Lysosomal TMEM165 controls cellular ion homeostasis and survival by mediating lysosomal Ca2+ import and H+ efflux

Abstract

The proper function of lysosomes depends on their ability to store and release calcium. While several lysosomal calcium release channels have been described, how lysosomes replenish their calcium stores in placental mammals has not been determined. Using genetic depletion and overexpression techniques combined with electrophysiology and visualization of subcellular ion concentrations and their fluxes across the lysosomal membrane, we show here that TMEM165 imports calcium to the lysosomal lumen and mediates calcium-induced lysosomal proton leakage. Accordingly, TMEM165 accelerates the recovery of cells from cytosolic calcium overload thereby enhancing cell survival while causing a significant acidification of the cytosol. These data indicate that in addition to its previously identified role in the glycosylation of proteins and lipids in the Golgi, a fraction of TMEM165 localizes on the lysosomal limiting membrane, where its putative calcium/proton antiporter activity plays an essential role in the regulation of intracellular ion homeostasis and cell survival.

Fig. 1. TMEM175 acidifies the perilysosomal area.

a Schematic presentation of Lyso-mKeima (top) and representative confocal images of live HeLa-Lyso-mKeima cells stained with Lysotracker® Green (bottom). r, Pearson’s colocalization coefficient (n = 20). Scale bars, 10 μm. b The standard curve for fluorescence intensity (FLI) ratios (Ex 585 nm/405 nm; Em 620 nm) of mKeima at pH ranging from 6.0 to 7.5. See Supplementary Fig. 1d for corresponding images. c Cytosolic and perilysosomal pH values of indicated cell lines. d Representative (n = 3) immunoblots of indicated proteins in HeLa cells treated with control (CTL) or TMEM175 siRNAs for 72 h. e Perilysosomal pH analyzed by Lyso-mKeima (left) and lysosomal pH analyzed by LysoSensor^TM Yellow/Blue FLI (right) in HeLa cells treated with indicated siRNAs for 72 h. f Relative volumes of acidic compartments in HeLa cells treated with Con A for 1 h, stained with Lysotracker green, and analyzed by flow cytometry (left), and mKeima FLI in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with either DMSO or 10 nM Con A for 1 h (right). See Supplementary Fig. 1h for flow cytometry gating. g Representative images of live HeLa-Lyso-mKeima cells treated with DMSO or 100 μM Arachidonic acid for 15 min (left) and corresponding mKeima FLIs (right). h Relative mKeima FLIs in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with either DMSO or 100 μM Arachidonic acid for the last 20 min. Bars, SD of three independent experiments with 10 (g) 30 (b, c, e-left, f-right, h) or ≥10000 (e-right, f-left) randomly chosen cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by one-way Anova with Tukey’s multiple comparisons (e, f left), two-way Anova with Dunnett’s multiple comparison (c, f right, h), or two-tailed, homoscedastic t-test (g).

Fig. 2. Ca²⁺ mobilizing agents induce TMEM175-independent lysosomal H⁺ leakage.

a Representative images of live HeLa-Lyso-mKeima cells treated with DMSO or ebastine (left) and corresponding mKeima FLIs (right). b Kinetics of mKeima FLI in HeLa cells treated with 6 μM terfenadine for 60 min. c Relative mKeima FLIs in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with either DMSO or 6 μM terfenadine for the last 20 or 40 min. d Relative mKeima FLIs in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with 15 μM ebastine (left) or 6 μM terfenadine (right) for the last 20, 40 or 60 min. e Relative Fluo-4-AM (left), mKeima (middle) and SypHer3 (right) FLIs in HeLa cells treated with 6 μM terfenadine, 500 nM thapsigargin, 10 μM ML-SA1 or 1 μM ATP for the last 5 min and analyzed by flow cytometry. See Supplementary Fig. 4g for gating of the cells for flow cytometry analyses. f Relative mKeima FLI in HeLa-Lyso-mKeima cells pretreated for 30 min with 20 μM BAPTA-AM and treated with 500 nM thapsigargin for the last 5 min. g Relative mKeima FLIs in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with 10 μM ML-SA1 (left) or 500 nM thapsigargin (right) for the last 5 min. Bars, SD of three independent experiments with 10 (a), 67 (b), 30 (c, d, f, g), or ≥10000 (e) randomly chosen cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by two-tailed, homoscedastic t-test (a), one-way Anova with Tukey’s multiple comparisons (b) or two-way Anova with Dunnett’s multiple comparisons (c–g).

Fig. 3. Ca²⁺-induced lysosomal H⁺ leakage is mediated by TMEM165.

a Representative (n = 3) immunoblots of indicated proteins in HeLa cells treated with indicated siRNAs for 72 h. b Relative mKeima FLI in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with 15 μM ebastine (left) or 6 μM terfenadine (right) for the last 20, 40 or 60 min. c Cytosolic pH analyzed by pHrodo-AM FLI in HeLa cells treated with indicated siRNAs for 72 h and with 15 μM ebastine or 6 μM terfenadine for 2 h. d Relative mKeima FLI in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with 10 μM ML-SA1 (left) or 500 nM thapsigargin (right) for the last 0–300 sec. e Perilysosomal pH analyzed by Lyso-mKeima FLI ratio (585/405 nm) in HeLa cells treated with indicated siRNAs for 72 h (left) or in TMEM165 overexpressed cells (right). f Representative (n = 3) immunoblots of indicated proteins in wild type (WT), TMEM165 overexpressed cell lines (top) and TMEM165-KO cell lines (bottom). g Lysosomal pH analyzed by LysoSensor^TM Yellow/Blue FLI in HeLa TMEM165-KO cells (left) and TMEM165 overexpressed cells (right). h Cytosolic pH analyzed by pHrodo-AM FLI in HeLa TMEM165-KO cells (left) and TMEM165 overexpressed cells (right). i Representative (n = 3) immunoblots of indicated proteins in mCherry- and TMEM165-mCherry-trasfected HeLa clones. Bars, SD of three independent experiments with 30 (b–e, h) or ≥15000 (g) randomly chosen cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by one-way Anova with Tukey’s multiple comparison (e-left, h-right), two-way Anova with Dunnett’s multiple comparison (b–d) or two-tailed, homoscedastic t-test (e-right, g, h-left).

Fig. 4. TMEM165 preserves lysosomal Ca²⁺stores.

Relative Fluo-4-AM FLIs (a) and pH-corrected Fura-2-AM FLI ratios (b, see Supplementary Fig. 4a, b for the basis of pH corrections) in indicated HeLa and MDA-MB-468 cells treated with 300 μM GPN for 300 sec. siRNA transfection was performed 72 h before the treatment (a). Bars, SD of three independent experiments with ≥10000 (a) or ≥15000 (b) cells analyzed in each sample. Ca²⁺ signals in HEK293 cells transiently transfected with TRPML1-GCaMP6s and mCherry (control, n = 19), TMEM165-WT-mCherry (n = 17) or TMEM165-R126C-mCherry (n = 17) (c) or indicated siRNAs (n = 4) from a representative experiment (d). Thin and bold lines represent Ca²⁺ signal curves in response to 10 μM ML-SA1 in individual cells and the mean values of all cells analyzed, respectively. The bar charts represent mean values for the area under the curve corresponding to relative fluorescence unit (RFU) x seconds. Bars, SEM of 17–19 (c) and 4 (d) independent experiments with 3–10 cells in each sample. e Representative images of live HEK293 cells transfected as in d and stained with CellMask^TM green plasma membrane stain, LysoTracker^TM Deep Red and Hoechst-33342 (left) and same cells treated with 1 µM apilimod for 16 h, fixed, and stained with LAMP1 antibodies and DAPI (right). Asterisks indicate colocalization of LysoTracker/LAMP1 and TMEM165. Scale bars, 10 µm. f Schematic presentation of Lyso-GCaMP8s. g Relative Lyso-GCaMP8s FLIs in indicated HeLa (left and middle) and MDA-MB-468 (right) cells. siRNA transfection was performed 72 h before the analyses (left). Bars, SD of three independent experiments with ≥10000 cells analyzed in each sample. h Lyso-GCaMP8s (top) and Lyso-mKeima FLIs in HeLa-WT (left) and HeLa-TMEM165-KO (right) cells transfected with Lyso-GCaMP8s/Lyso-mKeima 48 h earlier and treated three times with 10 µM ML-SA1 for 5 min. Cells were allowed to recover in fresh medium for 7 min between the treatments. Mean values of 30 cells in a representative (n = 3) experiment are shown. i Mean heights of ML-SA1-induced perilysosomal Ca²⁺ peaks shown in (h). Bars, SD of three independent experiments with 30 randomly chosen cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by two-way Anova with Dunnet’s (a, b, g, i) or Tukey’s (c, d) multiple comparison.

Fig. 5. TMEM165 protects cells against cytosolic Ca²⁺ overload.

Cytosolic calcium concentrations analyzed by Fluo-4-AM in WT and TMEM165-KO MDA-MB-468 clones treated with 10 μM ML-SA1 (a) or 500 nM thapsigargin (b) for 0–300 sec or with 15 μM ebastine for 1 h (c). Death of indicated MDA-MB-468 cell clones treated with indicated concentrations of thapsigargin (d), ebastine (e, left) or terfenadine (e, right) for 24 h. Cells were stained with propidium iodide (dead cells) and Hoechst-33342 (total cells) and cell death was analyzed by Celigo Imaging Cytometer. f Cytosolic [Ca²⁺] in WT and TMEM165-KO HeLa clones treated with DMSO or 15 µM ebastine for 1 h and analyzed by Fluo-4-AM. g Death of indicated HeLa clones treated with 0–15 µM ebastine for 24 h and analyzed as in (d). h Cytosolic [Ca²⁺] in indicated HeLa clones treated with DMSO or 15 µM ebastine for 1 h and analyzed by Fluo-4-AM. i Death of indicated HeLa cell clones treated with 6 µM terfenadine or 15 µM ebastine for 24 h. Cells were stained with SYTOX Green (dead cells) and Hoechst-33342 (total cells) and cell death was analyzed by Celigo Imaging Cytometer. j Galectin 1 puncta (leaky lysosomes) in indicated HeLa clones treated with DMSO or 15 μM ebastine for the last 16 h. Bars, SD of three independent experiments with 30 (a, b), ≥5000 (c, f, h) or ≥15000 (d, e, i) cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by two-way Anova with Dunnett’s multiple comparisons.

Fig. 6. TMEM165 is present on the lysosomal limiting membrane.

a Representative (n = 3) confocal images of live HeLa-TMEM165-mCherry cells transfected with a Golgi marker EGFP-RAB6 (top), or stably expressing Lyso-GFP (middle and bottom). Yellow arrows point to vacuolin-enlarged lysosomes with both Lyso-GFP and TMEM165-mCherry on limiting membranes. DNA is visualized by Hoechst staining. White squares mark the area shown in enlarged images. r, Pearson’s colocalization coefficient (n = 20). Scale bar, 10 μm. b Representative (n = 3) confocal images of live HeLa-TMEM165-mCherry-Lyso-EGFP cells with indicated treatments or genetic alteration. r, Pearson’s colocalization coefficient (n = 20). Scale bars, 10 μm. c Representative (n = 3) immunoblots of indicated proteins from purified lysosomes (Lys), light membrane fractions (LMF) and whole cell lysates (WC) of HeLa cells. When indicated, 10 nM concanamycin A (ConA) was added to all buffers used for the extraction. d Representative (n = 3) immunoblots of indicated proteins in WT and TMEM165-KO MDA-MB-468 clones treated with 1 μM MnCl₂ for indicated times (left), and relative quantification of fully glycosylated LAMP2 (right). e Cytosolic Ca²⁺ concentration analyzed by Fluo-4-AM FLI in WT and TMEM165- KO MDA-MB-468 clones treated with DMSO or 15 μM ebastine for the last 1 h of the 48 h treatment with 1 μM MnCl₂. Bars, SD of three independent experiments with at least 5000 cells analyzed in each sample. f Cytosolic pH analyzed by pHrodo-AM FLI in WT and TMEM165- KO MDA-MB-468 clones treated with DMSO, 6 μM terfenadine, or 15 μM ebastine for the last 1 h of the 48 h treatment with 1 μM MnCl₂. Bars, SD of three independent experiments with 30 randomly chosen cells analyzed in each sample. ***P < 0.001 as analyzed by one-way Anova with Tukey’s multiple comparison (d right) or two-way Anova with Dunnett’s multiple comparison (e, f).

Fig. 7. TMEM165 mediates H⁺ inward and Ca²⁺ outward currents.

a Voltage step protocol employed for current recordings. b Examples of macroscopic current recordings conducted for TMEM165-WT-mCherry (green), TMEM165-R126C-mCherry mutant (R126C, red), and mCherry control (black) transiently expressed in HEK293 cells. Measurements were performed using indicated pH and Ca²⁺ concentrations in the pipette/bath. c Current densities plotted against respective voltages for TMEM165-WT-mCherry (left, green squares, n = 6) and TMEM165-R126C-mCherry (right, red triangles, n = 6) and the control (black circles, n = 12). d Representative (n = 3) live images of HEK293 cells transiently transfected with TMEM165-WT- or TMEM165-R126C-mCherry. Arrows, plasma membrane; scale bars, 10 µm. e Examples of macroscopic current recordings conducted for TMEM165-WT-mCherry using indicated pH and Ca²⁺ concentrations in the pipette/bath. f Quantitative analyses of current densities (I/C_m) plotted against respective voltages for TMEM165-WT-mCherry at indicated pH and Ca²⁺ concentrations in the pipette/bath solution with asymmetrical Ca²⁺ and pH (green squares, n = 6) or asymmetrical Ca²⁺ and symmetrical pH (blue reverted triangles, n = 4), symmetrical Ca²⁺ and symmetrical pH (magenta circles, n = 3), and symmetrical Ca²⁺ and asymmetrical pH (mustard circles, n = 4). g, h Current densities plotted against respective voltages for TMEM165-WT-mCherry at indicated pH and Ca²⁺ concentrations in the pipette/bath solution with asymmetrical Ca²⁺ and pH with (green squares, n = 6) and without (mustard circles, n = 9, right) Mg²⁺ or symmetrical Ca²⁺ and asymmetrical pH without Mg²⁺ (cyan circles n = 7, left). Bars, mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 as analyzed by two-tailed, homoscedastic t-test using GraphPad Prism 10.2.3.