenoid.

Isolation of chloroplast envelopes from Chlamydomonas. Lipid and polypeptide composition

Abstract A membrane fraction enriched in chloroplast envelopes was isolated from the alga Chlamydomonas reinhardii. The envelope preparations contained the envelope marker enzyme, galactosyltransferase, with a specific activity about 26- and 37-fold greater than that of the intact chloroplasts and of the starting cells, respectively. Both the envelope fraction and intact chloroplasts incorporated labelled galactose mostly into digalactosyldiglyceride (DGDG), with little incorporation into monogalactosyldiglyceride (MGDG), in marked contrast to what had been observed in higher plants. The envelope preparation was characterized in terms of its composition in polar lipids and polypeptides, and certain features were found in common with the corresponding membranes of higher plants. The ratio of galactolipids MGDG/DGDG was typically lower in the envelopes, compared to the thylakoid fraction. Analysis of the proteins by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed a polypeptide pattern that was distinct from those of the thylakoids or of the soluble fraction of the chloroplasts. The major component of the envelope fraction was a 29-hilodalton polypeptide, similar to the main envelope polypeptide found in higher plants and believed to be the phosphate translocator.

Isolation of biochemically active chloroplasts from chlamydomonas

Abstract Chloroplasts from the cell wall mutant cw-15-2 of Chlamydomonas reinhardii were isolated by disruption of the cells in the Yeda press and fractionation through step gradients of Percoll. The resulting chloroplast fraction contained 80–85% intact chloroplasts. Electron micrographs of thin sections of the chloroplast fraction showed some cytoplasmic impurities, although almost no cytoplasmic ribosomes were detected by analysis of the ribosomal subunits. The isolated chloroplasts are active in photosynthetic O 2 -evolution and CO 2 -fixation, with the highest rates obtained in the presence of ATP. The chloroplast fraction also showed high rates of light-dependent in organello protein synthesis, with labelling of discrete chloroplast proteins known to be synthesized in the chloroplasts.

GROWTH-CONTROLLED OSCILLATION IN ACTIVITY OF HISTONE H1 KINASE DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Synchronous cultures of the cell wall‐less mutant Chlamydomonas reinhardtii Dangeard cw 15 were grown under different mean irradiances and different illumination regimes, which produced cell cycles that differed in the number of daughter cells released from one mother cell, in the length of the cell cycle, and in the growth rate. During the cell cycle, the cells reached several commitment points whose number and timing differed according to the particular pattern of the cell cycle. The cell volume was used as a growth parameter and increased in a stepwise manner. Each of the steps consisted of periods of both fast and slow growth. Growth usually stopped when the cells attained a volume twice that of the preceding step. Reaching particular commitment points was coupled with the position of these points in the enlargement of cell volume. Changes in the activity of histone H1 kinase were noted during the cell cycles of all experimental variants, and the activities were compared with the timing of various commitment points. It was found that kinase activity varied markedly within a single cell cycle, attaining maximal values when the cellular volume had doubled. Each peak in kinase activity slightly preceded the commitment to an individual sequence of reproductive events. In addition to the oscillations related to cell growth, a peak of kinase activity always occurred toward the end of the cell cycle when multiple rounds of DNA replication, mitosis, and cell division occurred.

Covalent binding of 3-azido-monuron to thylakoids of DCMU-sensitive and -resistant strains of Chlamydomonas reinhardtii

Abstract Six different mutants of Chlamydomonas reinhardtii resistant to DCMU ( N -(3,4-dichlorophenyl)- N ′-dimethylurea; Diuron) have been isolated and are described. Five of them, including one of the so-called secondary mutants which are resistant only as whole cells, show uniparental inheritance of the resistance. Thylakoids isolated from all mutants as well as from the parent strain were covalently photolabelled with the radioactive DCMU analogue 3-[ 14 C]azido-monuron ( N -(3-azido-4-chlorophenyl)- N ′-dimethylurea). In the parent strain, and in the secondary mutants, two polypeptides of 32 kDa and 41 kDa were specifically labelled. In the primary mutants, where the Photosystem II activity of isolated thylakoids was resistant to DCMU, the 32 kDa protein was not labelled. The photolabelled 32 kDa protein was identified as the ‘rapidly labelled 32 kDa herbicide-binding polypeptide’ by comparison of its proteolytic fingerprints with those of [ 35 S]methionine-labelled, in organello synthesized 32 kDa protein. The 3-azido-monuron was found to be covalently bound to a small proteolytic fragment of about 8 kDa.

Sulfolipid associated with the light-harvesting complex associated with photosystem II apoproteins of Chlamydomonas reinhardii

Abstract In the light-harvesting chlorophyll a/b- protein complex (LHC II) of the green alga Chlamydomonas reinhardii three immunologically cross-reacting LHC II-apoproteins of about 24, 25 and 29 kDa can be distinguished by SDS gel electrophoresis. When digested with S. aureus V8 protease, their proteolytic fragmentation patterns are characteristically different. The very hydrophobic apoproteins have been isolated on a preparative scale by extraction of crude thylakoids with organic solvents and subsequent purification by gel chromatography. Further analysis of the purified, colourless apoproteins by transesterification with sodium methoxide and gas liquid chromatography showed that per 1 mol of protein, they contain about 4 mol of fatty acids, 80% of them being palmitic acid. Separation by TLC revealed that the presence of palmitic acid in the LHC II-apoproteins is not due to covalent binding, but to a specific association of these proteins with sulfoquinovosyldiglycerol (SQDG). Sulfoquinovose was also identified by HPLC of the dansylhydrazine derivative. Also LHC II-apoproteins were isolated from the chlorophyll b- deficient mutant pg-113 , from which no LHC II-complex can be prepared. The tightly bound lipids of the mutant apoproteins had almost the same composition as those of the wild type.

SYNTHESIS OF EF-TU AND DISTRIBUTION OF ITS MRNA BETWEEN STROMA AND THYLAKOIDS DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDII

In Chlamydomonas reinhardii the elongation factor EF-Tu is encoded in the chloroplast DNA. We identified EF-Tu in the electrophoretic product pattern of chloroplast-made proteins and showed that this protein is only synthesized in the first half of the light period in synchronized cells. The newly synthesized EF-Tu contributed little to the almost invariable content of EF-Tu in chloroplasts during the light period of the cell cycle. However, increasing cell volume and the lack of EF-Tu synthesis in the second half of the light period led to a decrease in the concentration of EF-Tu in chloroplasts. At different times in the vegetative cell cycle, the RNA was extracted from whole chloroplasts and from free and thylakoid-bound chloroplast polysomes. The content of mRNA of EF-Tu in chloroplasts and the distribution between stroma and thylakoids were determined. During the light period, the content of the mRNA for EF-Tu varied in parallel to the rate of EF-Tu synthesis. However, in the dark, some mRNA was present even in the absence of EF-Tu synthesis. Most of the mRNA was bound to thylakoids during the whole cell cycle. This suggests that synthesis of EF-Tu is associated with thylakoid membranes.

In-vitro translation of different mRNA-containing fractions of Chlamydomonas chloroplasts

Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.

Sterol mutants of Chlamydomonas reinhardtii: Characterisation of three strains deficient in C24(28) reductase

Abstract Three mutants of Chlamydomonas reinhardtii (strain arg7cw15 ) were obtained using the strategy of insertional mutagenesis by random plasmid integration with subsequent selection for resistance against the polyene antibiotic nystatin. Sterols were isolated by precipitation with digitonin, fractionated by both normal and argentation TLC, and then analysed by GLC and GC-MS. All the mutants accumulated ergosta-5,7,22,24(28)-tetraenol, ergosta-5,7,24(28)-trienol, ergosta-7,24(28)-dienol, stigmasta-5,7,22,24(28)-tetraenol, stigmasta-5,7,24(28)-trienol, stigmasta-8,24(28)-dienol and stigmasta-7,24(28)-dienol, while ergosterol and 7-dehydroporiferasterol which are the only major sterol components of the original strain were absent in the mutants. It is concluded that all these mutants are impaired in this C24(28) reductase which catalyses the reduction of the C24(28) tetraenol to the corresponding 24-alkyl sterol. There is strong evidence that the same enzyme acts on both the C 28 and C 29 sterol series. This view is also supported by Southern blot hybridisation analysis revealing that in all three mutants, plasmid insertion occurred at the same site indicating the disruption of the same gene. Due to the insertional nature of the mutations, the strains can be used for cloning the corresponding gene.

Isolation and characterization of polysomes from thylakoid membranes of Chlamydomonas reinhardii

Chloroplast polysomes that were originally bound to thylakoid membranes were isolated from the cell wall mutant CW-15 from Chlamydomonas reinhardii. Polysomes were isolated from synchronously grown cells harvested in the middle of the third light period, when the ratio of chloroplast to cytoplasmic polysomes was maximal. Thylakoid membranes were isolated from a chloroplast fraction and polysomes were released by Triton X-100. Analyses of subunits on sucrose gradients showed that the polysomes consisted predominantly of the 70 S-type ribosomes. The detached polysomes as well as polysomes still bound to the thylakoid membrane were active in in vitro protein synthesis when supplemented with Escherichia coli-soluble factors. The in vitro activity was inhibited by chloramphenicol and aurintricarboxylic acid, but not by cycloheximide.