ATP13A2 (PARK9) is a late endo-lysosomal transporter of unknown function that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome, a parkinsonism with dementia 1 and early-onset Parkinson's disease (PD) 2. ATP13A2 offers protection against genetic and environmental risk factors of PD, whereas loss of ATP13A2 compromises lysosomal function 3. The lysosomal transport function of ATP13A2 remained unclear, but here, we establish ATP13A2 as a lysosomal polyamine exporter with highest affinity for spermine. Polyamines stimulate the activity of purified ATP13A2, while disease mutants are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes cellular polyamine uptake via endocytosis and transports polyamines into the cytosol, which highlights a role for endo-lysosomes in cellular polyamine uptake. At high concentrations, polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with loss of ATP13A2 or its orthologues. Thus, defective lysosomal polyamine export is a new mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration. Our findings further shed light on the molecular identity of the elusive mammalian polyamine transport system. ATP13A2 is a P5B-ATPase belonging to the family of P-type ATPases, which couple ATP hydrolysis to substrate transport while transiently forming a catalytic phospho-intermediate 4. ATP13A2 is generally described as a heavy metal transporter 5 , but Ca 2+ 6 and the polyamine spermidine (SPD) 7,8 were also proposed. To screen for the transported substrate(s) of ATP13A2, we measured ATPase activity in the presence of various candidate substrates in solubilized microsomal membrane fractions of SH-SY5Y cells that overexpress human ATP13A2 wild type (WT) (WT-OE) or comparable levels of the catalytically dead D508N mutant (D508N-OE) 9,10. ATPase activity of ATP13A2 WT was significantly stimulated by the polyamines SPD and spermine (SPM) (Fig. 1a), whereas SPM had no effect on the D508N mutant (Extended Data Fig. 1a). MnCl2, ZnCl2, FeCl3, CaCl2, diamines, monoamines and amino acids exerted no effect (Extended Data Fig. 1a-3 d). The polyamines SPM, N 1-acetylspermine and SPD were able to stimulate ATPase activity in a concentration-dependent manner (Fig. 1b, Extended Data Fig. 1e) with the highest apparent affinity for SPM (Extended Data Table 1). The catalytic auto-phosphorylation and/or dephosphorylation reactions of P-type ATPases occur in response to binding of the transported substrate 4. ATP13A2 forms a phospho-intermediate on the D508 residue in the absence of SPM supplementation 9,10 , whereas SPM leads to a dose-dependent reduction in ATP13A2 phospho-enzyme levels (Fig. 1c), which is not seen with ornithine (Extended Data Fig. 1f). The dephosphorylation rate following a chase with non-radioactive ATP increased in the presence of...

The various splice variants of the three SERCA-and the two SPCA-pump genes in higher vertebrates encode P-type ATPases of the P 2A group found respectively in the membranes of the endoplasmic reticulum and the secretory pathway. Of these, SERCA2b and SPCA1a represent the housekeeping isoforms. The SERCA2b form is characterized by a luminal carboxy terminus imposing a higher affinity for cytosolic Ca 2þ compared to the other SERCAs. This is mediated by intramembrane and luminal interactions of this extension with the pump. Other known affinity modulators like phospholamban and sarcolipin decrease the affinity for Ca 2þ . The number of proteins reported to interact with SERCA is rapidly growing. Here, we limit the discussion to those for which the interaction site with the ATPase is specified: HAX-1, calumenin, histidine-rich Ca 2þ -binding protein, and indirectly calreticulin, calnexin, and ERp57. The role of the phylogenetically older and structurally simpler SPCAs as transporters of Ca 2þ , but also of Mn 2þ , is also addressed.

Parkinson disease (PD) is a disabling, incurable disorder with increasing prevalence in the western world. In rare cases PD is caused by mutations in the genes for PINK1 (PTEN induced kinase 1) or PRKN (parkin RBR E3 ubiquitin protein ligase), which impair the selective autophagic elimination of damaged mitochondria (mitophagy). Mutations in the gene encoding LRRK2 (leucine rich repeat kinase 2) are the most common monogenic cause of PD. Here, we report that the LRRK2 kinase substrate RAB10 accumulates on depolarized mitochondria in a PINK1-and PRKN-dependent manner. RAB10 binds the autophagy receptor OPTN (optineurin), promotes OPTN accumulation on depolarized mitochondria and facilitates mitophagy. In PD patients with the two most common LRRK2 mutations (G2019S and R1441C), RAB10 phosphorylation at threonine 73 is enhanced, while RAB10 interaction with OPTN, accumulation of RAB10 and OPTN on depolarized mitochondria, depolarization-induced mitophagy and mitochondrial function are all impaired. These defects in LRRK2 mutant patient cells are rescued by LRRK2 knockdown and LRRK2 kinase inhibition. A phosphomimetic RAB10 mutant showed less OPTN interaction and less translocation to depolarized mitochondria than wild-type RAB10, and failed to rescue mitophagy in LRRK2 mutant cells. These data connect LRRK2 with PINK1-and PRKN-mediated mitophagy via its substrate RAB10, and indicate that the pathogenic effects of mutations in LRRK2, PINK1 and PRKN may converge on a common pathway.

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