Extended Data Figure 10: DENR activity is higher in proliferating cells compared to quiescent cells.

a, b, DENR–MCT-1 binding is not different in proliferating versus quiescent S2 cells. a, Immunoprecipitation of endogenous DENR from proliferating or quiescent S2 cells shows similar amounts of co-immunoprecipitating endogenous MCT-1 in the two conditions. b, A stably transfected S2 cell line bearing a copper-inducible pMT-V5-MCT-1 construct was grown in proliferative or quiescent conditions, as in Extended Data Fig. 7, and then induced to express MCT-1. V5-tagged MCT-1 was immunoprecipitated and endogenous DENR was detected by immunoblotting. An anti-Flag immunoprecipitation was performed as a negative control. Near-equal levels of DENR protein co-immunoprecipitated with MCT1 in both proliferative and quiescent conditions. c, Expression levels of DENR and eIF2D do not depend on each other and do not change in proliferating versus quiescent cells. Immunoblot of S2 cells treated with DENR, eIF2D or control (GFP) dsRNA, in proliferative or quiescent conditions. Two different amounts of the control samples were loaded to quantitatively assess knockdown efficiencies. A non-specific band on eIF2D blot is marked with an asterisk. d, d′, Abolishing phosphorylation on T118 and S119 blunts MCT-1 activity. d, Luciferase assay using a stuORF reporter in S2 cells depleted of endogenous MCT-1 via dsRNA targeting its 3′ UTR, and re-constituted to express wild-type or mutated forms of MCT-1 (using constructs with an exogenous 3′ UTR), identifies T118 and S119 as important phosphorylation sites. Four sites were tested—T118, S119, T82 and T125—for all of which phosphorylations on endogenous MCT-1 were observed by publicly available proteome-wide mass spectrometry analyses (PhosphoPep and PhosphoSite.org). Whereas wild-type MCT-1 is able to restore expression of the stuORF reporter, a T118A/S119A mutant form of MCT-1 cannot. Testing each site individually identifies S119 as the more important of the two. In contrast, mutating T82 and T125 to alanine does not impair their ability to promote stuORF translation. t-test **P < 0.01. Error bars indicate s.d. d′, MCT-1(T118A/S119A) is expressed and stable. Immunoblot of S2 cells transfected with control plasmid or plasmid expressing wild-type Flag–MCT-1 or Flag–MCT-1(T118A/S119A) shows that the mutant MCT-1 is expressed at roughly equivalent levels as the wild-type protein. e, f, Inhibition of Erk, PI(3)K, Akt or TORC1 does not have an effect on stuORF translation. Luciferase assay with a stuORF reporter in S2 cells treated with inhibitors for Erk (U0126, f), TOR complex 1 (rapamycin, e), Akt (Akt Inhibitor VIII, e) or PI(3)K (Wortmannin, e) shows that inhibition of any of these kinases has little to no effect on stuORF translation. g, Knockdown of Drosophila cdc2 (CG10498) does not reduce expression of a stuORF reporter. S2 cells were treated with either GFP dsRNA or CG10498 dsRNA, and then transfected with a stuORF reporter. GFP dsRNA was applied to 90 wells in a 96-well plate and the values displayed represent the average and s.d. (error bars). CG10498 knockdown results for two separate wells are shown.