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. 2024 Jun 5;43(14):2979–3008. doi: 10.1038/s44318-024-00132-2

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A) Schematic representation showing the positions of non-CL binding related mutations in Aac2. (B) Expression of WT and indicated FlagAac2 mutants. Kgd1 and Tom70 served as loading controls (n = 3, biological replicates). (C) Growth phenotype of Aac2 CL-binding mutants. Serial dilutions of indicated cells were spotted onto fermentable (YPD) and respiratory (YPEG) media and incubated at 30 or 37 °C for 3–5 days (n = 6, biological replicates). (D–F) ADP/ATP exchange: The efflux of matrix ATP was detected as in Fig. 4. The measurement was performed in the presence of 5 mM malate and 5 mM pyruvate (n = 6, biological replicates). (D) The linear part of the initial velocity following the addition of ADP at indicated concentrations was plotted (mean with SEM). Curve fitting was performed by nonlinear regression. (E) The linear part of velocity when 33 μM ADP was added is shown as scatter plots (mean with SEM). (F) Fitted K_m and V_max values were obtained by the Michaelis–Menten equation from the replicated experiments (mean). Significant differences were obtained by one-way ANOVA with Tukey’s multiple comparisons test (vs. WT); ****p < 0.0001. (G) Native MS analysis obtained the fractional population of WT and indicated FlagAac2 mutants associated with CATR and one to three CL molecules. Mean with SEM (n = 6: 2 biological replicates with 3 technical replicates). Significant differences were obtained by one-way ANOVA with Tukey’s multiple comparisons test (* vs. FlagAac2; † vs. FlagAac2+CATR + 1CL); */† p < 0.05, **/†† p < 0.01, ***/††† p < 0.001, ****/†††† p < 0.0001. (H) Aac2 tertiary structure: 100 μg of mitochondria from WT and the indicated mutants were mock-treated or instead incubated with either 40 μM CATR and then solubilized with 1.5% (w/v) digitonin. The extracts were resolved by 6 to 16% blue native-PAGE and immunoblotted for FlagAac2 (n = 4, biological replicates). Source data are available online for this figure.