Yme1l substrate quantification under Leucine and Glutamine starvation as well as Torin treatment
Metabolic reprogramming of mitochondria occurs during development, cell differentiation and in cancer and is coupled to changes in mitochondrial mass and shape. Here, we demonstrate 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 ensures spheroid and xenograft growth of pancreatic ductal adenocarcinoma (PDAC) cells. Similar mitochondrial proteome changes occur in tumor tissues of PDAC patients, suggesting that YME1L is relevant to the pathophysiology of specific solid tumors. Our results identify the mTORC1-LIPIN1-YME1L axis as a novel post-translational regulator of mitochondrial proteostasis at the interface
Sample Processing Protocol
Protein Digestion 40 µl of heated (70°C) 2% SDC (Sodium deoxycholate, 30970, Sigma Aldrich) lysis buffer (100 mM Tris pH = 8.5) was added to the PBS-washed cell pellet and incubated for 15 min at 90°C. Samples were sonicated (Sonoplus mini20, MS 2.5 Sonotrode Bandelin) for 30 sec, using 100 % Power, 1s pulse) to disrupt cells and dissolve proteins. Lysates were cleared by centrifugation (10 min, 12.000 x g, RT). Protein concentration was determined using the 660 nm Protein Assay (Pierce, Thermo Fisher Scientific, 22660). 40 µg protein were dehydrated and alkylated for 45 min at 45°C in the dark using 10 mM TCEP (646547, Sigma Aldrich) and 15 mM CAA (108448, Sigma Aldrich). 1µg of Lys-C (Wako) and Trypsin (Sigma Aldrich) were added and samples were incubated for 16 h at 37°C. Samples were acidified by adding 10 % TFA to a final concentration of 2%. Precipitated SDC was removed by centrifugation (15 min, 12.000 x g, RT) and peptides were desalted using the StageTip (material: SDB-RPS, Affinisep) technique. To generate the spectra library, 1 mg of pooled samples (all conditions pooled 1:1) was digested as described above. Desalting was performed using SepPak 50mg (Waters) and peptides were separated using a Dionex 3000 chromatography system. The buffer systems consisted out of two buffers. A) 10 mM Ammonium hydroxide and B) 90 % acetonitrile and 10 mM Ammonium hydroxide. Peptides were separated according to their hydrophobicity using a Waters XBridge BEH C18 130 Å, 3.5 µm ID beads (length=250 mm, inner diameter = 3 mm). In total, 40 fractions were collected (1/60s). Fractions were concentrated using a SpeedVac and dissolved in 40 µL of 2 % FA, 2.5 % acetonitrile. For LC-MS/MS measurement 2 µg peptides per fraction were injected. Mass spectrometry LC-MS/MS instrumentation consisted out of an Easy nLC-1200 (Thermo Fisher) coupled via a nanospray ionization source to a QExactive HF-x (Thermo Fisher) mass spectrometer. Liquid chromatography gradients were identical for MS/MS spectra library and DIA measurements using two different solvents A and B (Buffer A: 0.1 % formic acid and Buffer B: 0.1 % formic acid in 80% acetonitrile). The in-house packed column inner diameter was 75 µm and filled with PoroShell C18 2.7 µm beads and was kept in a column oven at 50°C (PRSO-V2, Sonation). The buffer B percentage was linearly raised from 5 % to 29 % within 30 minutes and further increased to 55 % within 7 min. The column was washed at 95% B for 7 min. Samples were measured in random order. MS/MS Library – Data dependent acquisition For the MS/MS spectra library, the QExactive HF-x operated in a Top22 data-dependent mode. MS1 spectra were acquired in a mass range of 350-1750 m/z, using an AGC target of 3E6 and a resolution at 200 m/z of 60,000. MS/MS spectra were acquired at 15,000 resolution using an AGC target of 5E5 and a maximal injection time of 22ms. Data independent acquisition For DIA measurements, MS1 spectra were acquired using a resolution of 60,000, an AGC target of 1E6. For MS/MS independent spectra acquisition, 48 windows were acquired at an isolation m/z range of 9 Th and the isolation windows overlapped by 1 Th. The isolation centre range covered a mass range of 385 – 1043 m/z. Fragmentation spectra were acquired at a resolution of 15,000 at 200 m/z using a maximal injection time of 22 ms and stepped normalized collision energies (NCE) of 24 ,27, 30.
Data Processing Protocol
To generate the MS/MS Spectra library, acquired raw files were analysed by MaxQuant (184.108.40.206) and the implemented Andromeda search engine using default settings. MS/MS spectra were correlated against the Uniprot mouse reference proteome (June 2018 downloaded). The FDR was controlled by the implemented decoy algorithm and set to 1 % at the peptide-spectrum match and protein level. Evidence.txt and msms.txt output files were loaded into Spectronaut 13 containing 173,464 identified peptide sequences (10,520 protein groups). Acquired DIA raw files were analysed by Spectronaut 13 (Biognosis) using default settings (q value < 0.01). Experiments (Torin treatment, Glutamine and Leucine starvation) were analysed separately. Search sne files can be opened and viewed with Spectronaut Viewer and are publicly available (see data availability).
Hendrik Nolte, Max Planck Institute for Biology of Ageing, 50931 Cologne, Germany
Thomas Langer, Max Planck Institute for Biology of Ageing Joseph-Stelzmann-Str. 9b D-50931 Cologne, Germany ( lab head )