Characterization of Biotin and 3-Methylcrotonyl-Coenzyme A Carboxylase in Higher Plant Mitochondria

Abstract

Mitochondria from green pea (Pisum sativum) leaves were purified free of peroxisomes and chlorophyll contamination and examined for their biotin content. The bulk of the bound biotin detected in plant mitochondria was shown to be associated with the matrix space to a concentration of about 13 micromolar, and no free biotin was detected. Western blot analysis of mitochondrial polypeptides using horseradish peroxidase-labeled streptavidin revealed a unique biotin-containing polypeptide with a molecular weight of 76,000. This polypeptide was implicated as being the biotinylated subunit of 3-methylcrotonyl-coenzyme A (CoA) carboxylase. Fractionation of pea leaf protoplasts demonstrated that this enzyme activity was located largely in mitochondria. The 3-methylcrotonyl-CoA carboxylase activity was latent when assayed in isotonic media. with MCase, which catalyzes the ATP-dependent carboxylation of the 3-methyl carbon of the branched acyl-CoA substrate to give 3-methylglutaconyl-CoA. MATERIALS AND METHODS ReagentsAll biochemicals were obtained from Sigma Chimie SARL, La Verpilliere, France. NaH'4CO3 (53.1 Ci/mol) was purchased from Amersham. Other chemicals were analytical grade. Plant MaterialsPea (Pisum sativum L., var Douce Provence) plants were grown from seeds in soil for 15 d under a 12-h photoperiod ofwhite light from fluorescent tubes (10-40 ,gE/m2/s) at 18°C.The plants were watered every day with tap water. Approximately 4 to 5 kg of fully expanded leaves were used for the isolation of mitochondria.Potato tubers (Solanum tuberosum L.) were obtained from a local market.In previous publications, Journet et al. (16) We also demonstrate that all the biotin present in the matrix space of the plant mitochondrion is associated 'Abbreviations: MCase, 3-methylcrotonyl-CoA carboxylase; PFP, PPi:fructose-6-phosphate-1 -phosphotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Preparation of MitochondriaMitochondria were isolated and purified from potato tubers as described by Douce et al. (6) by using self-generating Percoll gradients. The mitochondria were found in a broad brownred band near the bottom of the tube, whereas the "microsomal" fraction, containing the yellow envelope surrounding the amyloplasts, remained near the top of the tube. The mitochondria were subsequently concentrated by differential centrifugation. To remove all the contaminating peroxisomes the purification procedure was repeated once (i.e. a second time through Percoll). Under these conditions, we have verified that catalase activity with substrate concentrations up to 200 ,M was almost negligible.Mitochondria were isolated and purified from green pea leaves as described by Douce et al. (6), using self-generating Percoll gradients and a linear gradient of0 to 10% (w/v) PVP-25 (Kca 25, Serva) (top to bottom). The mitochondria were found in a tight white band near the bottom of the tube, whereas the thylakoids remained near the top of the tube. The mitochondria were subsequently concentrated by differential centrifugation. The ...

Characterization of the cDNA and Gene Coding for the Biotin Synthase of Arabidopsis thaliana

“…These studies should reveal how biotin becomes available for biotinylation of biotin-containing enzymes that are compartmentalized in different subcellular compartments. For example, methylcrotonyl-COA carboxylase is in the mitochondria (Baldet et al, 1992;Song et al, 1994), and acetyl-COA carboxylase isozymes are present in chloroplasts (Choi et al, 1995) and cytosol (Alban et al, 1994;Shorrosh et al, 1994;Yanai et al, 1995). In addition, such studies should reveal the mechanism(s) by which plants accumulate most of their biotin as protein-free biotin (Shellhammer and Meinke, 1990;Baldet et al, 1993a;Wang et al, 1995), whereas bacteria use a11 of their synthesized biotin for biotinylation of biotin-containing proteins (Cronan, 1989).…”

Dual Targeting of Arabidopsis HOLOCARBOXYLASE SYNTHETASE1: A Small Upstream Open Reading Frame Regulates Translation Initiation and Protein Targeting

“…Proteins were resolved by SDS-PAGE and electroblotted to nitrocellulose membrane. Biotin-containing polypeptides were detected with a system analogous to western blotting using streptavidin conjugated with horseradish peroxidase (Baldet et al, 1992). The presence of streptavidin-biotin complexes was analyzed by chemiluminescence.…”

Induction of β‐methylcrotonyl‐coenzyme A carboxylase in higher plant cells during carbohydrate starvation: evidence for a role of MCCase in leucine catabolism

“…After 4 days of starvation, values nearly 6-times higher than in control cells were obtained. Plant MCCase has been clearly characterized as a mitoc~ondrial enzyme [12]. We thus purified mitochondria from control and starved sycamore cells to investigate the evolution of the biotinylated subunit of the MCCase, which can be easily detected on Western blots using horseradish peroxidase-labelled streptavidin.…”

“…Polypeptides were transferred electrophoretically onto nitrocellulose sheets, essentially according to Towbin et al [24]. Biotin-containing polypeptides were detected using horseradish peroxidase-labelled streptavidin as described previously [12].…”

“…Gerbling and Gerhardt [9] suggested that an extraperoxisomal pathway is involved in leucine catabolism, since they reported that peroxisomes are unable to carry out the biotin-dependent carboxylation of I]-methylcrotonyl-CoA. Interestingly plant MCCase has been purified and located in mitochondria [12,13]. MCCase, present in all plant organs, is usually the most prevalent of the biotin-containing enzymes [14], and its activity can be regulated by biotinylation of the apoenzyme [15].…”