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L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis.

ABSTRACT: L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyses the last enzymatic step of the ascorbate biosynthetic pathway in plants. GLDH is localised to mitochondria and several reports have shown that GLDH is associated with complex I of the respiratory chain. In a gldh knock-out mutant, complex I is not detectable, suggesting that GLDH is essential for complex I assembly or stability. GLDH has not been identified as a genuine complex I subunit, instead, it is present in a smaller, lowly abundant version of complex I called complex I*. In addition, GLDH activity has also been detected in smaller protein complexes within mitochondria membranes. Here, we investigated the role of GLDH during complex I assembly. We identified GLDH in complexes co-localising with some complex I assembly intermediates. Using a mutant that accumulates complex I assembly intermediates, we confirmed that GLDH is associated with the complex I assembly intermediates of 400 and 450 kDa. In addition, we detected accumulation of the 200 kDa complex I assembly intermediate in the gldh mutant. Taken together, our data suggest that GLDH is an assembly factor of the membrane arm of complex I. This function appears to be independent of the role of GLDH in ascorbate synthesis, as evidenced by the ascorbate-deficient mutant vtc2-1 accumulating wild-type levels of complex I. Therefore, we propose that GLDH is a dual-function protein that has a second, non-enzymatic function in complex I assembly as a plant-specific assembly factor. We propose an updated model for complex I assembly that includes complex I* as an assembly intermediate.

SUBMITTER: Schimmeyer J

PROVIDER: S-EPMC4689740 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis.

Schimmeyer Joram J Bock Ralph R Meyer Etienne H EH

Plant molecular biology 20151031 1-2

L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyses the last enzymatic step of the ascorbate biosynthetic pathway in plants. GLDH is localised to mitochondria and several reports have shown that GLDH is associated with complex I of the respiratory chain. In a gldh knock-out mutant, complex I is not detectable, suggesting that GLDH is essential for complex I assembly or stability. GLDH has not been identified as a genuine complex I subunit, instead, it is present in a smaller, lowly abundant v ...[more]

PMID: 26520835

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Project description:Pepper fruit is one of the highest vitamin C sources of plant origin for our diet. In plants, ascorbic acid is mainly synthesized through the L-galactose pathway, being the L-galactono-1,4-lactone dehydrogenase (GalLDH) the last step. Using pepper fruits, the full GalLDH gene was cloned and the protein molecular characterization accomplished. GalLDH protein sequence (586 residues) showed a 37 amino acids signal peptide at the N-terminus, characteristic of mitochondria. The hydrophobic analysis of the mature protein displayed one transmembrane helix comprising 20 amino acids at the N-terminus. By using a polyclonal antibody raised against a GalLDH internal sequence and immunoblotting analysis, a 56kDa polypeptide cross-reacted with pepper fruit samples. Using leaves, flowers, stems and fruits, the expression of GalLDH by qRT-PCR and the enzyme activity were analyzed, and results indicate that GalLDH is a key player in the physiology of pepper plants, being possibly involved in the processes which undertake the transport of ascorbate among different organs. We also report that an NO (nitric oxide)-enriched atmosphere enhanced ascorbate content in pepper fruits about 40% parallel to increased GalLDH gene expression and enzyme activity. This is the first report on the stimulating effect of NO treatment on the vitamin C concentration in plants. Accordingly, the modulation by NO of GalLDH was addressed. In vitro enzymatic assays of GalLDH were performed in the presence of SIN-1 (peroxynitrite donor) and S-nitrosoglutahione (NO donor). Combined results of in vivo NO treatment and in vitro assays showed that NO provoked the regulation of GalLDH at transcriptional and post-transcriptional levels, but not post-translational modifications through nitration or S-nitrosylation events promoted by reactive nitrogen species (RNS) took place. These results suggest that this modulation point of the ascorbate biosynthesis could be potentially used for biotechnological purposes to increase the vitamin C levels in pepper fruits.

Dataset Information

L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis.

Publications

L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis.

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