Distribution of Folate Derivatives and Enzymes for Synthesis of 10-Formyltetrahydrofolate in Cytosolic and Mitochondrial Fractions of Pea Leaves

Abstract

Leaf extracts of 14-d-old pea (Pisum safivum L. cv Homesteader) seedlings were examined for folate derivatives and for 10-formyltetrahydrofolate synthetase (SYN), 5,lO-methenyltetrahydrofolate cyclohydrolase (CYC), and 5,l O-methylenetetrahydrofolate dehydrogenase (DHY) activities. Microbiological and enzyme assays showed that leaf folates SYN, CYC, and DHY were predominantly cytosolic. Extracts of Percoll gradient-purified mitochondria contained less than 1 % of total leaf folate and less that 170 of each enzyme activity. Fractionation of whole-leaf homogenates resulted in the copurification of DHY and CYC (subunit 38 kD) and the isolation of a SYN protein (subunit 66 kD). Polyclonal antibodies were raised against purified cytosolic DHY-CYC (DHY-CYC-Ab) and cytosolic SYN (SYN-Ab), respectively. lmmunoblots showed that DHY-CYC-Ab cross-reacted with a mitochondrial protein band (38 kD). l w o mitochondrial protein bands (subunit M, = 40,000 and 44,000) crossreacted with SYN-Ab. lmmunoaffinity chromatography (DHY-CYC-Ab as the immobile ligand) indicated that the bulk of mitochondrial SYN activity was not associated with mitochondrial DHY or CYC. When 9-d-old etiolated pea seedlings were exposed to light for up to 3 d, the specific enzyme activities of DHY-CYC in whole-leaf extracts rose 2-fold and more DHY-CYC-Ab cross-reacting protein was detected. In contrast, the specific activity of SYN fel1 from 5 to 1 pnol min-' mg-' protein and less SYN-Ab cross-reacting protein was detected. l h e data suggest that in pea leaves, the bulk of one-carbon-substituted tetrahydrofolates and enzymes for the generation of 1 O-formyltetrahydrofolate are extra-mitochondrial.

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

“…Analysis of the subcellular distribution of folate-synthesizing enzymes in pea leaves established that mitochondria play a major role in H 4 FGlu n synthesis (7). Other studies supported the mitochondrial localization of DHFS (15) and suggested the presence of FPGS in the cytosol (16). Organisms that lack DHFS activity and require exogenous folate for growth (animals and bacteria such as Lactobacillus) possess a monofunctional FPGS.…”

“…7). Also, the presence of DHFR-TS in the plastids (31) and FPGS in the cytosol (16) have been reported.…”

Cloning and Characterization of Mitochondrial 5-Formyltetrahydrofolate Cycloligase from Higher Plants

“…5-CHO-H 4 PteGlu n normally represents only 3-10% of the total cellular folate pool in mammals and yeast (4,7), whereas values of 14 -40% are reported for leaves and other metabolically active plant tissues (16,17). Moreover, 5-CHO-H 4 PteGlu n makes up 50% of the mitochondrial folate pool in pea leaves (17), which is much more than in mammalian mitochondria (18 -20).…”

“…Moreover, 5-CHO-H 4 PteGlu n makes up 50% of the mitochondrial folate pool in pea leaves (17), which is much more than in mammalian mitochondria (18 -20). The high 5-CHO-H 4 PteGlu n content of leaves and their mitochondria is of special interest given the massive SHMT-mediated glycine3serine fluxes that occur during photorespiration in leaf mitochondria (11), because in these conditions SHMT could both form 5-CHO-H 4 PteGlu n and be inhibited by it.…”

Enhanced Formaldehyde Detoxification by Overexpression of Glutathione-Dependent Formaldehyde Dehydrogenase from Arabidopsis

“…Most formaldehyde does not exist in vivo in a free state but is bound to endogenous nucleophiles, such as glutathione or tetrahydrofolate (Sardi and Tyihak, 1994;Chen et al, 1997). The main sources of formaldehyde in plants are dissociation of 5,10-methylenetetrahydrofolate and oxidation of methanol, derived mainly from pectin demethylation (Fall and Benson, 1996).…”