A Fluorescence-Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes

Abstract

Several mitochondrial proteins, such as adenine nucleotide translocase (ANT), aspartate/glutamate carrier, dicarboxylate carrier, and uncoupling proteins 2 and 3, are suggested to have dual transport functions. While the transport of charge (protons and anions) is characterized by an alteration in membrane conductance, investigating substrate transport is challenging. Currently, mainly radioactively labeled substrates are used, which are very expensive and require stringent precautions during their preparation and use. We present and evaluate a fluorescence-based method using Magnesium Green (MgGrTM), a Mg2+-sensitive dye suitable for measurement in liposomes. Given the different binding affinities of Mg2+ for ATP and ADP, changes in their concentrations can be detected. We obtained an ADP/ATP exchange rate of 3.49 ± 0.41 mmol/min/g of recombinant ANT1 reconstituted into unilamellar liposomes, which is comparable to values measured in mitochondria and proteoliposomes using a radioactivity assay. ADP/ATP exchange calculated from MgGrTM fluorescence solely depends on the ANT1 content in liposomes and is inhibited by the ANT-specific inhibitors, bongkrekic acid and carboxyatractyloside. The application of MgGrTM to investigate ADP/ATP exchange rates contributes to our understanding of ANT function in mitochondria and paves the way for the design of other substrate transport assays.

ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport

“…After adding ADP to the bulk solution, we measured the release of 3 H-ATP with time ( Supplementary Figure S2 ). The determined ADP/ATP exchange rate depends on ANT1 content (k ANT = 5.53 ± 0.74 mmol/min/g) and corresponds well to the reported results for ANT reconstituted into liposomes ( Supplementary Table S1 ) [ 26 , 27 , 28 , 29 ].…”

“…To evaluate whether recombinant murine ANT1 was correctly refolded in proteoliposomes, we performed ADP/ATP transport measurements using proteoliposomes initially filled with radioactively labeled 3 H-ATP [ 26 ]. After adding ADP to the bulk solution, we measured the release of 3 H-ATP with time ( Supplementary Figure S2 ).…”

“…We further investigated the controversially discussed ANT1 involvement in the basal leak [ 20 , 21 ]. For this, we formed planar bilayer lipid membranes from proteoliposomes reconstituted with recombinant ANT1 [ 26 ]. Figure 1 demonstrates that the total specific conductances, G m and G 0 , of bilayer membranes made from DOPC, DOPE, and cardiolipin were similar in the presence and absence of ANT1 (G m =10.0 ± 2.0 nS/cm 2 and G 0 =8.4 ± 2.3 nS/cm 2 ) if no purine nucleotides (PN) were added.…”

“…Cloning, purification and reconstitution of murine ANT1 followed a previously established protocol [ 26 ]. The protein concentration in proteoliposomes was measured with the Micro BCA TM Protein Assay Kit (Thermo Fisher Scientific, Prod.…”

“…ANT-mediated exchange of ADP/ATP was measured radioactively using 3 H-ATP (Prod. #NET420250UC, Perkin Elmer, Waltham, MA, USA) following the protocol as described elsewhere [ 26 ] ( Supplementary Figure S2 ).…”