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 approximately 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.

Figure 1.

Phenotypical Characterization of atm3-1 and atm3-2 Mutants. (A) Mo enzyme activities (NR, SO, AO, and XDH) in leaves of the sta1/atm3-1 mutant and the C24 wild type (NR, n = 4; SO, n = 6; bars represent se of the means). For comparison of AO and XDH activities, leaf extracts were electrophoresed on native polyacrylamide gels and either stained for AO activity or for XDH activity. For each, one representative gel of six experiments is shown. (B) T-DNA insertion site in the Arabidopsis GABI-Kat atm3-2 mutant. (C) RT-PCR–based detection of full-length ATM3 trancripts in Columbia-0 (Col-0) wild types and atm3-2 mutant plants (n = 3). Actin2 primers were used to demonstrate successful reverse transcription of mRNA. (D) Phenotypical comparison of atm3-1 and atm3-2 mutants. (E) Mo enzyme activities (NR, SO, AO, and XDH) in leaves of the GABI-Kat atm3-2 mutant and the Col-0 wild type (NR, n = 4; SO, n = 3; bars represent seof the means). For AO and XDH activities, one representative gel of four experiments is shown.

Figure 2.

Immunodetection of Molybo Enzymes in atm3-1 and atm3-2 Mutants. Total extracts from leaves of atm3-1 and atm3-2 mutants and their respective wild types (C24 and Col-0, respectively) were analyzed for their Mo enzyme contents using anti-NR antibodies, anti-SO antibodies, anti-XDH antibodies, or anti-AAO1 antibodies. XDH and AO activity staining (single lanes in right-most panel) of the same native polyacrylamide gels as used for immunoanalysis served as markers for XDH and AO proteins, respectively.

Figure 3.

ABA Contents in atm3-1 Mutants after Drought Stress. For drought stress treatment, atm3-1 mutants and Col-0 wild types were removed from the soil and placed in covered Petri dishes for the indicated periods (n = 6; bars represent se of the means).

Figure 4.

Amounts of Moco Intermediates in Leaves of atm3-1 and atm3-2 Mutants. MPT/Moco amounts in leaf extracts of atm3-1 (A) and atm3-2 (B) mutants were measured as FormA dephospho. cPMP amounts in leaf extracts of atm3-1 (C) and atm3-2 (D) mutants were measured as CompoundZ. Data represent averages from four experiments. Bars represent se of the means.

Figure 5.

Immunodetection of CNX2 and CNX3 in Mitochondria of Wild-Type Plants. Using specific anti-CNX2 (A) and anti-CNX3 (B) antibodies, cross-reacting proteins were detected exclusively in mitochondria. The purity of the fractions was confirmed by probing the same membrane for cytosolic NBP35 protein, chloroplastidic PetC, and mitochondrial Porin (C). In the presence of iodoacetamide, anti-CNX2 (D) and anti-CNX3 (E) antibodies recognized proteins of ∼36 and ∼18 kD, respectively, that correspond to the masses of mature CNX2 and CNX3 monomers without their respective NH₂-terminal signal sequences.

Figure 6.

Localization of CNX2 and CNX3 in the Mitochondrial Matrix. Mitochondria were subfractionated into a fraction consisting of OMVs and inner membrane space (IMS), a fraction consisting of inner membrane (IM), OMV, and the matrix fraction. All fractions (10 μ g each) were immunoanalyzed for the presence of CNX2 and CNX3 proteins using anti-CNX2 and anti-CNX3 antibodies, respectively. The purity of the mitochondrial fractions was confirmed by probing the same membrane for the mitochondrial proteins porin (outer membrane) and Isu1 (matrix). AO activity staining of these fractions allowed estimation of the degree of contamination with cytosolic proteins.

Figure 7.

Biochemical Characterization of the sir1/cnx5 Mutant. Leaf extracts of Arabidopsis sir1/cnx5 mutants with a deficiency in step 2 of Moco biosynthesis were analyzed for their cPMP contents (A) and MPT/Moco contents (B), as well as for the activities of NR (C), SO (D), AO (E), and XDH (F). cPMP and MPT/Moco were measured as CompoundZ (n = 3) and FormA dephospho (n = 3), respectively. NR and SO activities are given as mean ± sd(n = 4). For AO and XDH, activities of one representative gel of four experiments are shown.

Figure 8.

Determination of cPMP in Mitochondria of atm3-1 and cnx5 Mutants. cPMP amounts in total and mitochondrial (mito) extracts from leaves of C24 wild types and atm3-1 mutants ([A]; n = 4), and Col-0 wild types and sir1/cnx5 mutants ([B]; n = 3). Bars represent se of the means.

Figure 9.

Reconstitution of Moco Biosynthesis by Coincubation of atm3-1 Protoplasts with cPMP. Protoplasts of Arabidopsis C24 wild type and the atm3-1 mutant were coincubated with 2.5 μ g of purified cPMP for the indicated periods. Cells were harvested by centrifugation, broken by sonication, and subjected to MPT/Moco (FormA dephospho) analysis. MPT/Moco amounts were calculated to 1 mg of total protein in the protoplast extracts (n = 3; bars represent se of the means).