Lipid signalling drives proteolytic rewiring of mitochondria by YME1L

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Abstract

Reprogramming of mitochondria provides cells with the metabolic flexibility required to adapt to various developmental transitions such as stem cell activation or immune cell reprogramming, and to respond to environmental challenges such as those encountered under hypoxic conditions or during tumorigenesis1,2,3. Here we show that the i-AAA protease YME1L rewires the proteome of pre-existing mitochondria in response to hypoxia or nutrient starvation. Inhibition of mTORC1 induces a lipid signalling cascade via the phosphatidic acid phosphatase LIPIN1, which decreases phosphatidylethanolamine levels in mitochondrial membranes and promotes proteolysis. YME1L degrades mitochondrial protein translocases, lipid transfer proteins and metabolic enzymes to acutely limit mitochondrial biogenesis and support cell growth. YME1L-mediated mitochondrial reshaping supports the growth of pancreatic ductal adenocarcinoma (PDAC) cells as spheroids or xenografts. Similar changes to the mitochondrial proteome occur in the tumour tissues of patients with PDAC, suggesting that YME1L is relevant to the pathophysiology of these tumours. Our results identify the mTORC1–LIPIN1–YME1L axis as a post-translational regulator of mitochondrial proteostasis at the interface between metabolism and mitochondrial dynamics.

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Fig. 1: YME1L-mediated proteolysis is required for spheroid growth.
Fig. 2: mTORC1 acutely regulates YME1L-dependent proteolysis and mitochondrial import.
Fig. 3: mTORC1 regulates mitochondrial PE in a LIPIN1-dependent manner.
Fig. 4: YME1L-mediated proteolysis is required for PDAC cell growth.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the following dataset identifiers: PXD011750 (DDA data—hypoxia/normoxia WT and Yme1l1−/− MEF mitochondria, https://www.ebi.ac.uk/pride/archive/projects/PXD011750) and PXD014405 (MEF MS/MS library and DIA data, https://www.ebi.ac.uk/pride/archive/projects/PXD014405). Transcriptomic data from hypoxia/normoxia WT MEF cells have been deposited to the GEO omnibus (accession number GSE133753). These datasets are presented in Supplementary Tables 16. Uncropped immunoblot images are available in Supplementary Fig. 1 and all Source Data are available online.

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Acknowledgements

We thank D. Ehrentraut for expert technical assistance; L. Raatz and M. Heitmann for assisting with xenograft analysis and tissue protein extraction; and A. Wilbrand-Hennes and U. Cullmann of the CECAD proteomics facility for technical assistance. RNA sequencing was performed at the Cologne Center for Genomics and Data Analysis in the MPI Biology of Ageing bioinformatics core facility. We thank J. Altmüller, M. Franitza, F. Metge and J. Boucas for technical and bioinformatic assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (La918/15-1; SFB1218/A1), the German-Israel-Project (DIP; RA1028/10-1) and the Max-Planck-Society to T.L.; EMBO and Alexander von Humboldt fellowships to T.M. (GA-2013-609409, ALTF 1220-2014); and a fellowship of the Japan Society for the Promotion of Science (JSPS) for research abroad, The Osamu Hayaishi Memorial Scholarship for Study Abroad and grants from the Uehara Memorial Foundation to Y.O. We are grateful for the following gifts: Tsc2/ MEFs from C. Demetriades and D. Kwiatkowski, Atg5−/− MEFs from K. Winklhofer, WT (S/S) and EIF2αS51A knock-in MEFs from R. Kaufman, 4E-BP DKO, iRaptor and iRictor MEFs from H. McBride and pUC57-LbNOX from V. Mootha (Addgene plasmid # 75285).

Author information

The study was conceived and designed by T.M., Y.O. and T.L.; T.M. performed the analysis in hypoxia and spheroids; Y.O. performed lipid analysis and in vitro reconstitution experiments; proteomic experiments were performed and evaluated by T.M., B.L., F.C.M., H.N. and M.K.; metabolomic experiments were performed by T.M. and N.Z.; T.T. performed lipid transfer assays; H.-G.S. supported data interpretation; Y.Z., J.L. and C.B. provided support for clinical materials and performed the xenograft assay; T.M., M.H. and J.R. performed and evaluated import and labelling experiments; R.S., M.P., S.H. and J.C.B. assisted with in vivo studies and supported data interpretation; T.M., Y.O. and T.L. wrote the manuscript, which was edited by all authors.

Correspondence to Thomas Langer.

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The authors declare no competing interests.

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Peer review information Nature thanks Thomas Becker, Markus Ralser and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Characterization of YME1L-dependent spheroid growth.

a, b, Spheroid surface area (a) and ATP levels (b) of HEK293 cell lines after 7 days of culture (a, n = 5 independent experiments; b, n = 3 independent experiments; # is number of spheroids per condition). c, d, Spheroid surface area (c) and ATP content (d) of WT and YME1L/ HeLa cells after 7 days (n = 3 independent experiments; # is number of spheroids per condition). e, Quantification of the NAD/NADH ratio in WT and YME1L/ HEK293 cells cultured in normoxia (21% O2) or hypoxia (0.5% O2) for 24 h (n = 3 independent experiments). f, Spheroid surface area of WT and YME1L/ HEK293 cells and of YME1L/ HEK293 cells expressing Lb-NOX (NADH oxidase from Lactobacillus brevis)47 after 7 days (n = 1 experiment; # is number of spheroids per condition). The expression of Lb-NOX was determined by SDS–PAGE and immunoblotting of control and Lb-NOX-expressing WT and YME1L/− HEK293 cells in normoxia (21% O2) or hypoxia (0.5% O2) for 24 h. g, The dependency of WT and YME1L/ HEK293 cells on glucose oxidation (left) and fatty acid oxidation (right) was monitored using the XF Fuel Flex Assay. Dependencies are calculated as a percentage of combined glucose, fatty acid and glutamine oxidation (n = 4 independent experiments). hj, Quantification of cellular metabolites from HEK293 cells (h, i) and MEFs (j). Metabolite abundance was normalized to those of WT cells (n = 5 independent experiments). Mean ± s.e.m.; one-way ANOVA with Dunnett’s multiple comparisons test (a, b); two tailed t-test (c, d, g); two-way ANOVA with Sidak’s multiple comparisons test (e). Source data

Extended Data Fig. 2 HIF1α drives YME1L-dependent proteolysis and occurs independently of mitophagy.

a, SDS–PAGE and immunoblot analysis of WT and YME1L/ HEK293 cells expressing YME1L or YME1L(E543Q) cultured for 7 days as monolayers (M) or spheroids (S) (n = 1 experiment). b, Representative immunoblot of WT and YME1L/ HEK293 cells and YME1L/ HEK293 cells expressing YME1L or YME1L(E543Q) cultured in normoxia (21% O2) or hypoxia (0.5% O2) for 16 h (n = 4 independent experiments). c, SDS–PAGE and immunoblot analysis of the indicated cell lines treated with control (Scr) or Yme1l siRNA and incubated in normoxia (21% O2) or hypoxia (0.5% O2) for 16 h (n = 1 experiment). d, SDS–PAGE and immunoblot analysis of WT and YME1L/ HEK293 cells treated with Co(II)Cl2 (200 µM) for 24 h or DFP (1 mM) for 24 h. Quantification of PRELID1 and TIMM23 protein levels is shown (n = 3 independent experiments; fc, fold change). e, Quantified PRELID1 and TIMM17A protein levels in HeLa cells transfected with Gfp or Hif1α esiRNA cultured in normoxia or hypoxia for 24 h (example blot shown in Fig. 1 f; n = 3 independent experiments; fc, fold change). f, SDS–PAGE and immunoblot analysis of WT and Atg5/ MEFs cultured in hypoxia (0.5% O2) for the indicated time. Quantification of PRELID1 and TIMM23 protein levels is shown (n = 3 independent experiments; fc, fold change). Mean ± s.e.m.; two-way ANOVA with Sidak’s multiple comparisons test (e). Source data

Extended Data Fig. 3 YME1L reshapes the mitochondrial proteome independently of OMA1.

a, Box plot analysis of the log2 ratio distribution comparing hypoxia and normoxia in WT and Yme1l/ MEFs of total proteins and mitochondrial proteins. P values calculated by Wilcoxon sum-rank test. Centre lines denote medians, box limits denote 25th and 75th percentiles; whiskers denote maxima and minima (1.5 times the interquartile range). Data located outside the maxima or minima were denoted as outliers and removed. b, Volcano plot representation of proteins determined by quantitative mass spectrometry after isolation of mitochondria from WT and Yme1l/ MEFs cultivated under normoxic conditions (dataset as in Fig. 1g, n = 5 independent experiments, two-tailed t-test). Filled plots indicate proteins that differ significantly between Yme1l/ and WT MEFs at a permutation-based estimated FDR < 0.05. Among these, mitochondrial proteins (according to Gene Ontology Cellular Component) are highlighted in blue. Mitochondrial proteins enriched in Yme1l/ compared to WT are putative YME1L substrates (class I or II). c, d, Volcano plots of mitochondrial protein changes in hypoxia versus normoxia from WT (c) or Yme1l/ (d) MEFs (dataset as in Fig. 1g, n = 5 independent experiments, two-tailed t-test). Class I YME1L substrates are highlighted in red. e, Z-score of log2-transformed LFQ intensities of class I YME1L substrates in WT, Oma1/ and Oma1/Yme1l/ MEFs treated as in Fig. 1g (n = 5 independent experiments). f, SDS–PAGE and immunoblot analysis of WT, Yme1l/ and Oma1/ MEFs cultured in normoxia (21% O2) or hypoxia (0.5% O2) for 24 h. # denotes nonspecific cross-reaction (n = 1 experiment). g, Spheroid surface area of the indicated HEK293 cell lines after 7 days (means from 16 spheroids shown, n = 1 experiment). Source data

Extended Data Fig. 4 mTORC1 acutely regulates YME1L-dependent proteolysis and mitochondrial import.

ac, SDS–PAGE and immunoblot analysis of WT and YME1L−/− HEK293 cells cultured in hypoxia (0.5% O2) (a), glutamine-depleted medium (b) or in the presence of 400 nM Torin1 (c) for the indicated times (representative immunoblots from n = 3 independent experiments, quantification shown in Fig. 2b). d, Immunofluorescence of WT and YME1L/ HeLa cells treated with Torin1 (400 nM) for 4 h. Cells were immunostained with TIMM17A- and ATP5β-specific antibodies. Quantification of mean fluorescence intensity per cell is shown (TIMM17A mean from three independent experiments, two-way ANOVA with Sidak multiple comparisons test; ATP5β mean from two independent experiments). # is number of cells per condition; au, arbitrary unit; scale bar, 10 µm. e, Immunoblot of WT, Raptor and Rictor knockout (KO) MEFs. Quantified PRELID1 protein levels are shown (Raptor, n = 4 independent experiments; Rictor, n = 3 independent experiments; mean ± s.e.m.; two-tailed t-test). fc, fold change. # denotes nonspecific cross-reaction. f, HEK293 cells were cultured in glutamine-depleted medium (Starve) for 16 h and then cultured in non-essential amino acid (NEAA)-containing medium for the indicated time. Cell lysates were analysed by SDS–PAGE and immunoblotting (n = 1 experiment). g, HEK293 cells were cultured in serum-depleted medium for 16 h and treated with insulin (100 nM) for the indicated time. Cell lysates were analysed by SDS–PAGE and immunoblotting (n = 1 experiment). h, i, [35S]-SOD2 (h) and [35S]-LIAS (i) were imported into mitochondria isolated from WT and YME1L−/− HEK293 cells treated with or without 400 nM Torin1 for 18 h. Import was stopped after 1.5, 3 and 4.5 min in the presence or absence of membrane potential (∆ψ). Mitochondria were analysed by SDS–PAGE and autoradiography (representative blots from n = 2 (h) and n = 3 (i) independent experiments). Arrows indicate the mature form of SOD2. j, [35S]-LIAS and [35S]-SOD2 were imported into mitochondria isolated from WT and YME1L−/− HEK293 cells incubated in normoxia (21% O2) or hypoxia (0.5% O2) for 18 h. Import was stopped after 5, 10 and 15 min. Mitochondria were analysed by SDS–PAGE and levels of imported [35S]-LIAS and [35S]-SOD2 were observed by autoradiography (n = 1 experiment). Source data

Extended Data Fig. 5 mTORC1 regulates YME1L-mediated proteolysis in a post-translational manner.

a, b, YME1L degradation of substrates does not depend on eIF2α phosphorylation and integrated stress response (ISR) activation. SDS–PAGE and immunoblot analysis of WT (S/S) and EIF2αS51A knock-in (A/A) MEFs, which cannot activate the ISR48, treated with Torin1 for 4 h (a; n = 3 independent experiments, mean ± s.e.m.) or cultured in normoxia (21% O2) or hypoxia (0.5% O2) for 24 h (b; n = 3 independent experiments). c, d, YME1L degradation of substrates does not depend on mTORC1 regulation of translation initiator factor 4 binding proteins (4E-BPs). SDS–PAGE and immunoblot analysis of WT and Eif4ebp1/Eif4ebp2/ (4E-BP DKO) MEFs treated with Torin1 for 4 h (c; n = 4 independent experiments, mean ± s.e.m.) or cultured in normoxia (21% O2) or hypoxia (0.5% O2) for 16 h (d; n = 3 independent experiments). e, f, We observed a slight reduction in newly synthesized TIMM17A in Torin1-treated WT HEK293 cells, consistent with previous reports49. After treatment with DMSO or Torin1 for 2 h, cells were incubated in labelling medium containing [35S]-methionine for the indicated time before lysis and immunoprecipitation with an antibody targeting TIMM17A. Input (5% of total) and immunoprecipitates (IP) were analysed by SDS–PAGE and autoradiography. [35S]-TIMM17A (indicated by an arrow) was quantified (f). Total TIMM17A protein level was determined by immunoblotting (n = 3 independent experiments, mean ± s.e.m.). au, arbitrary unit. gi, TIMM17A synthesis and the majority of global translation was restored in Torin1-treated MEFs that lack 4E-BP proteins. This confirms that the reduced synthesis of TIMM17A upon mTORC1 inhibition reflects 4E-BP-dependent attenuation of translation49. After treatment with DMSO or Torin1 for 2 h, cells were incubated in labelling medium containing [35S]-methionine for 60 min before lysis and immunoprecipitation with an antibody targeting TIMM17A. TIMM17A levels (h) were analysed and quantified as in f (n = 3 independent experiments, mean ± s.e.m.). Total protein synthesis was determined by the intensity of all bands in input lanes (i) (n = 2 independent experiments). The synthesis rate of TIMM17A was quantified in WT and Eif4ebp1/Eif4ebp2/ (4E-BP DKO) MEFs. au, arbitrary unit. jl, Post-translational degradation of YME1L substrates monitored by [35S]-methionine pulse-chase experiment in HEK293 cells. After labelling for 1 h in [35S]-methionine containing medium, cells were incubated for 4 h and 6 h in radioactive-free medium in the presence and absence of Torin1. [35S]-labelled TIMM17A and STARD7 were immunoprecipitated and their levels determined by SDS–PAGE and autoradiography. Quantification of the fraction of [35S]-TIMM17A (k) and [35S]-STARD7 (l) remaining after 4 and 6 h chase is shown. Torin1 treatment accelerated TIMM17A and STARD7 degradation (n = 3 independent experiments for 4 h, n = 4 independent experiments for 6 h; mean ± s.e.m.; two tailed t-test). Source data

Extended Data Fig. 6 Decreased mitochondrial PE promotes YME1L-mediated proteolysis.

a, b, Phospholipid analysis of mitochondrial fractions from HeLa cells treated with Torin1 for 4 h (n = 3 independent experiments); a, relative distribution; b, absolute abundance. c, Acyl chain composition of mitochondrial PE from HeLa cells treated with Torin1 for 4 h (n = 3 independent experiments). d, Phospholipid analysis of mitochondrial fractions from WT, PRELID3B/ and PRELID3B/ HeLa cells expressing PRELID3B–Flag or PRELID3B(T57K)–Flag (n = 3 independent experiments). e, Quantification of protein levels from Fig. 2h (n = 4 independent experiments). f, g, Immunoblot and mitochondrial phospholipid analysis of scrambled control (Scr) or Prelid3b siRNA-transfected HeLa cells. Quantification of indicated protein levels is shown (immunoblot, n = 6 independent experiments; phospholipid analysis, n = 3 independent experiments). h, NBD-PS transfer by PRELID3B (black circles), PRELID3B(T57K) (red circles) or without addition of protein (white circles). PRELID3B(T57K) contains a mutation in the PS-binding site, which abolishes PS transfer but does not interfere with the assembly of PRELID3B into lipid transfer complexes. Average of n = 3 independent experiments. i, SDS–PAGE analysis of recombinant PRELID3B–TRIAP1 complexes and the T57K variant (40 pmol) by CBB staining (n = 1 experiment). j, Quantification of protein levels from Fig. 2i (n = 5 independent experiments). k, l, Phospholipid analysis of the mitochondrial fraction from HeLa cells treated with scrambled control (Scr) or Pisd siRNA (n = 3 independent experiments) (k), WT and Tsc2−/− MEFs (n = 3 independent experiments) (l). Mean ± s.e.m.; two tailed t-test (a, b, f, g, jl), one-way ANOVA with Dunnett’s multiple comparisons test (d, e). fc, fold change. Source data

Extended Data Fig. 7 PE regulates YME1L-mediated proteolysis.

a, b, HeLa cells pretreated with ethanolamine (Etn, 200 µM) for 24 h were treated with Torin1 for 4 h. Phospholipids in mitochondrial fractions were analysed by mass spectrometry (a) (n = 3 independent experiments). Cell lysates were analysed by immunoblotting and indicated protein levels were quantified (n = 5 independent experiments) (b). c, d, HEK293 cells were treated with Torin1 and/or 100 µM LPE for 4 h. Phospholipids in mitochondrial fractions were analysed by mass spectrometry (c) (n = 3 independent experiments). Cell lysates were analysed by immunoblotting and indicated protein levels were quantified (n = 4–5 independent experiments) (d). e, f, HeLa cells transfected with scrambled control (Scr) or Pisd siRNA were treated with 100 µM LPE for 24 h. Cell lysates were analysed by immunoblotting and indicated protein levels were quantified (n = 5 independent experiments) (e). PE levels in mitochondrial fractions were determined by mass spectrometry (n = 3 independent experiments) (f). g, h, YME1L (WT or E543Q) and OPA1ΔC (g) or TIMM17A (h) reconstituted in liposomes was incubated in the presence or absence of ATP at 37 °C for the indicated times. Samples were analysed by immunoblotting (representative data from n = 3 independent experiments). i, j, YME1L (WT or E543Q) and OPA1ΔC (i) or TIMM17A (j) reconstituted in liposomes containing different amounts of PE were incubated in the presence of ATP at 37 °C for the indicated times. Samples were analysed by immunoblotting (representative data from n = 4 independent experiments). Mean ± s.e.m.; two-way ANOVA with Tukey’s multiple comparisons test (ad), with Holm-Sidak’s multiple comparisons test (e, f). fc, fold change. Source data

Extended Data Fig. 8 Depletion of LIPIN1 preserves YME1L-mediated proteolysis after inhibition of mTORC1.

a, HeLa cells transfected with the indicated esiRNAs were treated with Torin1 for 4 h before immunoblotting. Representative immunoblot and quantification of TIMM17A protein levels are shown (n = 3 independent experiments; fold change (fc) from DMSO control). b, c, Immunoblot analysis of HeLa cells transfected with Gfp or Lipin1 esiRNA (b) or scrambled control (Scr) or Lipin1 siRNA (c) treated with Torin1 for 4 h. Quantification of PRELID1 and TIMM17A protein levels is shown (n = 7 independent experiments (b), n = 6 independent experiments (c)). d, Immunoblot analysis of MEFs transfected with Gfp or Lipin1 esiRNA and treated with Torin1 for 4 h. Quantification of TIMM17A protein levels is shown (n = 5 independent experiments). e, Immunoblot analysis of HeLa cells transfected with the indicated esiRNAs and treated with Torin1 for 4 h (representative data from six independent experiments). f, The mTORC1–LIPIN1–YME1L regulatory axis. Upon inhibition of mTORC1, dephosphorylation of LIPIN1 stimulates its PA-phosphatase activity and leads to a decrease in PA. The depletion of PA inhibits CCTα and therefore limits the supply of PS to mitochondria. Reduced PS transfer to the inner membrane limits accumulation of PE, stimulating YME1L-mediated proteolysis. Mean ± s.e.m.; two-way ANOVA with Tukey’s multiple comparisons test (bd). Source data

Extended Data Fig. 9 YME1L-mediated proteolysis is enhanced in PDAC and supports the growth of PDAC cells.

a, b, SDS–PAGE and immunoblot analysis of pancreatic tissue from patients with PDAC (a) and liver tissue from patients with HCC (b). T, tumour tissue; NT, corresponding adjacent non-tumour tissue. Quantification of indicated protein levels is shown below (n = 6 independent patient samples). YME1L substrate proteins are highlighted in red, other mitochondrial proteins shown in dark grey. The immunoblots from two other patients are shown in Fig. 4a, b. c, Cellular ATP levels in spheroids of the indicated cell lines cultured for 6 days (n = 3 independent experiments for HPAF-II, HepG2 and Huh7, n = 4 independent experiments for PANC1; # is number of spheroids; fc, fold change). d, e, Immunoblot analysis of spheroids from HPAF-II (d) and Huh7 (e) cells treated with Gfp or Lipin1 esiRNA, quantified in Fig. 4e, f. n = 1 immunoblot. f, Representative immunoblot of HPAF-II cells expressing shGfp and shYme1l (n = 3 independent samples). g, Weights of xenograft tumours derived from HPAF-II cells treated with Gfp or Yme1l shRNA 28 days after injection (Gfp n = 7 mice; Yme1l n = 8 mice). Mean ± s.e.m.; two tailed unpaired t-test (ac, g). Source data

Extended Data Table 1 Class I and class II YME1L substrates

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MacVicar, T., Ohba, Y., Nolte, H. et al. Lipid signalling drives proteolytic rewiring of mitochondria by YME1L. Nature 575, 361–365 (2019) doi:10.1038/s41586-019-1738-6

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