Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K_m value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K_m values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V_max of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.     

Purification of Hydrogenase from Chlamydomonas reinhardtii

A method is described which results in a 2750-fold purification of hydrogenase from Chlamydomonas reinhardtii, yielding a preparation which is approximately 40% pure. With a saturating amount of ferredoxin as the electron mediator, the specific activity of pure enzyme was calculated to be 1800 micromoles H₂ produced per milligram protein per minute. The molecular weight was determined to be 4.5 × 10⁴ by gel filtration and 4.75 × 10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an abundance of acidic side groups, contains iron, and has an activation energy of 55.1 kilojoules per mole for H₂ production; these properties are similar to those of bacterial hydrogenases. The enzyme is less thermally stable than most bacterial hydrogenases, however, losing 50% of its activity in 1 hour at 55°C. The K_m of purified hydrogenase for ferredoxin is 10 micromolar, and the binding of these proteins to each other is enhanced under slightly acidic conditions. Purified hydrogenase also accepts electrons from a variety of artificial electron mediators, including sodium metatungstate, sodium silicotungstate, and several viologen dyes. A lag period is frequently observed before maximal activity is expressed with these artificial electron mediators, although the addition of sodium thiosulfate at least partially overcomes this lag.     

Isolation,Purification, and Characterization of Mitochondria from Chlamydomonas reinhardtii

Mitochondria were isolated from autotrophically grown Chlamydomonas reinhardtii cell-wall-less mutant CW 92. The cells were broken by vortexing with glass beads, and the mitochondria were collected by differential centrifugation and purified on a Percoll gradient. The isolated mitochondria oxidized malate, pyruvate, succinate, NADH, and [alpha]-ketoglutarate. Respiratory control was obtained with malate (2.0) and pyruvate (2.2) but not with the other substrates. From experiments with KCN and salicylhydroxamic acid, it was estimated that the capacity of the cytochrome pathway was at least 100 nmol O2 mg-1 protein min-1 and the capacity of the alternative oxidase was at least 50 nmol O2 mg-1 protein min-1. A low sensitivity to oligomycin indicates some difference in the properties of the mitochondrial ATPase from Chlamydomonas as compared to higher plants.     

Partial Purification of Intact Chloroplasts from Chlamydomonas reinhardtii

Partially purified intact chloroplasts were prepared from batch cultures of both wild type (Wt) and a mutant strain of Chlamydomonas reinhardtii. Protoplasts were generated from log phase cultures of Wt (137c) and the phosphoribulokinase-deficient mutant F60 by incubation of the cells in autolysine. These protoplasts were suspended in an osmoticum, cooled, and then subjected to a 40 pounds per square inch pressure shock using a Yeda pressure bomb. The resulting preparation was fractionated on a Percoll step gradient which separated the intact chloroplasts from both broken chloroplasts and protoplasts.

The chloroplast preparation was not significantly contaminated with the cytoplasmic enzyme activity phosphoenolpyruvate carboxylase (>5%), and contained (100%) stromal enzyme activity ribulose-1,5-bisphosphate carboxylase. The chloroplast preparation is significantly contaminated by mitochondria, as determined by succinate dehydrogenase activity. Chloroplasts prepared from Wt cells retained CO₂-dependent O₂ photoevolution at rates in excess of 60 micromoles per milligram chlorophyll per hour, an activity which is severely inhibited by the addition of 10 millimolar KH₂PO₄. The chloroplasts are osmotically sensitive as determined by ferricyanide-dependent O₂ photoevolution.

     

Purification and Characterization of UDP-Arabinopyranose Mutase from Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells are surrounded by a mixture of hydroxyprolin-rich glycoproteins consisting of L-arabinose, D-galactose, D-glucose, and D-mannose residues. The L-arabinose residue is thought to be attached by a transfer of UDP-L-arabinofuranose (UDP-Araf), which is produced from UDP-L-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). UAM was purified from the cytosol to determine the involvement of C. reinhardtii UAM (CrUAM) in glycoprotein synthesis. CrUAM was purified 94-fold to electrophoretic homogeneity by hydrophobic and size-exclusion chromatography. CrUAM catalyzed the reversible conversion between UDP-Arap and UDP-Araf and exhibited autoglycosylation activity when UDP-D-[¹⁴C]glucose was added as substrate. Compared to the properties of native and recombinant CrUAM overexpressed in Escherichia coli, native CrUAM showed a higher affinity for UDP-Arap than recombinant CrUAM did. This increased affinity for UDP-Arap might have been caused by post-translational modifications that occur in eukaryotes but not in prokaryotes.     

Purification and properties of L-aspartate aminotransferase of Chlamydomonas reinhardtii

An enzyme which catalyzes the transamination of L-aspartate with 2-oxoglutarate has been purified 400-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. An apparent relative molecular mass of 138,000 was estimated by gel filtration. The enzyme is a dimer consisting of two identical subunits of Mr 65,000 each as deduced from PAGE/SDS studies. A stoichiometry of two molecules pyridoxal 5-phosphate/enzyme molecule was calculated. The enzyme has an isoelectric point of 8.48 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-aspartate. L-Aspartate or pyridoxal 5-phosphate, but not 2-oxoglutarate, protected the enzyme from heat inactivation. The purified enzyme was able to transaminate, although to a low extent, L-phenylalanine and L-tyrosine with 2-oxoglutarate, and L-serine, L-alanine and L-glutamine with oxaloacetate. L-Aspartate aminotransferase exhibited hyperbolic kinetics for 2-oxoglutarate and oxaloacetate, and nonhyperbolic behaviour for L-aspartate and L-glutamate. Apparent Km values were 0.55 mM for 2-oxoglutarate, 0.044 mM for oxaloacetate, 2.53 mM for L-aspartate and 3.88 mM for L-glutamate. Transamination of L-aspartate in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from Chlamydomonas reinhardtii

Chloroplastic NADP-isocitrate dehydrogenase isoenzyme (NADP-IDH₂; EC 1.1.1.42) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by a procedure which included affinity chromatography on Red-Sepharose as the key step. The 70-kDa isoenzyme was found to be a dimer formed by 40-kDa subunits. Antibodies raised against a recombinant tobacco cytosolic NADP-IDH cross-reacted strongly with the cytosolic NADP-IDH₁ and weakly with the NADP-IDH₂ isoenzyme from this alga. NADPH and GTP were found to inhibit both isoenzymes, whereas intermediates of the tricarboxylic acid cycle, glycolysis or reductive pentose phosphate cycle had no significant effect. The simultaneous presence of isocitrate and Mn²⁺ protected NADP-IDH₂ against thermal inactivation or inhibition by reagents specific for arginine or lysine.     

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii y-1, newly synthesized chlorophyll a/b-binding apoproteins are degraded when chlorophylls are not present for assembly of stable light-harvesting complexes. A protease was purified from the membrane fraction of degreened y-1 cells, which digested chlorophyll a/b-binding proteins in membranes from C. reinhardtii pg-113, a protease-deficient strain. This protease was active with p-nitroanilides of nonpolar amino acids (Leu and Phe), but not of basic amino acids (Lys and Arg). The apparent molecular weight of the enzyme is 38,000 ± 2,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. Typical inhibitors of the major classes of proteases were ineffective with this enzyme. Protease activity was constant from pH 7.5 to 9; a plot of log V versus pH suggested that deprotonation of an ionizable group with a pK value of 6.0 to 6.5 is required for activity. The protease was inactivated by diethylpyrocarbonate and by photooxidation sensitized by rose bengal. These results suggested that a histidyl residue is required for catalysis. Although very sensitive to photodynamic conditions in vitro, the enzyme was not inactivated in vivo when cells were exposed to light.     

Purification and properties of L-alanine aminotransferase from Chlamydomonas reinhardtii.

An enzyme which catalyzes the transamination of L-alanine with 2-oxoglutarate has been purified 157-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50 degrees C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5-phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-alanine. Pyridoxal 5-phosphate protected activity against heat inactivation and, to a minor extent, L-alanine and 2-oxoglutarate, but not L-glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5-phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only L-alanine and L-glutamate with glyoxylate out of ten amino acids tested. L-Alanine aminotransferase exhibited hyperbolic kinetic for 2-oxoglutarate, pyruvate, and L-glutamate, and nonhyperbolic behaviour for L-alanine. Apparent Km values were 0.054 mM for 2-oxoglutarate, 0.52 for L-glutamate, 0.24 mM for pyruvate, and 2.7 mM for L-alanine. Transamination of L-alanine in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.     

Chlamydomonas reinhardtii

Recombinant proteins have become more and more important for the pharmaceutical and chemical industry. Although various systems for protein expression have been developed, there is an increasing demand for inexpensive methods of large-scale production. Eukaryotic algae could serve as a novel option for the manufacturing of recombinant proteins, as they can be cultivated in a cheap and easy manner and grown to high cell densities. Being a model organism, the unicellular green alga Chlamydomonas reinhardtii has been studied intensively over the last decades and offers now a complete toolset for genetic manipulation. Recently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its ability for biotechnological applications.     

Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonas reinhardtii.

Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form.     

Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii

A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.     

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

We have developed a rapid method for isolation of the Photosystem I (PS1) complex from Chlamydomonas reinhardtii using epitope tagging. Six histidine residues were genetically added to the N-terminus of the PsaA core subunit of PS1. The His₆-tagged PS1 could be purified with a yield of 80–90% from detergent-solubilized thylakoid membranes within 3 h in a single step using a Ni-nitrilotriacetic acid (Ni-NTA) column. Immunoblots and low-temperature fluorescence analysis indicated that the His₆-tagged PS1 preparation was highly pure and extremely low in uncoupled pigments. Moreover, the introduced tag appeared to have no adverse effect upon PS1 structure/function, as judged by photochemical assays and EPR spectroscopy of isolated particles, as well as photosynthetic growth tests of the tagged strain. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn²⁺ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (K_i, 0.14 mm) and NADPH was a noncompetitive inhibitor (K_i, 0.42 mm) with respect to NAD⁺. Citrate and adenine nucleotides at concentrations less than 1 mm had no effect on the activity, but 10 mm citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but l-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.IDH catalyzes the oxidative decarboxylation of isocitrate to produce 2-oxoglutarate. According to the specificity for the electron acceptor, two enzymes with IDH activity are known, NAD-IDH (EC 1.1.1.41) and NADP-IDH (EC 1.1.1.42) (Chen and Gadal, 1990a).In photosynthetic organisms NADP-IDH has been detected in the cytosol, chloroplasts, mitochondria, and peroxisomes. Cytosolic NADP-IDH has been purified from higher plants (Chen et al., 1988) and eukaryotic algae (Martínez-Rivas et al., 1996), and its cDNA has been cloned from alfalfa (Shorrosh and Dixon, 1992), soybean (Udvardi et al., 1993), potato (Fieuw et al., 1995), and tobacco (Gálvez et al., 1996). This 80-kD isoenzyme is a dimer, and it is likely to be involved in the synthesis of NADPH for biosynthetic purposes in the cytosol (Chen et al., 1988), in the synthesis of 2-oxoglutarate for ammonium assimilation (Chen and Gadal, 1990b), and in the cycling, redistribution, and export of amino acids (Fieuw et al., 1995). Chloroplastic NADP-IDH has been studied in higher plants (Gálvez et al., 1994) and eukaryotic algae (Martínez-Rivas and Vega, 1994). It is a 154-kD dimer that has been proposed to be involved in the supply of NADPH for biosynthetic reactions in the chloroplast when photosynthetic NADPH production is low (Gálvez et al., 1994). The mitochondrial NADP-IDH of higher plants may have a physiological role in the production of NADPH, which can be converted to NADH by a transhydrogenase or used to reduce glutathione in the mitochondrial matrix (Rasmusson and Møller, 1990). NADP-IDH activity has also been detected in peroxisomes from spinach leaves (Yamazaki and Tolbert, 1970).NAD-IDH is localized exclusively in the mitochondria in association with the TCA cycle. This enzyme has been purified from several nonphotosynthetic eukaryotes such as fungi (Keys and McAlister-Henn, 1990; Alvarez-Villafañe et al., 1996) and animals (Giorgio et al., 1970), in which it appears to be a 300-kD octamer. Its key regulatory role in the TCA cycle is well documented. The NAD-IDH from yeast is activated by AMP and citrate (Hathaway and Atkinson, 1963), whereas the animal enzyme is activated by ADP and citrate (Cohen and Colman, 1972). In addition, the NAD-IDH cDNAs have been cloned from yeast (Cupp and McAlister-Henn, 1991, 1992) and animals (Nichols et al., 1995; Zeng et al., 1995). In these organisms, the enzyme is composed of two (yeast) or more (animals) different subunits encoded by different genes.To our knowledge, no NAD-IDH from photosynthetic organisms has yet been purified to homogeneity, mainly because of the low stability of the enzyme (Oliver and McIntosh, 1995). However, partial purifications have been reported from pea (Cox and Davies, 1967; Cox, 1969; McIntosh and Oliver, 1992), potato (Laties, 1983), spruce (Cornu et al., 1996), and the eukaryotic microalga Chlamydomonas reinhardtii (Martínez-Rivas and Vega, 1994). Matrix and membrane forms of the enzyme have been detected in potato (Tezuka and Laties, 1983) and pea (McIntosh, 1997). Although it is an allosteric enzyme that exhibits sigmoidal kinetics with respect to isocitrate (Cox and Davies, 1967; McIntosh and Oliver, 1992) and is activated in vitro by ABA (Tezuka et al., 1990), the regulatory importance of NAD-IDH in photosynthetic organisms is still under debate.To elucidate the regulatory significance of NAD-IDH in photosynthetic organisms and its apparent contribution to the 2-oxoglutarate supply for ammonium assimilation, we have purified and characterized the NAD-IDH from C. reinhardtii.     

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis     

Algal Lipid Bodies: Stress Induction,Purification, and Biochemical Characterization in Wild-Type and Starchless Chlamydomonas reinhardtii

When the unicellular green soil alga Chlamydomonas reinhardtii is deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in the sta6 mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid “popped-cell” microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are ∼50% saturated (C₁₆ and C₁₈) fatty acids and ∼50% unsaturated fatty acids, half of which are in the form of oleic acid (C_18:1). The FFA are ∼50% C₁₆ and ∼50% C₁₈. The LB-derived TAG yield from a liter of sta6 cells at 10⁷ cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production in C. reinhardtii has the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.There is keen interest in the development of technologies that harvest lipids from microalgae and convert them into diesel fuel (7, 11, 24, 43). A widely adopted approach is to identify algal species that are ascertained to be oleaginous (e.g., Neochloris, Nannochloropsis, and Dunaliella species) and focus on extracting and processing their oils (43). We have adopted a complementary approach, which is to start with the unicellular green soil alga Chlamydomonas reinhardtii, widely regarded as nonoleaginous (48), and utilize the highly developed molecular and genetic tools available for this organism (22) to investigate how its oil production might be enhanced. Such information could then, in the future, be applied to any algae that may prove promising for industrial application.Algal oil production is commonly quantitated by extracting cells with organic solvents, characterizing the lipid fraction, and expressing the lipid yield as the percent dry weight of the starting material (24, 19, 43). Many cellular components (e.g., pigments, cell walls, and starch) contribute to dry weight, and these may change in quantity and/or composition during the cell cycle and/or during fluctuations in growth conditions for a given alga; they are also likely to differ among different algal lineages. Therefore, this convention provides an ambiguous metric for assessing how much oil a given cell produces when genetic background and/or culture conditions are manipulated and may yield misleading between-species comparisons.A related difficulty with this convention is that the extract is expected to contain lipids from numerous sources, e.g., plasma membranes, endomembranes, chloroplasts, and lipid bodies (LBs) dedicated to the storage of triacylglycerides (TAG) and fatty acids (FA). Although all of these sources are of potential value for fuel production, it is problematic to evaluate the output of a particular pathway when the outputs from multiple pathways are being pooled.We have therefore elected to focus on the biosynthesis of LBs (also referred to as lipid droplets or oil bodies), since this process has the hallmark of a discrete and inducible pathway amenable to genetic and culture condition manipulation (37), and we have developed a simple and direct microscopic assay for quantitating LB production per cell that is independent of dry weight.The assay was used to study two parameters. (i) Increases in extractable TAG in numerous algal species, including C. reinhardtii (65), when cells are starved for nitrogen (N) have been reported previously (3, 4, 25, 26, 33, 43, 44, 49, 53, 57, 59). We demonstrate that, in C. reinhardtii, this increase correlates with a robust increase in LB production. (ii) N starvation has also been shown to stimulate a pronounced increase in the biosynthesis of starch granules in C. reinhardtii (31, 35, 45), prompting us to ask whether LB production might be enhanced if starch production was eliminated. We report that the cw15 sta6 starch null strain (66) deleted in ADP glucose phosphorylase (an essential enzyme in the starch biosynthetic pathway) produces twice as much LB-associated TAG as its parental cw15 strain under nitrogen stress, suggesting that carbon skeletons normally converted into starch are instead able to flow into the starvation-induced TAG biosynthetic pathway when the starch option is unavailable.We have also developed a simple LB purification procedure that has permitted analysis of LB composition. The bodies contain ∼90% TAG, ∼10% free FA (FFA), and minor levels of charged glycerolipids (CGLs). Protein levels are also minor, and no proteins with the expected features of “dedicated” LB proteins, like the oleosins or caleosins of plant seeds, were identified.     

Purification and characterization of Chlamydomonas reinhardtii chloroplast glutamyl-tRNA synthetase, a natural misacylating enzyme

Glutamyl-tRNA synthetase from Chlamydomonas reinhardtii was purified by sequential column chromatography on DEAE-cellulose, phosphocellulose, Mono Q, and Mono S. The apparent molecular mass of the protein when analyzed under both denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 62,000 Da; this indicates that the active enzyme is a monomer. The purified glutamyl-tRNA synthetase was identified as the chloroplast enzyme by its tRNA charging specificity. Reversed-phase chromatography of unfractionated C. reinhardtii tRNA resolved four peaks of glutamate acceptor RNA when assayed with the purified enzyme. The enzyme can also glutamylate Escherichia coli tRNA(2Glu), but not cytoplasmic tRNA(Glu) from yeast or barley. In addition, the enzyme misacylates chloroplast tRNA(Gln) with glutamate. A similar mischarging phenomenon has been demonstrated for the barley chloroplast enzyme (Sch?n, A., Kannangara, C.G., Gough, S., and S?ll, D. (1988) Nature 331, 187-190) and for Bacillus subtilis glutamyl-tRNA synthetase (Proulx, M., Duplain, L., Lacoste, L., Yaguchi, M., and Lapointe, J. (1983) J. Biol. Chem. 258, 753-759).