Fig. 2: TM4SF19, a lysosomal membrane protein, inhibits V-ATPase V1-V0 holoenzyme assembly and lysosomal acidification by interacting with ATP6V0B. | Nature Communications

Fig. 2: TM4SF19, a lysosomal membrane protein, inhibits V-ATPase V1-V0 holoenzyme assembly and lysosomal acidification by interacting with ATP6V0B.

From: TM4SF19-mediated control of lysosomal activity in macrophages contributes to obesity-induced inflammation and metabolic dysfunction

Fig. 2

A Representative fluorescence images of RAW264.7 cells overexpressing GFP-tagged TM4SF19 (green) co-stained with the indicated markers (red) (lysosomal associated membrane protein 1(LAMP1), protein disulfide-isomerase (PDI)) (n = 6 biologically independent replicates). Scale bar = 5 μm. B Immunoprecipitation analysis of Myc-tagged TM4SF19 overexpressing in RAW264.7 cells (representative results from 3 independent experiments). C, D Immunoblot analysis of TM4SF19, ATP6V1B2, ATP6V0B, LAMP1, and β-actin protein levels in membrane and cytosolic fractions of WT and TM4SF19 KO BMDM (C) (n = 6) and TM4SF19 overexpressing RAW264.7 cells (OE) and negative control cells (Mock) (D) (n = 6 biologically independent replicates). E V-ATPase activity of lysosomal fractions from TM4SF19 KO and WT BMDM or TM4SF19 overexpressing RAW264.7 cells and negative controls (n = 6 biologically independent replicates). F Representative images of BMDM obtained from WT and TM4SF19 KO mice, stained with LysoSensor Yellow/Blue dextran. Yellow fluorescence represents an acidic lysosomal environment, and blue fluorescence represents a neutral lysosomal environment (n = 6 biologically independent replicates). Scale bar = 100 μm. G, H Long-term live imaging (G) and imaging of fixed cells (H): WT and TM4SF19 KO BMDMs (stained with DiO: green) cocultured with dying adipocytes (stained with BODIPY: red). Scale bar = 100 μm (G). The arrows indicate CtB (green)-stained BMDM that phagocytosed LipidTOX (red)-labeled lipids (LipidTOX+CtB+). Scale bar = 20 μm (H). The intensity analysis was conducted on each well, with one center field analyzed as six biologically independent replicates (n = 6). I An illustration of the working model where TM4SF19 interacts with V0 domain of V-ATPase, and inhibits the assembly of V1/V0 complex, resulting in reduced lysosomal acidification. Data are presented as mean values ± SEM. p-values were determined by the unpaired two-sided Student’s t-test (C–H). Source Data are provided as a Source Data File.

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