Roland Douce

Purification of plant mitochondria by isopycnic centrifugation in density gradients of Percoll.

Abstract Mitochondria from potato tubers have been separated from contaminating organelles and membrane vesicles on self-generated Percoll gradients and in a relatively short time. The Percoll-purified mitochondria devoid of carotenoids and galactolipids showed no contamination with intact plastids, microbodies, or vacuolar enzymes. Percoll-purified mitochondria exhibited intact membranes and a dense matrix. The intactness of purified mitochondrial preparations was ascertained by the measurement of KCN-sensitive ascorbate cyt c-dependent O2 uptake. When compared with washed mitochondria, Percoll-purified mitochondria showed improved rates of substrate oxidation, respiratory control, and ADP:O ratios. The recovery of the cyt oxidase was 70–90% and on a cyt oxidase basis the rate of succinate oxidation by unpurified mitochondria was equal to that recorded for Percoll-purified mitochondria. The great flexibility of purification procedure involving silica sols was extended from mitochondria to the isolation of intact peroxisomes.

[37] Isolation of plant mitochondria: General principles and criteria of integrity

Publisher Summary This chapter describes the criteria for the assessment of mitochondrial integrity, including (1) the respiratory rate in the presence of added ADP compared to the rate obtained following its expenditure, (2) the ADP/O ratio, and (3) the latency of matrix enzymes, such as fumarate hydratase mitochondria are measured. The chapter also discusses isolation of chlorophyll-free mitochondria from pea leaves. Because the report of a procedure is to obtain fully functional and intact mitochondria from leaves of higher plants, several techniques for the purification of the mitochondria must free from contaminating thylakoid membranes. The methods used have included discontinuous Percoll gradient centrifugation of both mechanically prepared crude mitochondria and those from broken protoplasts, a combination of phase-partition and Percoll gradient centrifugation, and linear sucrose gradient centrifugation.

Structure and Function of the Plastid Envelope

Publisher Summary In higher plants, the process of photosynthesis occurs within specific membrane bounded organelles called “chloroplasts.” All the chloroplasts exhibit three major structural regions— namely, highly organized internal sac-like flat compressed vesicles called “thylakoids,” an amorphous background rich in soluble proteins called “stroma,” and a pair of outer membranes known as the “envelope.” The envelope essentially renders functional and structural integrity to the chloroplast. This chapter discusses the structure, isolation, chemical composition, and origin of the higher plant chloroplast envelope. The chapter examines the multiple functions of this important membranous system involved in the regulation of the inflow of raw materials for photosynthesis and the outflow of photosynthetic products. The chloroplast envelope of higher plants is a permanent structure and consists of two morphologically and topologically distinct membranes separated by a region about 10–20 nm thick, which appears electron-translucent. The structure of both envelope membranes is consistent with the lipid-globular protein mosaic model of membrane structure as proposed by Singer and Nicolson.

Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts

The prenylquinone content and biosynthetic capabilities of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts were analyzed. Both envelope membranes contain prenylquinones, and in almost similar amounts (on a protein basis). However, the outer envelope membrane contains more alpha-tocopherol than the inner one although this prenylquinone is the major one in both fractions. On the contrary, plastoquinone-9 is present in higher amounts in the inner envelope membrane than in the outer one. In addition, it has been demonstrated that all the enzymes involved in the last steps of alpha-tocopherol and plastoquinone-9 biosynthesis, i.e., homogentisate decarboxylase polyprenyltransferase, S-adenosyl-methionine:methyl-6-phytylquinol methyltransferase, S-adenosyl-methionine: alpha-tocopherol methyltransferase, homogentisate decarboxylase solanesyltransferase, S-adenosyl-methionine:methyl-6-solanesylquinol methyltransferase, and possibly 2,3-dimethylphytylquinol cyclase, are localized on the inner envelope membrane. These results demonstrate that the inner membrane of the chloroplast envelope plays a key role in chloroplast biogenesis, and especially for the synthesis of the two major plastid prenylquinones.

Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase/7,8‐dihydropteroate synthase localized in mitochondria

In pea leaves, the synthesis of 7,8‐dihydropteroate, a primary step in folate synthesis, was only detected in mitochondria. This reaction is catalyzed by a bifunctional 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase/7,8‐dihydropteroate synthase enzyme, which represented 0.04–0.06% of the matrix proteins. The enzyme had a native mol. wt of 280–300 kDa and was made up of identical subunits of 53 kDa. The reaction catalyzed by the 7,8‐dihydropteroate synthase domain of the protein was Mg2+‐dependent and behaved like a random bireactant system. The related cDNA contained an open reading frame of 1545 bp and the deduced amino acid sequence corresponded to a polypeptide of 515 residues with a calculated Mr of 56 454 Da. Comparison of the deduced amino acid sequence with the N‐terminal sequence of the purified protein indicated that the plant enzyme is synthesized with a putative mitochondrial transit peptide of 28 amino acids. The calculated Mr of the mature protein was 53 450 Da. Southern blot experiments suggested that a single‐copy gene codes for the enzyme. This result, together with the facts that the protein is synthesized with a mitochondrial transit peptide and that the activity was only detected in mitochondria, strongly supports the view that mitochondria is the major (unique?) site of 7,8‐dihydropteroate synthesis in higher plant cells.

Methionine catabolism in Arabidopsis cells is initiated by a γ-cleavage process and leads to S-methylcysteine and isoleucine syntheses

Despite recent progress in elucidating the regulation of methionine (Met) synthesis, little is known about the catabolism of this amino acid in plants. In this article, we present several lines of evidence indicating that the cleavage of Met catalyzed by Met γ-lyase is the first step in this process. First, we cloned an Arabidopsis cDNA coding a functional Met γ-lyase (AtMGL), a cytosolic enzyme catalyzing the conversion of Met into methanethiol, α-ketobutyrate, and ammonia. AtMGL is present in all of the Arabidopsis organs and tissues analyzed, except in quiescent dry mature seeds, thus suggesting that AtMGL is involved in the regulation of Met homeostasis in various situations. Also, we demonstrated that the expression of AtMGL was induced in Arabidopsis cells in response to high Met levels, probably to bypass the elevated Km of the enzyme for Met. Second, [13C]-NMR profiling of Arabidopsis cells fed with [13C]Met allowed us to identify labeled S-adenosylmethionine, S-methylmethionine, S-methylcysteine (SMC), and isoleucine (Ile). The unexpected production of SMC and Ile was directly associated to the function of Met γ-lyase. Indeed, we showed that part of the methanethiol produced during Met cleavage could react with an activated form of serine to produce SMC. The second product of Met cleavage, α-ketobutyrate, entered the pathway of Ile synthesis in plastids. Together, these data indicate that Met catabolism in Arabidopsis cells is initiated by a γ-cleavage process and can result in the formation of the essential amino acid Ile and a potential storage form for sulfide or methyl groups, SMC.

Tetrahydrofolate biosynthesis in plants: Molecular and functional characterization of dihydrofolate synthetase and three isoforms of folylpolyglutamate synthetase in Arabidopsis thaliana

Tetrahydrofolate coenzymes involved in one-carbon (C1) metabolism are polyglutamylated. In organisms that synthesize tetrahydrofolate de novo, dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) catalyze the attachment of glutamate residues to the folate molecule. In this study we isolated cDNAs coding a DHFS and three isoforms of FPGS from Arabidopsis thaliana. The function of each enzyme was demonstrated by complementation of yeast mutants deficient in DHFS or FPGS activity, and by measuring in vitro glutamate incorporation into dihydrofolate or tetrahydrofolate. DHFS is present exclusively in the mitochondria, making this compartment the sole site of synthesis of dihydrofolate in the plant cell. In contrast, FPGS is present as distinct isoforms in the mitochondria, the cytosol, and the chloroplast. Each isoform is encoded by a separate gene, a situation that is unique among eukaryotes. The compartmentation of FPGS isoforms is in agreement with the predominance of γ-glutamyl-conjugated tetrahydrofolate derivatives and the presence of serine hydroxymethyltransferase and C1-tetrahydrofolate interconverting enzymes in the cytosol, the mitochondria, and the plastids. Thus, the combination of FPGS with these folate-mediated reactions can supply each compartment with the polyglutamylated folate coenzymes required for the reactions of C1 metabolism. Also, the multicompartmentation of FPGS in the plant cell suggests that the transported forms of folate are unconjugated.

The galactolipid:galactolipid galactosyltransferase is located on the outer surface of the outer membrane of the chloroplast envelope

1. INTRODUCTION It is well known that the plastid envelope is in- volved in the synthesis of galactolipids (for a re- view see [l]). For instance it has been demon- strated that at least two distinct enzymes res- ponsible for the synthesis of monogalactosyldiacyl- glycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are associated with chloroplast envelope membranes. The first enzyme, UDP-galactose:di- acylglycerol galactosyltransferase, catalyses the in- corporation of galactose from UDP-galactose into MGDG [3-51:

The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution.

Acetohydroxy acid isomeroreductase catalyzes the conversion of acetohydroxy acids into dihydroxy valerates. This reaction is the second in the synthetic pathway of the essential branched side chain amino acids valine and isoleucine. Because this pathway is absent from animals, the enzymes involved in it are good targets for a systematic search for herbicides. The crystal structure of acetohydroxy acid isomeroreductase complexed with cofactor NADPH, Mg2+ ions and a competitive inhibitor with herbicidal activity, N‐hydroxy‐N‐isopropyloxamate, was solved to 1.65 Å resolution and refined to an R factor of 18.7% and an R free of 22.9%. The asymmetric unit shows two functional dimers related by non‐crystallographic symmetry. The active site, nested at the interface between the NADPH‐binding domain and the all‐helical C‐terminus domain, shows a situation analogous to the transition state. It contains two Mg2+ ions interacting with the inhibitor molecule and bridged by the carboxylate moiety of an aspartate residue. The inhibitor‐binding site is well adjusted to it, with a hydrophobic pocket and a polar region. Only 24 amino acids are conserved among known acetohydroxy acid isomeroreductase sequences and all of these are located around the active site. Finally, a 140 amino acid region, present in plants but absent from other species, was found to make up most of the dimerization domain.

Purification and Kinetic Properties of Serine Acetyltransferase Free of O-Acetylserine(thiol)lyase from Spinach Chloroplasts

Serine acetyltransferase, a key enzyme in the L-cysteine biosynthetic pathway, was purified over 300,000-fold from the stroma of spinach (Spinacia oleracea) leaf chloroplasts. The purification procedure consisted of ammonium sulfate precipitation, anion-exchange chromatography (Trisacryl M DEAE and Mono Q HR10/10), hydroxylapatite chromatography, and gel filtration (Superdex 200). The purified enzyme exhibited a specific activity higher than 200 units mg-1 and a subunit molecular mass of about 33 kD upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Moreover, the purified serine acetyltransferase appeared to be essentially free of O-acetyleserine(thiol)lyase, another enzyme component in the L-cysteine biosynthetic pathway. A steady-state kinetic analysis indicated that the mechanism of the enzyme-catalyzed reaction involves a double displacement. The apparent Km for the two substrates, L-serine and acetyl-coenzyme A, were 2.29 [plus or minus] 0.43 and 0.35 [plus or minus] 0.02 mM, respectively. The rate of L-cysteine synthesis in vitro was measured in a coupled enzyme assay using extensively purified O-acetylserine(thiol)lyase and serine acetyltransferase. This rate was maximum when the assay contained approximately a 400-fold excess of O-acetylserine(thiol)lyase over serine acetyltransferase. Measurements of the relative level of O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma indicated that the former enzyme was present in much larger quantities than the latter. Thus, the activity ratio for these two enzymes [O-acetylserine(thiol)lyase activity/serine acetyltransferase activity] measured in the stromal protein extract was 345. This strongly suggested that all the O-acetylserine(thiol)lyase and serine acetyltransferase activities in the stroma are involved in bringing a full synthesis of L-cysteine in the chloroplast.