Journal: Nature Cell Biology

Article Title: Malonyl-CoA is a conserved endogenous ATP-competitive mTORC1 inhibitor

doi: 10.1038/s41556-023-01198-6

Figure Lengend Snippet: a , Microscale thermophoresis experiment using purified TORC1 (containing GFP–Tor1) from yeast cells, confirming binding of Mal-CoA to TORC1; n = 3 independent experiments. b , Mal-CoA does not affect mTORC1 complex stability. Endogenous mTOR was immunoprecipitated in the presence or absence of 1 mM Mal-CoA (added directly in the lysates 5 min previous to addition of the antibody). Co-immunoprecipitation of Raptor and mLST8 assayed by immunoblotting; n = 2 independent experiments. c , Mal-CoA inhibits mTORC1 independently from mTOR malonylation. IVKs as in Fig. using SBP-tagged WT or K1218R mutant mTOR and HA–Raptor in the presence or absence of 5 mM Mal-CoA. Reactions omitting ATP were used as negative controls; n = 2 independent experiments. d , e , Mal-CoA does not inhibit Snf1 in vitro. d , IVK assays as in Fig. , but using purified Snf1 and His-tagged Mig1 as substrate, with the indicated concentrations of Mal-CoA. e , Quantification of Snf1 activity; n = 3 independent experiments. f , g , Src IVK assay as in Fig. using Glo1 as a substrate, with the indicated amounts of Mal-CoA. g , Quantification of Src activity; n = 3 independent experiments. h , i , Stability of HA-tagged Tor1 R2105/2107A mutant in vivo. i , Quantification of Tor1 protein levels normalized to Adh1; n = 3 independent experiments. j , In vitro stability of HA-tagged Tor1 R2105/2107A mutant purified from yeast cells via TAP-mediated pulldown of Tco89; n = 2 independent experiments. k , The human SBP-tagged mTOR R2168A/R2170A mutant (mTOR RR/AA ) is relatively stable and binds other mTORC1 components similarly to WT mTOR. Streptavidin pulldown of WT or mTOR RR/AA detecting binding to HA–Raptor and endogenous mLST8; n = 2 independent experiments. l , The mTOR RR/AA mutant lacks catalytic kinase activity in vitro. IVKs as in Fig. using SBP-tagged WT or mTOR RR/AA and HA–Raptor in the presence or absence of 5 mM Mal-CoA. Reactions omitting ATP were used as negative controls; n = 2 independent experiments. Data are the mean ± s.d. ( a ) or mean ± s.e.m. (all other graphs). *** P < 0.0005. Source numerical data and unprocessed blots are provided.

Article Snippet: Briefly, bacterially purified His-tagged human Glo1 recombinant protein was mixed with 600 ng recombinant GST-tagged Src (0200-0000-1, ProQinase) in 1×kinase assay buffer (50 mM HEPES pH 7.4, 6 mM MgCl 2 , 6 mM β-glycerophosphate and 1 mM DTT) and 500 μM ATP in a total volume of 25 μl with the indicated concentrations of Mal-CoA, and incubated for 1 h at 30 °C.

Techniques: Microscale Thermophoresis, Purification, Binding Assay, Immunoprecipitation, Western Blot, Mutagenesis, In Vitro, Activity Assay, In Vivo