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Mitochondria contact lipid droplets through the mitochondrial import complex binding to lipid metabolism enzyme Ayr1

Nature Cell Biology volume 28, pages 436–448 (2026)Cite this article

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Abstract

Mitochondria play central roles in the energetics and metabolism of eukaryotic cells. Their outer membrane is essential for protein transport, membrane dynamics, signalling and metabolic exchange with other cellular compartments. The mitochondrial import (MIM) complex functions as main translocase for importing the precursors of more than 90% of integral outer-membrane proteins. Here we report that the MIM complex performs a second major function in lipid-droplet homeostasis. Lipid droplets are crucial in cellular lipid metabolism and as storage organelles for neutral lipids. The lipid metabolism enzyme Ayr1 captures the MIM complex, promoting the formation of mitochondria–lipid droplet contact sites. MIM and Ayr1 enhance the lipid droplet number in cells. Ayr1 binds to MIM via its single hydrophobic segment in a substrate-mimicry mechanism but remains bound and is not released into the outer membrane. The functional diversity is mediated by different MIM complexes: MIM–Ayr1 for recruiting lipid droplets and MIM–preprotein for protein insertion into the outer membrane. Our work uncovers translocase capture as a mechanism for functional conversion of a membrane protein complex from protein insertion to lipid metabolism.

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Fig. 1: Ayr1 associates with the MIM complex.
Fig. 2: Ayr1 modulates lipid homeostasis.
Fig. 3: Ayr1 promotes contact-site formation between mitochondria and lipid droplets.
Fig. 4: The MIM complex recruits Ayr1 to mitochondria and modulates lipid-droplet homeostasis.
Fig. 5: The hydrophobic segment of Ayr1 functions in mitochondrial localization and lipid-droplet homeostasis.
Fig. 6: Ayr1 binds to the MIM complex via a substrate-mimicry mechanism.

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Data availability

The mass spectrometry data have been deposited to the ProteomeXchange Consortium88 via the PRIDE partner repository89. The data are available (http://proteomecentral.proteomexchange.org/) using the dataset identifiers PXD062106 (Mim2HA affinity purification), PXD062073 (Ayr1-overexpressing mitochondria) and PXD070899 (Ayr1S18A-, Ayr1G20,22A-overexpressing mitochondria). Further data supporting the reported findings are in the Supplementary Information. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank J. Schiessl, R. Mahlberg, H. Lamby and J. Puchalski for technical support. Work in this study has also been performed in partial fulfilments of the requirements for the doctoral theses of S.H., A.G., I.S. and S.B. M.H. is a member of CiM-IMPRS, the joint graduate school of the Cells-in-Motion Interfaculty Centre, University of Münster, Germany and the International Max Planck Research School–Molecular Biomedicine, Münster, Germany. This study was supported by the Deutsche Forschungsgemeinschaft/DFG (BE 4679/2-2 project ID 269424439, SFB 1218 B11 project ID 269925409, BE 4679/9-1 project ID 528247081, priority program SPP 2453 BE 4679/11-1 project ID 541555098 to T.B.; BR 6283/5-1 project ID 529716110, BR 6283/6-1 project ID 541596792 to F.d.B.; PF 202/9-1 project ID 394024777 to N.P.; FOR5815 P6, project ID 538651361, SFB 1557 P03, project ID 467522186 to M.B.; WI 4506/1-1 project ID 406757425, SFB 1381 project ID 403222702 to N.W.; project ID 450216812, project ID 409673687, SFB1381 project ID 403222702, SFB1177 project ID 259130777, GRK 2606 project ID 423813989 to C.K.; SFB 1381 project ID 403222702 to C.M.; priority program SPP 2453 WA 1598/7-1 project ID 541758684, GRK 2234/2 project B7 to B.W.) and Germany’s Excellence Strategy (CIBSS–EXC-2189–project ID 390939984 to N.P., C.K., C.M. and N.W.) and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme under grant agreement number 769065. This work reflects only the authors’ view and the European Union’s Horizon 2020 research and innovation programme is not responsible for any use that may be made of the information it contains. The mass spectrometer of the Core Facility ‘Analytical Proteomics’, University of Bonn was funded by the DFG (project ID 386936527).

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  1. Lars Ellenrieder

    Present address: Novartis Pharma AG, Basel, Switzerland

Authors and Affiliations

  1. Institute of Biochemistry and Molecular Biology, Faculty of Medicine, University of Bonn, Bonn, Germany

    Sandra Heinen, Vitasta Tiku, Isabelle Steymans, Sara Bonini, Nicole Okon, Jiyao Song, Matthias Eckhardt, Dominic Winter, Fabian den Brave & Thomas Becker

  2. Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Alexander Grevel, Lars Ellenrieder, Mariya Licheva, Sylvia Poppe, Chris Meisinger, Nils Wiedemann, Claudine Kraft & Nikolaus Pfanner

  3. Faculty of Biology, University of Freiburg, Freiburg, Germany

    Alexander Grevel & Mariya Licheva

  4. Institute of Cell Dynamics and Imaging, University of Münster, Münster, Germany

    Marie Hugenroth & Maria Bohnert

  5. Cells in Motion Interfaculty Centre, University of Münster, Münster, Germany

    Marie Hugenroth & Maria Bohnert

  6. Department of Biochemistry II, Theodor-Boveri-Institute, Biocenter, Faculty of Chemistry and Pharmacy, University of Würzburg, Würzburg, Germany

    Silke Oeljeklaus & Bettina Warscheid

  7. Institute of Neuroanatomy, Faculty of Medicine, University of Bonn, Bonn, Germany

    Jessica Lambertz & Lars Fester

  8. CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Chris Meisinger, Nils Wiedemann, Claudine Kraft & Nikolaus Pfanner

  9. BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

    Chris Meisinger, Nils Wiedemann & Nikolaus Pfanner

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  1. Sandra Heinen
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Contributions

S.H., V.T., A.G., M.H., L.E., I.S., M.L., M.E., S.B., S.O., N.O., J.L., S.P., J.S. and T.B. performed the experiments and analysed them together with L.F., C.M., B.W., N.W., D.W., C.K., F.d.B., M.B., N.P. and T.B. T.B. and N.P. designed and supervised the project. S.H. and A.G. prepared the figures with help from N.P. and T.B. T.B. and N.P. wrote the Article. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Nikolaus Pfanner or Thomas Becker.

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Extended data

Extended Data Fig. 1 Characterization and localization of Ayr1 and analysis of ∆ayr1 mitochondria.

a,b, Wild-type (WT), Tom40HA and Sam35His mitochondria were lysed, subjected to affinity purification and analysed by immunodetection with the indicated antisera. Load 2%, elution 50%. c, Yeast cells expressing mtBFP (blue), GFP-tagged Ayr1 (white) and mCherryErg6 (magenta) were analysed by fluorescence microscopy. d, The indicated amounts of WT and ayr1∆ mitochondria were analysed by SDS–PAGE and immunodetection with the indicated antisera. e, Protein complexes of WT and ayr1∆ mitochondria were separated by blue native electrophoresis and analysed by immunodetection with the indicated antisera. Unprocessed gels are provided.

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Extended Data Fig. 2 Characterization of the Ayr1-overexpressing mutant strain.

a, The phospholipid composition of isolated wild-type (WT) and Ayr1 overexpression mitochondria was determined by mass spectrometry. The mean values of the relative abundances of the major phospholipid classes from three biological replicates with their standard deviation (S.D.) are shown. LP, lysophospholipids, PS, phosphatidylserine, PI, phosphatidylinositol, PC, phosphatidylcholine, PE, phosphatidylethanolamine, CL, cardiolipin, PA, phosphatidic acid. b, Steady-state levels of WT and Ayr1 overexpression cells expressing Tom70–VN and Faa4–VC. c, Top, fluorescence image of yeast WT or dnm1∆ cells expressing mtGFP (white) with or without overexpression of the indicated Ayr1 variants are shown. Bottom, quantification of the number of cells with the indicated types of mitochondrial networks were quantified. Depicted are mean values of three biological replicates with 100 cells each and their corresponding standard error of the mean (S.E.M.). d, Growth analysis of the serial dilutions of the indicated strain at 30 °C on full medium containing either glycerol (YPG) or glucose (YPD) as carbon source. Source numerical data and unprocessed gels are provided.

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Extended Data Fig. 3 Characterization of Ayr1S18A and Ayr1G20,22A mutants.

a, Import of the indicated Ayr1 variants into wild-type (WT) and HAMim2 mitochondria followed by affinity purification. Load 2 %, elution 100%. b, The indicated amounts of mitochondria isolated from WT or ayr1∆ cells overexpressing the indicated Ayr1 variants were analysed by SDS–PAGE and immunodetection with the indicated antisera. c, Steady-state levels of WT and cells overexpressing the indicated Ayr1 variants and expressing Tom70–VN and Faa4–VC. d, The ratio of selected lipid droplet proteins to Ayr1 in the mitochondrial fraction were determined by proteomic analysis in cells expressing Ayr1 (compare Fig. 3a), Ayr1S18A and Ayr1G20,22A. Depicted are mean values from three biological replicates with their corresponding standard error of the mean (S.E.M.). Compare Supplementary Table 2 for the complete list of detected proteins. Source numerical data and unprocessed gels are provided.

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Extended Data Fig. 4 Characterization of Mim1 mutant strains.

a, The indicated amounts of mitochondria (protein content) from wild-type (WT) and mim1∆ cells11,72 were analysed by SDS–PAGE and immunodetection with the indicated antisera. b, Quantification of Pet10 in the mitochondrial fraction isolated from WT and mim1∆ cells11,72. The mean values of three biological replicates with their standard error of the mean (S.E.M.) are shown. For comparison between two groups, an unpaired t-test was used. The asterisks in the figures indicate the level of significance according to the following thresholds: p > 0.05 (ns), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****). c, Steady-state levels of WT and Mim1 overexpression cells expressing Tom70–VN and Faa4–VC. Source numerical data and unprocessed gels are provided.

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Extended Data Fig. 5 Characterization of yeast cells expressing Ayr1–Tom5TMD.

a, The indicated amounts of mitochondria isolated from wild-type (WT) or Ayr1–Tom5TM overexpressing cells were analysed by SDS–PAGE and immunodetection with the indicated antisera. b–d,35S-labelled Ugo1 (b), Tom6 (c) or Tom20 (d) was imported into mitochondria isolated from WT or cells overexpressing either Ayr1–Tom5TM for the indicated time periods and analysed by blue native electrophoresis and autoradiography. Right panels, the assembly into the mature 140 kDa complex (Ugo1) or TOM complex (Tom6, Tom20) of 4 (Ugo1), 5 (Tom6) and 3 (Tom20) technical replicates were quantified. Depicted are mean values with the standard error of the mean (S.E.M.). e, Steady-state levels of WT and Ayr1 overexpression cells expressing Tom70–VN and Faa4–VC. Source numerical data and unprocessed gels are provided.

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Extended Data Fig. 6 Characterization of yeast cells expressing Ayr1dmGPAT4.

a, Linear model of Ayr1 constructs. Rossmann fold, the NADPH-binding motif, the hydrophobic segment (HyS) are indicated. b, Steady-state levels of wild-type (WT) and Ayr1dmGPAT4 overexpression cells. c, Mitochondria from WT, Ayr1, and Ayr1GPAT4 overexpressing cells were lysed and subjected to co-immunoprecipitation via anti-Mim1 or pre-immune serum (PI). Bound proteins were detected by immunodetection with the indicated antisera. Load 2%, elution 50%. d, Left, WT and Ayr1GPAT4 overexpressing cells were stained with the lipid droplet-specific dye BODIPY 493/503 (white) and analysed by fluorescence microscopy. White scale bar corresponds to 10 µm. Right, the number of lipid droplets per cell was determined. Depicted are mean values of three biological replicates with 200 cells each and their corresponding standard error of the mean (S.E.M.). e, Fluorescent images of the split Venus assay in WT and Ayr1dmGPAT4 overexpressing cells. Right panels, quantification of the fluorescence intensity in the split Venus assay of WT, Ayr1 overexpression strains. Depicted are mean values of three biological replicates with 100 cells each and their corresponding standard error of the mean (S.E.M.). f, Left, fluorescence image of yeast WT or Ayr1dmGPAT4 cells expressing mtGFP (white) with or without overexpression of the indicated Ayr1 variants are shown. Right, quantification of the number of cells with tubular mitochondrial network was quantified. Depicted are mean values of three biological replicates with 100 cells each and their corresponding standard error of the mean (S.E.M.). g, Growth analysis of the serial dilutions of the indicated strain at 30 °C on full medium containing either glycerol (YPG) or glucose (YPD) as carbon source. d–f, For comparison of three or more groups, an ordinary one-way ANOVA followed by Tukey’s multiple comparison test was performed. The asterisks in the figures indicate the level of significance according to the following thresholds: p > 0.05 (ns), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****). Source numerical data and unprocessed gels are provided.

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Extended Data Fig. 7 Characterization of the membrane integration of Ayr1.

a, Sequence alignments of the carboxy-terminal hydrophobic part of Ayr1 proteins in fungi. b, Sequence alignments of the carboxy-terminal hydrophobic part of Fis1 and Gem1 proteins in fungi. c, Intact or osmotically swollen wild-type (WT) mitochondria were treated with proteinase K. Proteins were analysed by SDS–PAGE and immunodetection with the indicated antisera. d, Intact and osmotic swollen WT and Ayr1His mitochondria were incubated with anti-His antibodies and analysed by blue native electrophoresis and immunodetection with anti-Ayr1 antibodies. e, Cell fractionation of Ayr1, Ayr1P267S, Ayr1E272K and Ayr1P267S E272K overexpressing cells. The post-nuclear supernatant, the mitochondria-enriched fraction (P13), the microsome-enriched fraction (P100) and the cytosol-enriched fraction (S100) were analysed by immunodetection with the indicated antisera. f, Intact or osmotically swollen WT, Ayr1, Ayr1P267S, Ayr1E272K and Ayr1P267S E272K mitochondria were treated with proteinase K. Proteins were analysed by SDS–PAGE and immunodetection with the indicated antisera. g, Mitochondria from WT, Ayr1, and Ayr1P267S E272K overexpressing cells were lysed and subjected to co-immunoprecipitation via anti-Mim1 or pre-immune serum (PI). Bound proteins were detected by immunodetection with the indicated antisera. Load 2%, elution 50%. Source numerical data and unprocessed gels are provided.

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Extended Data Fig. 8 Protein import into mitochondria from Ayr1-overexpressing cells.

a–c, Import of Tom6 (a), Tom20 (b) and Tom22 (c) into mitochondria from wild-type (WT) or Ayr1-overexpressing cells. The import has been performed for the indicated time periods, analysed by blue native electrophoresis and autoradiography. Source numerical data and unprocessed gels are provided.

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Heinen, S., Tiku, V., Grevel, A. et al. Mitochondria contact lipid droplets through the mitochondrial import complex binding to lipid metabolism enzyme Ayr1. Nat Cell Biol 28, 436–448 (2026). https://doi.org/10.1038/s41556-026-01890-3

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