Adenine nucleotide translocator transports haem precursors into mitochondria

Abstract

Haem is a prosthetic group for haem proteins, which play an essential role in oxygen transport, respiration, signal transduction, and detoxification. In haem biosynthesis, the haem precursor protoporphyrin IX (PP IX) must be accumulated into the mitochondrial matrix across the inner membrane, but its mechanism is largely unclear. Here we show that adenine nucleotide translocator (ANT), the inner membrane transporter, contributes to haem biosynthesis by facilitating mitochondrial accumulation of its precursors. We identified that haem and PP IX specifically bind to ANT. Mitochondrial uptake of PP IX was inhibited by ADP, a known substrate of ANT. Conversely, ADP uptake into mitochondria was competitively inhibited by haem and its precursors, suggesting that haem-related porphyrins are accumulated into mitochondria via ANT. Furthermore, disruption of the ANT genes in yeast resulted in a reduction of haem biosynthesis by blocking the translocation of haem precursors into the matrix. Our results represent a new model that ANT plays a crucial role in haem biosynthesis by facilitating accumulation of its precursors into the mitochondrial matrix.

Figure 1. Identification of mitochodrial haem-binding proteins.

Purification of haem or PP IX-binding proteins. Haem- or PP IX-conjugated or unconjugated SG beads were incubated with mitochondrial extracts of rat liver. Bound proteins were eluted with Laemmli dye and subjected to SDS-PAGE, followed by silver staining (A) or Western blotting with anti-ANT antibody (B). C, FLAG-tagged ANT1, 2, or 3 was incubated with haem-conjugated (+) or unconjugated (−) SG beads. The eluates were separated by SDS-PAGE, followed by western blotting.

Figure 2. Docking model of ANT with haem.

(A) Lateral view of BtAAC1 structure docking with haem (red). Blue lines highlight the contact residues of BtAAC with haem. View of the transmembrane sector from the mitochondrial intermembrane space of BtAAC1 bound to haem (red) (B) or ADP (yellow) (C). (D) A superposition of ADP and haem on BtAAC. (E) The closed conformation of BtAAC1 with haem. (F) FLAG-tagged ANT1 wild type (WT) or series of the mutant of K22A, R79A and R279A, was incubated with haem-conjugated (+) or unconjugated (−) SG beads. The eluates were separated by SDS-PAGE, followed by western blotting.

Figure 3. Haem and its precursors accumulate into mitochondria via ANT.

(A) [³H]-labeled ADP was incubated with rat liver mitochondria in the presence or absence of haem or atractyloside for 0.5, 1, 2, 5, 10 min, and then the reaction was stopped by addition of atractyloside. (B) [³H]-labeled ADP was incubated with rat liver mitochondria in the presence of the indicated concentrations of haem, PP IX, or CP III on ice for 30 sec, and then the reaction was stopped by addition of atractyloside. (C) Lineweaver-Burk plot of ADP uptake into mitochondria in the presence of the indicated concentrations of haem for 30 sec. (D) Inhibition constant (K_i) for the inhibition of the mitochondria uptake of ADP or 2-oxoglutarate by haem, PP IX, or CP III. N.D., cannot be detected. (E) PP IX (50 µM) and Zn-acetate (50 µM) was incubated with mitochondria in the presence or absence of ADP, ATP or AMP on ice for 30 sec The generated Zn-PP IX was extract and detected by HPLC equipped with a fluorometric detector. Data represent mean±s.e.m. from four to six independent experiments (*, P<0.05; **, P<0.01; ***, P<0.005).

Figure 4. Analysis of haem biosynthesis in the ANT-deficient yeast strain.

(A) The amount of mitochondrial haem in the wild-type (WT), ΔAAC or ΔATP3 yeast strain was measured with a fluorometric detector as described in Materials and Methods . Lower panel shows Western blot analysis of mitochondrial extracts using anti-porin antibody. (B) HA-tagged catalase A activity of the WT, ΔAAC and ΔATP3 yeast strain. HA-catalase A was immunoprecipitated, and its catalase activity was determined (upper panel). Western blotting was performed using anti-HA antibody (lower panel). (C) Haem precursors (UP, uroporphyrin; 7, heptaporphyrin; 6, hexaporphyrin; 5, pentaporphyrin; CP I, coproporphyrin I; CP III, coproporphyrin III; PP IX, protoporphyrin IX) obtained from the WT, ΔAAC or ΔATP3 yeast strain were analyzed by C₁₈ reverse phase HPLC. (D) Quantification of (C). Data represent mean±s.e.m. from five independent experiments (*, P<0.05; **, P<0.01; ***, P<0.005).