A Novel Role for Arabidopsis Mitochondrial ABC Transporter ATM3 in Molybdenum Cofactor Biosynthesis  

Abstract

The molybdenum cofactor (Moco) is a prosthetic group required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Its biosynthesis in eukaryotes can be divided into four steps, of which the last three are proposed to occur in the cytosol. Here, we report that the mitochondrial ABC transporter ATM3, previously implicated in the maturation of extramitochondrial iron-sulfur proteins, has a crucial role also in Moco biosynthesis. In ATM3 insertion mutants of Arabidopsis thaliana, the activities of nitrate reductase and sulfite oxidase were decreased to ;50%, whereas the activities of xanthine dehydrogenase and aldehyde oxidase, whose activities also depend on iron-sulfur clusters, were virtually undetectable. Moreover, atm3 mutants accumulated cyclic pyranopterin monophosphate, the first intermediate of Moco biosynthesis, but showed decreased amounts of Moco. Specific antibodies against the Moco biosynthesis proteins CNX2 and CNX3 showed that the first step of Moco biosynthesis is localized in the mitochondrial matrix. Together with the observation that cyclic pyranopterin monophosphate accumulated in purified mitochondria, particularly in atm3 mutants, our data suggest that mitochondria and the ABC transporter ATM3 have a novel role in the biosynthesis of Moco.

The Molybdenum Cofactor

“…Loss of ATM3 function is associated not only with a deficiency in extramitochondrial Fe-S proteins but also with an accumulation of cPMP in mitochondria, which has the consequence that the cytosol becomes short of cPMP, thus leading to decreased Moco levels and molybdenum enzyme activities in the cell (36). The precise role of ATM3 is still unknown, however.…”

“…3). Although cPMP is hydrophobic enough to pass through biological membranes simply by diffusion, recent work on plants demonstrated that a specific transporter in the inner membrane of mitochondria is involved in the transport of cPMP into the cytosol (36). The respective transporter belongs to the ABC transporter family and is referred to as ATM3 in plants.…”

“…Subfractionation of mitochondria demonstrated that Cnx2 and Cnx3 reside in the matrix, where 5Ј-GTP is available as a substrate for cPMP synthesis and where Fe-S clusters are synthesized as the essential prosthetic group for Cnx2 (36). In contrast to the first step, all subsequent steps of Moco biosynthesis were demonstrated to be localized in the cytosol (37)(38)(39), and thus, after its synthesis in mitochondria, cPMP has to pass the mitochondrial membranes to enable its further processing to Moco (Fig.…”

An Essential Role for Tomato Sulfite Oxidase and Enzymes of the Sulfite Network in Maintaining Leaf Sulfite Homeostasis

“…The reaction assay contained 0.1 M Tris-HCl buffer, pH 8.48, 0.1 mM Na 2 SO 3 , 5 mM b-mercaptoethanol, 50 mM NaSCN, and 80 mg mL 21 desalted protein extract treated before the assay with 1 mM tungstate for 30 min at 4°C to disrupt SO activity that consumes sulfite. The sulfite-consuming activity was measured during 15 min at 37°C and was estimated as sulfite disappearance, as described for SO activity (Pachmayr, 1960;Lang et al, 2007;Khan et al, 2010;Teschner et al, 2010;Brychkova et al, 2012a) and employing Na 2 SO 3 as a standard in solution containing NaSCN. Sulfite-consuming activity was assayed also as SCN 2 disappearance by detecting Fe(SCN) 3 (Papenbrock and Schmidt, 2000a).…”

“…In brief, SO activity was measured as the disappearance of sulfite (Pachmayr, 1960;Lang et al, 2007;Khan et al, 2010;Teschner et al, 2010;Brychkova et al, 2012a). The desalted protein extracts were treated with 1 mM tungstate for 30 min at 4°C to inhibit SO activity (Brychkova et al, 2012a).…”