Fig. 4: CTLH substrates ZMYND19 and MKLN1 associate with lysosomes upon CTLH inhibition. | Nature Communications

Fig. 4: CTLH substrates ZMYND19 and MKLN1 associate with lysosomes upon CTLH inhibition.

From: The CTLH ubiquitin ligase substrates ZMYND19 and MKLN1 negatively regulate mTORC1 at the lysosomal membrane

Fig. 4: CTLH substrates ZMYND19 and MKLN1 associate with lysosomes upon CTLH inhibition.

a Confocal microscopy analysis of CTLH subcellular distribution. YCCEL1 expressing GFP-tagged MAEA for 24 h were stained with LysoTracker red. b Two regions of interest (ROI) marked in (a) were quantitated over an ~8 min timecourse to analyze for dynamic association between MAEA-GFP and lysosomes. c Schematic diagram of lysosomal immunopurification (LysoIP)72. HA-epitope tagged lysosomal transmembrane protein TMEM192 was stably expressed in YCCEL1. TMEM192HA decorated lysosomes were immunopurified with anti-HA magnetic beads. Created in BioRender. Guo, R. (2025) https://BioRender.com/580vurf. d Immunoblots of WCL vs LysoIP samples from YCCEL1 TMEM192-HA expressing cells that also expressed control or MAEA sgRNAs and that were treated with 0.5 μM alpelisib for 24 h. Lysosomal marker LAMP1, Golgi marker Golgin97, ER marker calreticulin, mitochondrial marker VDAC1, and cytosol marker GAPDH are shown as controls. HC, immunoglobulin heavy chain. e Immunoblots of WCL vs LysoIP samples from YCCEL1 transfected with in vitro transcribed (IVT) mRNAs encoding V5-tagged GFP, ZMYND19, or MKLN1. f Analysis of ZMYND19 and LAMP2 co-localization. Representative confocal microscopy images of YCCEL1 expressing control, MAEA, or ZMYND19 sgRNA. ZMYND19 and lysosomal marker LAMP2 were stained with Alexa Fluor 488 (green) or Alexa Fluor 595 (red) conjugated antibodies. g Immunoblot analysis of WCL vs LysoIP samples from YCCEL1 TMEM192-HA expressing cells that also expressed MAEA and ZMYND19 sgRNAs. HC: immunoglobulin heavy chain. h Analysis of MKLN1 and ZMYND19 subcellular localization. YCCEL1 with stable HA-ZMYND19 were gently lysed by Dounce homogenization and co-incubated with proteinase K (10 µg/ml) for 15 min on ice. As a positive control, Triton X-100, which disrupts lysosomal membranes, was added together with proteinase K. Shown are immunoblot analyses following the indicated proteinase K and Triton X-100 treatments, including for the intra-lysosomal resident protein Cathepsin L. i Schematic model. A cytosolic subpopulation of MKLN1-containing CTLH complexes targets lysosome-associated ZMYND19 and MKLN1 for proteasomal degradation. Immunoblot analysis and confocal microscopy are representative of at least n = 3 biologically independent replicates. Source data are provided as a Source Data file for (d), (e), (g), and (h). Created in BioRender. Guo, R. (2025) https://BioRender.com/jpdx7d4.

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