Search Results (9)

The carnitine/acylcarnitine carrier (CAC) is a crucial protein for cellular energy metabolism, facilitating the exchange of acylcarnitines and free carnitine across the mitochondrial membrane, thereby enabling fatty acid β-oxidation and oxidative phosphorylation (OXPHOS). Although CAC has not been crystallised, structural insights are derived from the mitochondrial ADP/ATP carrier (AAC) structures in both cytosolic and matrix conformations. These structures underpin a single binding centre-gated pore mechanism, a common feature among mitochondrial carrier (MC) family members. The functional implications of this mechanism are well-supported, yet the structural organization of the CAC, particularly the formation of dimeric or oligomeric assemblies, remains contentious. Recent investigations employing biochemical techniques on purified and reconstituted CAC, alongside molecular modelling based on crystallographic AAC dimeric structures, suggest that CAC can indeed form dimers. Importantly, this dimerization does not alter the transport mechanism, a phenomenon observed in various other membrane transporters across different protein families. This observation aligns with the ping–pong kinetic model, where the dimeric form potentially facilitates efficient substrate translocation without necessitating mechanistic alterations. The presented findings thus contribute to a deeper understanding of CAC’s functional dynamics and its structural parallels with other MC family members. Full article

Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein–lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane. Full article

Adenosine 5′ triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease. Full article

The ADP/ATP carrier (AAC) plays a central role in oxidative metabolism by exchanging ATP and ADP across the inner mitochondrial membrane. Previous experiments have shown the involvement of the matrix loops of AAC in its function, yet potential mechanisms remain largely elusive. One obstacle is the limited information on the structural dynamics of the matrix loops. In the current work, unbiased all-atom molecular dynamics (MD) simulations were carried out on c-state wild-type AAC and mutants. Our results reveal that: (1) two ends of a matrix loop are tethered through interactions between the residue of triplet 38 (Q38, D143 and Q240) located at the C-end of the odd-numbered helix and residues of the [YF]xG motif located before the N-end of the short matrix helix in the same domain; (2) the initial progression direction of a matrix loop is determined by interactions between the negatively charged residue of the [DE]G motif located at the C-end of the short matrix helix and the capping arginine (R30, R139 and R236) in the previous domain; (3) the two chemically similar residues D and E in the highly conserved [DE]G motif are actually quite different; (4) the N-end of the M3 loop is clamped by the [DE]G motif and the capping arginine of domain 2 from the two sides, which strengthens interactions between domain 2 and domain 3; and (5) a highly asymmetric stable core exists within domains 2 and 3 at the m-gate level. Moreover, our results help explain almost all extremely conserved residues within the matrix loops of the ADP/ATP carriers from a structural point of view. Taken together, the current work highlights asymmetry in the three matrix loops and implies a close relationship between asymmetry and ADP/ATP transport. Full article

The mitochondrial ADP/ATP carrier (AAC) exports ATP and imports ADP through alternating between cytosol-open (c-) and matrix-open (m-) states. The salt bridge networks near the matrix side (m-gate) and cytosol side (c-gate) are thought to be crucial for state transitions, yet our knowledge on these networks is still limited. In the current work, we focus on more conserved m-gate network in the c-state AAC. All-atom molecular dynamics (MD) simulations on a variety of mutants and the CATR-AAC complex have revealed that: (1) without involvement of other positive residues, the charged residues from the three Px[DE]xx[KR] motifs only are prone to form symmetrical inter-helical network; (2) R235 plays a determinant role for the asymmetry in m-gate network of AAC; (3) R235 significantly strengthens the interactions between H3 and H5; (4) R79 exhibits more significant impact on m-gate than R279; (5) CATR promotes symmetry in m-gate mainly through separating R234 from D231 and fixing R79; (6) vulnerability of the H2-H3 interface near matrix side could be functionally important. Our results provide new insights into the highly conserved yet variable m-gate network in the big mitochondrial carrier family. Full article

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2^M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2^M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations. Full article

Although the mitochondrial permeability transition pore (PTP) is presumably formed by either ATP synthase or the ATP/ADP carrier (AAC), little is known about their differential roles in PTP activation. We explored the role of AAC and ATP synthase in PTP formation in Saccharomyces cerevisiae using bisindolylpyrrole (BP), an activator of the mammalian PTP. The yeast mitochondrial membrane potential, as indicated by tetramethylrhodamine methyl ester signals, dissipated over 2–4 h after treatment of cells with 5 μM BP, which was sensitive to cyclosporin A (CsA) and Cpr3 deficiency and blocked by porin1/2 deficiency. The BP-induced depolarization was inhibited by a specific AAC inhibitor, bongkrekate, and consistently blocked in a yeast strain lacking all three AACs, while it was not affected in the strain with defective ATP synthase dimerization, suggesting the involvement of an AAC-associated pore. Upon BP treatment, isolated yeast mitochondria underwent CsA- and bongkrekate-sensitive depolarization without affecting the mitochondrial calcein signals, indicating the induction of a low conductance channel. These data suggest that, upon BP treatment, yeast can form a porin1/2- and Cpr3-regulated PTP, which is mediated by AACs but not by ATP synthase dimers. This implies that yeast may be an excellent tool for the screening of PTP modulators. Full article

ADP/ATP carriers (AACs) are mitochondrial transport proteins playing a strategic role in maintaining the respiratory chain activity, fueling the cell with ATP, and also regulating mitochondrial apoptosis. To understand if AACs might represent a new molecular target for cancer treatment, we evaluated AAC expression levels in cancer/normal tissue pairs available on the Tissue Cancer Genome Atlas database (TCGA), observing that AACs are dysregulated in most of the available samples. It was observed that at least two AACs showed a significant differential expression in all the available kidney cancer/normal tissue pairs. Thus, we investigated AAC expression in the corresponding kidney non-cancer (HK2)/cancer (RCC-Shaw and CaKi-1) cell lines, grown in complete medium or serum starvation, for investigating how metabolic alteration induced by different growth conditions might influence AAC expression and resistance to mitochondrial apoptosis initiators, such as “staurosporine” or the AAC highly selective inhibitor “carboxyatractyloside”. Our analyses showed that AAC2 and AAC3 transcripts are more expressed than AAC1 in all the investigated kidney cell lines grown in complete medium, whereas serum starvation causes an increase of at least two AAC transcripts in kidney cancer cell lines compared to non-cancer cells. However, the total AAC protein content is decreased in the investigated cancer cell lines, above all in the serum-free medium. The observed decrease in AAC protein content might be responsible for the decrease of OXPHOS activity and for the observed lowered sensitivity to mitochondrial apoptosis induced by staurosporine or carboxyatractyloside. Notably, the cumulative probability of the survival of kidney cancer patients seriously decreases with the decrease of AAC1 expression in KIRC and KIRP tissues making AAC1 a possible new biomarker of metabolic remodeling and survival in kidney cancers. Full article

Verticillium dahliae invades the roots of host plants and causes vascular wilt, which seriously diminishes the yield of cotton and other important crops. The protein AAC (ADP, ATP carrier) is responsible for transferring ATP from the mitochondria into the cytoplasm. When V. dahliae protoplasts were transformed with short interfering RNAs (siRNAs) targeting the VdAAC gene, fungal growth and sporulation were significantly inhibited. To further confirm a role for VdAAC in fungal development, we generated knockout mutants (ΔVdACC). Compared with wild-type V. dahliae (Vd wt), ΔVdAAC was impaired in germination and virulence; these impairments were rescued in the complementary strains (ΔVdAAC-C). Moreover, when an RNAi construct of VdAAC under the control of the 35S promoter was used to transform Nicotiana benthamiana, the expression of VdAAC was downregulated in the transgenic seedlings, and they had elevated resistance against V. dahliae. The results of this study suggest that VdAAC contributes to fungal development, virulence and is a promising candidate gene to control V. dahliae. In addition, RNAi is a highly efficient way to silence fungal genes and provides a novel strategy to improve disease resistance in plants. Full article