Fig. 7: ZMYND19 and MKLN1 block a late stage in mTORC1 activation, distal to lysosomal membrane recruitment.

a Analysis of MAEA depletion effects on amino acid stimulation-driven mTOR lysosomal subcellular localization. Representative confocal microscopy images of YCCEL1 expressing control or MAEA sgRNA and amino acid starved for 50 min and then cultured in amino acid-free media (top) or with amino acid addback for 10 min (bottom). mTOR and lysosomal marker LAMP2 were stained with Alexa Fluor 488 (green) or Alexa Fluor 595 (red) conjugated antibodies, respectively. b Quantification of co-localization of mTORC1 and LAMP2 in immunofluorescence images in (a). Shown are mean ± SD values from n = 25 independent replicates of YCCEL1 expressing MAEA sgRNA vs control sgRNA cells. Pearson’s correlation coefficients were analyzed with Fiji. P-values were calculated by two-tailed one-way ANOVA. c Analysis of whether TSC2 is necessary for mTORC1 inhibition upon MAEA depletion. Immunoblot analysis of WCL from YCCEL1 that expressed the indicated MAEA or TSC2 sgRNAs (#1 and #2). d Analysis of MAEA depletion effects on Raptor association with Rheb and with mTORC1 substrates S6K and 4E-BP1. Immunoblot analysis of 5% input versus anti-GFP immuno-purified complexes from 293T control or MAEA KO single cell clones that were transiently transfected with Rheb-V5, GFP, or Raptor-GFP expression vectors. e Analysis of MKLN1 and ZMYND19 co-expression effects on Raptor association with Rheb and with mTORC1 substrates S6K and 4E-BP1. Immunoblots of 5% input versus anti-GFP immuno-purified complexes from 293 T cells with stable Rheb-V5 expression and transiently transfected with GFP, Raptor-GFP, MKLN1, and ZMYND19 expression vectors. f Schematic model of CTLH, ZMYND19, and MKLN1 roles in mTORC1 regulation. A subpopulation of CTLH homes to lysosome regions. When MAEA is active, CTLH targets ZMYND19 and MKLN1 for proteasomal degradation. Upon loss of MAEA-dependent CTLH activity, ZMYND19 and MKLN1 associate at lysosome outer membrane sites, perhaps through ZMYND19 zinc-finger association with Raptor. Rather than blocking mTORC1 lysosomal recruitment, ZMYND19 and MKLN1 block mTORC1 association with Rheb and with its substrates S6K and 4E-BP1. Created in BioRender. Guo, R. (2025) https://BioRender.com/9oyxckk and https://BioRender.com/algs0ol. Immunoprecipitation and Immunoblot analysis are representative of at least n = 3 biologically independent replicates. Source data are provided as a Source Data file for (b–e).