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

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

From: Lipid signalling drives proteolytic rewiring of mitochondria by YME1L

Extended Data Fig. 5

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

Back to article page