Pathogenic Effect of Missense Mutations in ATP13A2 monitor the steady-state levels of ATP13A2, or with b-tubulin antibody (or Ponceau S stain) to demonstrate equivalent protein loading. Densitometric analysis indicates dramatically reduced steady-state levels of F182L, G504R and G877R compared with WT ATP13A2 in the Triton-soluble fraction. Bars represent ATP13A2 levels normalized to b-tubulin (mean6SEM; n = 3–4 experiments) expressed in arbitrary units. E, Western blot analysis of the detergent-soluble fraction from SH-SY5Y cells transiently expressing WT or mutant V5-tagged ATP13A2. Densitometric analysis of ATP13A2 levels normalized to b-tubulin reveals reduced levels of F182L, G504R and G877R compared with WT ATP13A2 (mean6SEM; n = 3 experiments). F, Detergent-soluble extracts from HEK-293T cell transiently expressing WT and KRS mutant V5-tagged human ATP13A2 were probed with anti-V5 antibody, or b-tubulin antibody as a loading control. KRS mutants exhibit markedly reduced steady-state levels and produce truncations due to the loss of three (3057delC) or six (1632_1653dup22) C-terminal transmembrane domains, or the in-frame skipping of exon 13 (1306+5GRA) that removes half of the third transmembrane domain [5]. Molecular weight markers are indicated in kilodaltons (kDa). *P,0.05, **P,0.01 or non- significant (ns) compared to WT ATP13A2 by one-way ANOVA with Newman-Keuls post-hoc analysis. doi:10.1371/journal.pone.0039942.g001 Homozygous missense mutations impair the protein stability of ATP13A2 Since homozygous missense mutations dramatically reduce the steady-state levels of ATP13A2, we next sought to determine whether these effects are due to impaired protein stability. HEK- 293T cells transiently expressing human ATP13A2 variants (WT, F182L, G504R and G877R) were treated with cycloheximide (CHX) to inhibit new protein synthesis and sampled at 1, 3, 6 and 8 hours post-treatment to estimate the rate of protein turnover by Western blotting. The levels of WT ATP13A2 are reduced by ,25% following treatment with CHX for 8 hours (Figure 2). In contrast, the F182L and G504R variants are dramatically reduced by ,90% after treatment with CHX for 8 hours with an estimated half-life of ,3 hours, whereas the G877R variant is modestly reduced by ,60% over 8 hours (Figure 2). The turnover of the F182L and G504R ATP13A2 variants correlates closely with their reduced steady-state protein levels (refer to Figure 1D and 1E), whereas the reduced steady-state protein levels of the G877R mutant result from a combination of reduced protein stability (Figure 2) and reduced mRNA expression levels (Figure S1). Our data demonstrate that homozygous missense mutations markedly impair the protein stability of ATP13A2 in support of a loss-of- function mechanism of action for these mutations. Homozygous missense mutations enhance the proteasomal degradation of ATP13A2 To understand the mechanism leading to the destabilization of ATP13A2 due to homozygous missense mutations, we assessed the degradation of ATP13A2 variants through the proteasomal and lysosomal pathways. HEK-293T cells transiently expressing human ATP13A2 variants (WT, F182L, G504R and G877R) were treated for 24 hours with the proteasomal inhibitor, MG132 (5 mM) or the lysosomal inhibitor, ammonium chloride (50 mM). The steady-state levels of Triton-soluble and Triton-insoluble ATP13A2 were monitored by Western blotting. The levels of WT, F182L, G504R and G877R ATP13A2 in the Triton-soluble fraction are markedly increased by lysosome inhibition (Figure 3) suggesting that ATP13A2 is normally degraded by the lysosomal pathway. Proteasomal inhibition results in an increase of F182L and G504R ATP13A2 in the Triton-soluble fraction that is more pronounced in the Triton-insoluble fraction compared to the WT and G877R proteins (Figure 3). Collectively, our data suggest that ATP13A2 is normally subjected to lysosomal degradation consis- tent with the known localization of this protein to lysosomes [5]. Furthermore, the homozygous missense mutations, F182L and G504R, promote the proteasomal degradation of ATP13A2 consistent with their reduced protein stability. Figure 2. Homozygous missense mutations enhance the protein turnover of ATP13A2. Disease-associated homozygous mutations (F182L, G504R and G877R) enhance the turnover of ATP13A2. HEK-293T cells transiently expressing V5-tagged human ATP13A2 (WT or mutant) were treated with cycloheximide (CHX, 200 mg/ml) at 24 h post-transfection, and cells were harvested at 0, 1, 3, 6 and 8 h thereafter. Equivalent detergent-soluble extracts were probed with anti-V5 antibody to monitor ATP13A2 turnover, or with anti-b-tubulin antibody to demonstrate equal protein loading. Densitometric analysis indicates the markedly increased turnover of F182L, G504R and G877R variants compared to WT ATP13A2. The levels of ATP13A2 were normalized to b-tubulin. Bars represent ATP13A2 levels normalized to b-tubulin (mean6SEM; n = 3–4 experiments) expressed as a percent of time point 909 for each variant. *P,0.05 or **P,0.01 compared to WT ATP13A2 within each time interval by one-way ANOVA with Newman-Keuls post-hoc analysis. doi:10.1371/journal.pone.0039942.g002 PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39942