and contributes to ATP production by NAD+.Figure 6(A) Schema of purification of lysosomes by centrifugation. We applied a low osmotic medium for density gradient centrifugation. After primary organelle enrichment by differential centrifugation from WT MEFs, the subcellular compartments were separated on a discontinuous iodixanol gradient. After membrane pellets were enriched (input), each organelle was purified by an OptiPrepTM density gradient. We separated the five fractions from top to bottom. After ultracentrifugation, five individual fractions became apparent at the individual interphases, and were collected, pelleted and used for subsequent analyses. (B) The western blots were analyzed for the presence of known organelle marker proteins as indicated. Lysosomal and mitochondrial fractions from WT MEFs were enriched in fractions 2 and 5, respectively. (C) Lysate of WT MEFs were immunoprecipitated with anti-GAPDH, anti-PGK1 and IgG antibodies. Immunoprecipitants were analyzed by western blotting with anti-PGK1 and anti-GAPDH antibody. (D) After adding various concentrations of NaCl to the lysosomal fraction of WT MEFs, the membrane pellet (bound) and supernatant (free) were subjected to immunoblotting with GAPDH and PGK1 antibodies. (E) Schematic representation showing that the lysosomal V-ATPase activity is regulated by the glycolytic enzymes, GAPDH and PGK1. The glycolytic GAPDH activity was measured with GAP as a substrate in the presence of NAD. PGK1 catalyzes the reaction of 1,3-bisphosphoglycerate (1,3-BPG) and ADP to form 3-phosphoglycerate (3-PG) and ATP. (F) After addition of GAP, NAD and Pi, ATP production in the lysosomal fraction was measured using a luciferin/luciferase kit. Iodoacetate (IA; 4 µM) was used to inhibit GAPDH activity. Three independent experiments were performed. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001. (G) Lysates were prepared from MEF cells expressing Tmem192-3×HA [the only enzyme that catalyzes PI(3,5) P2 production in the lysosomal membrane] that were treated with 1 µM YM201636 (PIKfyve inhibitor). The lysosome immunoprecipitation method isolates pure lysosomes with anti-HA antibody. Immunoblotting of protein markers of various subcellular compartments in purified immunoprecipitated lysosomes are shown. (H) The lysosomal fraction of WT MEF cells using the Dextran magnetic beads (MAG10) method contained GAPDH and PGK1. (I) After addition of GAP, NAD and Pi, ATP production in the lysosomal fraction isolated by MAG10 was measured using a luciferin/luciferase kit. Iodoacetate (IA; 4 µM) was used to inhibit GAPDH activity. Three independent experiments were performed. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, **p < 0.002. (J) Schema of this novel study. Mitochondrial translation deficiency suppresses HIF1α-Nmnat3-mediated NAD+ production. The glycolytic enzymes, GAPDH and PGK1, are associated with lysosomal vesicles, and NAD+ is required for ATP production around lysosomal vesicles. NAD+ content is essential for lysosomal maintenance.