Pathogenic Effect of Missense Mutations in ATP13A2 Figure 3. Homozygous missense mutations enhance the degradation of ATP13A2 by the proteasome. HEK-293T cells transiently expressing V5-tagged human ATP13A2 (WT, F182L, G504R and G877R) were treated with the proteasome inhibitor, MG132 (5 mM), or the lysosome inhibitor, ammonium chloride (NH4Cl, 50 mM), for 24 h. Triton-soluble and Triton-insoluble (RIPA-soluble) fractions were probed with anti-V5 antibody to monitor ATP13A2 levels, or with b-tubulin antibody (or Ponceau S stain) to demonstrate equivalent protein loading. Inhibition of the proteasome leads to the marked recovery of F182L and G504R protein variants compared to WT and G877R ATP13A2 in the Triton-soluble and - insoluble fractions. Molecular weight markers are indicated in kilodaltons (kDa). Blots are representative of at least four independent experiments. doi:10.1371/journal.pone.0039942.g003 Localization of ATP13A2 to endosomal and lysosomal vesicles in neurons ATP13A2 has been shown to localize to lysosomal membranes in mammalian cells [5,11,13,17]. To explore the subcellular localization of ATP13A2 in neurons, we assessed the co- localization of exogenous ATP13A2 with various vesicular markers in rat primary cortical neurons by confocal fluorescence microscopy. Cortical neurons were co-transfected with V5-tagged human ATP13A2 and fluorescent fusion proteins (LAMP1-RFP, RFP-Rab5A, GFP-Rab7A, GFP-Rab9A, GFP-Rab11A and GFP- LC3) at a 10:1 DNA molar ratio, and subjected to immunocy- tochemistry. Human ATP13A2 adopts a punctate distribution pattern within the soma and processes of cortical neurons consistent with an intracellular vesicular localization (Figure 4). ATP13A2 co-localizes to a large extent with LAMP1-RFP, a marker of lysosomal membranes, predominantly within the neuronal soma (Figure 4). We also explored the co-localization of ATP13A2 with additional vesicular compartments related to the lysosomal pathway. Human ATP13A2 markedly co-localizes with GFP-Rab7A- and GFP-Rab9A-positive late endosomal compart- ments and RFP-Rab5A-positive early endosomes but fails to appreciably co-localize with GFP-Rab11A-positive recycling endosomes or GFP-LC3-positive autophagosomes in cortical neurons (Figure 4). Our data demonstrate that ATP13A2 normally co-localizes with intracellular vesicular compartments in neurons, including lysosomes and early and late endosomes. Homozygous missense mutations disrupt the vesicular localization of ATP13A2 KRS mutations which produce truncated forms of ATP13A2 cause the mislocalization of ATP13A2 from intracellular vesicles to the ER where they are degraded by the ERAD pathway [5,13]. To determine whether disease-associated missense mutations potentially act through a similar mechanism, we explored the subcellular localization of human ATP13A2 variants expressed in rat primary cortical neurons by confocal fluorescence microscopy. WT and mutant (T12M, G533R D513N, A746T and G877R) ATP13A2 adopt a similar distribution to large vesicular mem- branes throughout the cytoplasm of neuronal soma (Figure 5). In contrast, the F182L and G504R mutants localize to smaller diffuse punctate structures similar to the truncated KRS mutants, 3057delC, 1632_1653dup22 and 1306+5GRA, consistent with mislocalization to the ER (Figure 5). To further explore the mislocalization of ATP13A2 missense mutants, we examined the co-localization of V5-tagged ATP13A2 variants with the exoge- nous lysosomal marker, LAMP1-RFP, or the endogenous ER marker, calreticulin, in human SH-SY5Y neural cells (Figure 6). WT and mutant (T12M, G533R D513N, A746T and G877R) ATP13A2 markedly co-localize with LAMP1, whereas in com- parison, the F182L and G504R mutants reveal markedly reduced co-localization with LAMP1 and increased co-localization with calreticulin thereby demonstrating mislocalization of these mutant proteins at least in part to the ER (Figure 6). Our data reveal that homozygous missense (F182L and G504R) or KRS mutations disrupt the normal localization of ATP13A2 to vesicular membranes in cortical neurons and neural cell lines, whereas additional heterozygous (T12M, G533R or A746T), homozygous (G877R) or non-functional (D513N) mutations do not appreciably influence the vesicular localization of ATP13A2. Heterozygous missense mutations impair the ATPase activity of ATP13A2 The majority of missense mutations in ATP13A2 are located within the second large intracellular loop that contains the ATPase enzymatic domain (refer to Figure 1A). To explore the effects of missense mutations on ATPase activity, we first prepared microsomal fractions containing vesicular membranes including lysosomes from HEK-293T cells transiently expressing human ATP13A2 variants, since we anticipate that ATP13A2 would be fully active within vesicular membranes. Compared to WT ATP13A2, the F182L, G504R and G877R mutants are not appreciably detected in microsomes even following combined proteasome and lysosome inhibition to prevent their degradation (Figure 7A). Heterozygous mutations (T12M, G533R and A746T) or the ATPase-deficient D513N mutant were normally enriched in microsomal fractions together with markers of lysosomal (LAMP1) and Golgi (giantin) membranes (Figure 7B). As a positive control, we employed human Secretory Pathway Ca2+-ATPase 1 (SPCA1/ ATP2C1), a related P-type ATPase localized to Golgi membranes [20,21], which could also be detected in microsomes (Figure 7B). PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39942