Extended Data Fig. 4: mTORC1 acutely regulates YME1L-dependent proteolysis and mitochondrial import.
From: Lipid signalling drives proteolytic rewiring of mitochondria by YME1L
a–c, 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).
