Mitochondria directly interact with the nuclear pore complex

Abstract Mitochondria regulate cellular processes through direct and indirect interactions with other organelles. A well-studied example has been contact with the endoplasmic reticulum at mitochondrial-associated endoplasmic reticulum membranes1, which control pathways including redox and calcium homeostasis2,3. Recent studies have also reported direct mitochondria–nuclear membrane contacts in cancer cells and yeast that promote pro-survival signalling4,5. Here we identify direct interactions between mitochondria and nuclear pores. Using two unbiased proteomic screens, GST pulldown and BioID, we found that VDAC1 was the top mitochondrial candidate that interacts with the filamentous nuclear pore protein RANBP2. In vitro RANBP2 CRISPR knockout, RANBP2 truncation or site-directed mutagenesis of RANBP2–VDAC1 interacting amino acids resulted in reduced mitochondria–nucleus proximity and decreased nuclear ATP and phosphocreatine levels. This was accompanied by a decline in the levels of the nuclear phosphoproteome and downregulation of pathways involved in histone modification, cellular differentiation and transcriptional regulation in vitro. Moreover, deletion of the RANBP2 C-terminal domain in vivo in mice resulted in embryonic lethality due to cardiac and neural crest differentiation defects. Collectively, these results describe a mechanism by which mitochondria directly interact with the nuclear pore complex, a phenomenon critical for regulation of nuclear energetics and cellular differentiation. Undoubtedly, additional roles of this interaction remain to be revealed. This is a preview of subscription content, access via your institution Access options Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription £17.99 / 30 days cancel any time Subscribe to this journal Receive 52 print issues and online access £199.00 per year only £3.83 per issue Buy this article - Purchase on SpringerLink - Instant access to the full article PDF. £ 29.95 Prices may be subject to local taxes which are calculated during checkout Data availability All large datasets have been deposited as raw files: RNA-seq (GSE325290), ChIP–seq (GSE324951), ATAC-seq (GSE324952) and proteomics (PXD065792 and PXD065793). Analysed, curated datasets, required for the conclusions described in this article, can be found in the Supplementary Information. Biological models (CRISPR-edited cell lines and mouse model) are available on request to the corresponding author. Source data are provided with this paper. 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H.A.S. is supported by grants from the NIH (NIH R01 HL147276-01 and NIH R01 HL149137-01), Cancer Prevention and Research Institute of Texas (RP160520), Hamon Center for Regenerative Science and Medicine, and Foundation Leducq (Redox Regulation of Cardiomyocyte Renewal). S.P. is supported by the NIH (R35GM148237), ARO (W911NF-23-1-0304) and NSF (2310610). R.S.-Y.F. is funded by Individual Research Grants from the National Medical Research Council (NMRC) of Singapore (MOH-001480-00, MOH-001325-00 and MOH-001685) and MOE Academic Research Fund (AcRF) Tier 3 (MOE-000333-00). C.J.M.L. is supported by the Singapore Ministry of Health’s National Medical Research Council under its Open-Fund Young Investigator Research Grant (MOH-001712-00). The following grants also contributed to this work: NIH grant 1S10OD021685-01A1 (JEOL 100+ electron microscope) to K. Luby-Phelps and NIH grant 1S10OD028630-01 (Nikon SoRa microscope) to K. Luby-Phelps. Access to the Abberior Facility Line STED microscope was provided by the UT Southwestern O’Donnell Brain Institute. We thank K. Luby-Phelps for assistance with STED imaging; J. Shelton and the UTSW Histology Core for their assistance with histological processing of embryos; the Next-Generation Sequencing Facility at CRI/UTSW for their expertise in generating sequencing data; and the UTSW Proteomics Core for assistance with proteomics experiments. Author information Authors and Affiliations Contributions H.A.S. designed and supervised the project, designed the experiments, wrote and revised the manuscript and obtained funding. I.M.-.M. designed, performed and analysed the experiments, wrote the manuscript and designed the figures. C.M.-V., E.C., M.T. and J.V. performed and analysed the phosphoproteomics data. S.M. and A.A. performed the BioID labelling experiments. M.S.A. performed the AlphaFold prediction. M.J.G. and F.S.-C. analysed the RNA-seq data. C.G.A.-N., C.J.M.L., S.K. and R.S.-Y.F. performed and analysed the ChIP–seq and ATAC-seq data. A.S. and R.D. obtained and analysed the metabolomics data. T.T. performed and analysed the circular dichroism experiments. P.P. and S.P. developed the biophysical model. A. Elnwasany, A.C.C., A.H.M.P. and L.S. assisted with the GSH pull-down experiments. F.X., S.R.A. and P.W. assisted with cloning and the protein expression experimental design. A.K.W. and C.X. performed the evolutionary/conservation analyses. M.K. performed the targeted proteomics analyses. J.A.E. provided the mitochondria–pore blue native data. S.T. and G.G.-A. assisted with mouse breeding and maintenance. N.U.N.N., C.-C.H., N.T.L., H.E.-f., A. Elghamry and A.M. assisted with experimental execution. L.S., A.A., J.A.E., M.T. and S.R.A. revised the study and provided feedback and discussion. Corresponding author Ethics declarations Competing interests The authors declare no competing interests. Peer review Peer review information Nature thanks Martin Hetzer, Luca Pellegrini, Sophie Trefely, Sean Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Additional information Extended data figures and tables Extended Data Fig. 1 Blue-Native mitochondrial and pore proteomics. A) Schematic of the subcomplexes of the nuclear pore complex. Number in boxes indicate the number of proteins belonging to each subcomplex. B) Quantification of RANBP2 peptides in control and complex I, complex III and complex IV-deficient purified mitochondria mass spec analysis. Extended Data Fig. 2 Phylogenetic and interactome of RANBP2. A) Major conserved protein domains in RANBP2 (human and mouse) and Nup358 (drosophila) annotated by Uniprot. Black lines represent total protein length for that species. B) Plot of omega (dN/dS) at every site along the filtered RANBP2 codon alignment. Red points are significance. Blue dashed line indicates omega = 1. Triangles indicate sites under positive selection above the graph limits. C) Shannon entropy across aligned RANBP2 (182 vertebrates) and Nup358 (164 arthropods) protein sequences. Colored boxes denote conserved interaction domains and the vertebrate-specific C-terminal domain. The dashed blue line indicates entropy threshold of 2, below which positions are considered conserved. Sites with >25% gaps among species were excluded, as these were typically species-specific insertions. D) Maximum-likelihood tree of full-length RANBP2 protein in chordates, with Strongylocentrotus purpuratus (echinoderm) as an outgroup. Branches and tips are colored by major clades. E) Maximum-likelihood tree of C terminal domain amino acid sequence in vertebrates. The tree shows low overall divergence and poor resolution among vertebrate lineages, consistent with a signal of extreme conservation. F) RANBP2 interactions based on STRING database. The schematic in the bottom indicates in which domain of RANBP2 the interactions take place. Extended Data Fig. 3 BioID stable cell lines characterization. A) Schematic of BioID stable lines generation and constructs used. B) Western-Blot analysis of total cell lysates after biotin treatment for MYC (left) and Streptavidin (right). Squares highlight the different BioID constructs bands. C) Streptavidin-beads immunoprecipitation and western-blot of TOM20-BioID clone (lane 1), RANBP2Ct-BioID clone (lane2) and control BioID clone (lane 3) against MYC (top) and Streptavidin (bottom) in input (left) and after immunoprecipitation (right). D) Venn diagram of the comparison between control and TOM20-BirA samples, showing 90 proteins exclusive labelled in TOM20 vs control. E) Cell distribution of the 90 proteins only labelled in presence of TOM20-BirA and not control-BirA. Each bar represents the number of proteins with the indicated location. Proteins with more than one location were counted once per each location. F) Abundance and location of the 16 proteins TOM20-BirA labelled that are exclusively located in the nucleus. G) Fold Change over BioID-Control of mitochondria-encoded proteins in CTD-BirA sample. Extended Data Fig. 4 VDAC1 and CTD mutants. A) AlphaFold prediction of resulting interaction between FD > AA CTD mutant (pink) and WT VDAC1 (blue). B) AlphaFold prediction of resulting interaction between FD > AA CTD mutant (pink) and ETT > LII VDAC1 mutant (green). C) 3D alignment of WT and LII VDAC1 mutant structures. D) 3D alignment of WT and AA CTD mutant structures. Extended Data Fig. 5 VDAC1 and RANBP2 KO 10T1/2 cell lines. A) Characterization of VDAC1 KO (VKO) cell line by Western Blot (left) and immunostaining (right). For staining, DAPI nuclear staining is shown in blue, VDAC1 staining in green and mitochondrial MitoTracker signal in red. Scale bar represents 10 μm. B) Imaris reconstruction and C) quantification of Contact Site Score in WT and VKO 10 T ½ cells. Scale bar represents 5 μm. Welch t-test, n = 13-19, **, p-value < 0.01. D) Crystal Violet Cell Growth assay in WT and RANBP2 KO 10T1/2 cells. Cells were analyzed at 24 h intervals. Graph represents values relative to t = 0. 2-way ANOVA, n = 3. E) PI staining and FACS analysis of cell cycle in WT and RANBP2 KO 10T1/2 cells. Cell cycle phases peaks were determined used automatic analysis built-in in FlowJo. 2-way ANOVA test, n = 4-5. For all graphs: * p-value < 0.05, ** p-value < 0.01. F) Confocal (top), imaris reconstruction (middle) and contact sites detection (bottom) in wild-type (WT) and Ranbp2 KO (RKO) cell lines in control conditions (top), after 4 h at 3% oxygen (center), and after 1 h at 40 ng/mL EGF. Mitochondria are stained with MitoTracker Orange (red), nuclear membrane with SYNE3 antibody (green) and nucleus with DAPI (blue). Scale bars represent 5 µm. G) Quantification of mitochondria-nucleus contact sites in in WT and RKO cell lines in control, hypoxia and EGF samples. Score was defined as number of Nesprin 3 (SYNE3) dots at distance zero of mitochondrial surface, normalized by total Nesprin 3 dots and total mitochondrial area. Welch 1-way ANOVA, n = 11-16. H) Immunostaining of PKA in WT and RANBP2 KO 10T1/2 cells with and without EGF treatment (40 ng/mL, 1 h). Scale bar represents 20 μm. Graph represents PKA nuclear intensity. Brown-Forsythe one-way ANOVA, n = 126-384. For all graphs: * p-value < 0.05, ** p-value < 0.01. Extended Data Fig. 6 RNASeq, H3K27me3 ChIPSeq and ATACSeq in RANBP2 KO cells. A) Limma pathway analysis of under-phosphorylated peptides, with fold change <0.8, in RKO vs WT cell lines. B) Panther pathway analysis of significantly downregulated genes in RKO vs WT cell lines. C) Representation of H3k27me3 and ATAC signals for functional categories Cardiac Development (GO terms 0003151, 0003205 and 0007507), Development and Cell Differentiation (GO terms 0051093, 0050793 and 0030154), and Neuronal Development (GO terms 0022008, 0007399 and 0007417). Each category is linked to the heatmaps (left) of genes involved that showed consistent signal among the three techniques (increased methylation, decreased chromatin accessibility, decreased expression). Extended Data Fig. 7 Mitochondrial proteome and metabolism in RANBP2 KO cell line. A) Glycolytic protein levels measured by targeted MS/MS proteomics. Mann-Whitney test, n = 3. B) Krebs’ Cycle protein levels measured by targeted MS/MS proteomics. Mann-Whitney test, n = 3. C) Total cellular ATP content in WT and RKO cells, as measured by HPLC. Unpaired t-test, n = 5. D) Total cellular creatine and phosphocreatine levels measured by mass spectrometry in WT and RKO cells. Mann-Whitney test, n=3. E and F) Fractional enrichment of topoisomers upon treatment of WT or RKO cell lines with 13C-Glucose after 6 or 24 h. Mann-Whitney test, n = 3. G) Metabolomic analysis by mass spectrometry in WT and RKO cells. Mann-Whitney test, n = 3. In A and B, data represent fold over WT (green dotted line). For all graphs, * p-value < 0.05. Extended Data Fig. 8 RANBP2 and VDAC1 mutant iPSC lines. A) Immunostaining of VDAC1 (green) and Mitotracker (red) in WT and VDAC1 mutant iPSC, showing proper mitochondrial localization of VDAC1 LII mutant. Scale bar represents 10 μm. B) PolyT-based FISH of mRNAs in WT and CTD-truncated iPSC. The graph shows the quantification of nuclear vs cytoplasmic intensity ration. Scale bar represents 10 μm. Welch t test, n = 6. C) Time-lapse images of WT and CTD-truncated iPSC expressing H2AX-GFP, showing normal mitosis (top), multipolar mitosis (middle) and segregation failure (bottom). Scale bar represents 20 μm. The graph shows the percentage of each type of mitosis detected. 2-way ANOVA, n = 4. D) Immunoprecipitation analysis of SUMOylated Ran in WT and CTD-truncated iPSC. The graph shows the quantification of IPed SUMO-Ran relative to total Ran in the input. Welch t test, n = 3. Extended Data Fig. 9 Proposed working model. The efficient transfer of mitochondria-derived energy molecules to the nucleus is mediated by VDAC1 and RANBP2-CTD interaction. The ATP regenerated by the action of nuclear creatine kinase (CK) is utilized in the nucleus for phosphorylation, replication, transcription, and chromatin dynamics, with implications in cell growth and differentiation. Schematic created in BioRender; Menendez-Montes, I. https://biorender.com/plzo9bu (2026). Extended Data Fig. 10 Mouse model of truncated RANBP2 C-terminal domain. A) Strategy to generate a C-terminal truncation of RANBP2 in vivo using CRISPR/Cas9 and a donor template. B) Sequencing of WT and KO bands showing efficient insertion of STOP codon ant 3′ UTR after the 4th RANBD1 domain. C) Genotyping of WT, heterozygous and KO mouse embryos for RANBP2 C-t deletion. D) TUNEL staining (green) and cTnT staining (red) on WT and CTD KO E10.5 embryonic heart sections. Scale bar represents 100 µm. E) WGA staining (green) on WT and CTD KO E10.5 embryonic heart sections (left) and quantification of cardiomyocyte cross-sectional area (right). Scale bar represents 25 µm. Unpaired t-test, n = 3. *p-value < 0.05. F) Representation of average BrdU %, heart size and cardiomyocyte size in published E9.5 and E10.5 WT embryos data and our WT and CTD KO embryos. Supplementary information Supplementary Figure 2 (download DOCX ) Omics pathway analysis in RKO cells. A) Limma pathway analysis of under-phosphorylated peptides, with fold change<0.8, in RKO vs WT cell lines. B) Panther pathway analysis of significantly downregulated genes in RKO vs WT cell lines Supplementary Tables (download ZIP ) Supplementary Tables 1–13 Supplementary Methods (download DOCX ) The Supplementary Methods file contains all the experimental methods information for the article Rights and permissions About this article Cite this article Menendez-Montes, I., Marin-Vicente, C., Mukherjee, S. et al. Mitochondria directly interact with the nuclear pore complex. Nature (2026). https://doi.org/10.1038/s41586-026-10588-3 Received: Accepted: Published: Version of record: DOI: https://doi.org/10.1038/s41586-026-10588-3