a–d, The ATPase activity of ATP13A2 was measured in solubilized microsomes (5 μg) collected from SH-SY5Y cells stably overexpressing wild-type ATP13A2 (WT-OE) in the presence of 100 nM CaCl 2 , MnCl 2 , ZnCl 2 or FeCl 3 and 100 μM SPD or SPM (D508N-OE as a negative control, wild type was referenced from Fig. 1b) (a) or in the presence of the indicated doses of inorganic ions and heavy metals CaCl 2 , MnCl 2 , ZnCl 2 or FeCl 3 (b), diamines (cadaverine, agmatine and the amino acid l-arginine) (c), monoamines (dopamine and histamine) (d) and acetylated polyamines (N1-acetylspermine, N8-acetylspermidine or N1-acetylspermidine) (e). As a reference for c–e, we plotted the dose–response curve of SPM from Fig. 1b. f, Microsomes (20 μg) collected from SH-SY5Y cells that overexpress ATP13A2 were incubated for 60 s with [γ-32P]ATP in the presence of 10 mM ornithine (ORN) or SPM (referenced from Fig. 1c). Left, a representative autoradiogram of the phosphoenzymes (EP); right, quantification. CON, control. g, The ATPase activity of purified ATP13A2 was assessed after 1 mM SPM was administered in the presence or absence of 0.25 mM orthovanadate (ORTH), a general P-type ATPase inhibitor (supplemented with 125 μM phosphatidic acid/PtdIns(3,5)P 2 ; conditions (−) and (−)/SPM refer to Fig. 1f). h, Purified ATP13A2 was incubated with [γ-32P]ATP in the presence of 1 mM SPM, and radioactivity of the phospho-intermediate was assessed by scintillation counting. i, Comparison of the pulse (5 μM, 15 min) chase (105 min, medium) BODIPY–SPM uptake in KO/WT and KO/D508N cell lines by confocal microscopy. Cells were subsequently stained with LAMP1 and imaged with the same laser settings by confocal microscopy. DAPI was used to visualize the nuclei. Scale bar, 5 μm. j, Line intensity plots of the indicated dashed lines in Fig. 2d. k, Analysis of the Pearson’s coefficient of LAMP1 and BODIPY–SPM for the images in Fig. 2d (KO/WT, 78 images; KO/D508N, 85 images). l, Mean fluorescence intensities (MFI) of BODIPY in DAPI-positive regions of samples shown in Fig. 2d (KO/WT, 233 nuclei; KO/D508N, 243 nuclei). Data are presented as the mean ± s.e.m. or mean with individual data points shown (points represent replicates), with n = 3 independent biological experiments. Analysis was carried out using one-way ANOVA with Dunnett’s (f) or Tukey’s (a, g) corrections, or by two-tailed t-tests (unpaired, h or Welch’s, k, l). Fitted lines are semi-log lines (b) or nonlinear allosteric sigmoidal association (c–e). For gel source data, see Supplementary Fig. 1.