Pathogenic Effect of Missense Mutations in ATP13A2 Figure 1. Disease-associated mutations reduce the steady-state levels of ATP13A2 protein. A, Predicted domain topology model of human ATP13A2. Wild-type (WT) protein consists of ten transmembrane-spanning domains. The second intracellular loop contains the predicted catalytic ATPase domain comprising four functional domains (A, actuator domain; P1, catalytic phosphorylation site domain; P2 and N, nucleotide binding domains). Locations of disease-associated missense mutations are indicated by stars with homozygous variants in bold. KRS frameshift mutations are indicated by orange and red arrows, with an in-frame exon skipping mutation shown by yellow shading. B, Western blot indicating the buffer solubility of V5-tagged human ATP13A2 transiently expressed in HEK-293T cells. C, Western blot revealing the effect of increasing temperature on the solubility of V5-tagged human ATP13A2 in cell extracts obtained by lysis in 1% Triton X-100 buffer. D, Triton-soluble or Triton-insoluble (RIPA- soluble) fractions derived from HEK-293T cells transiently expressing WT or mutant V5-tagged human ATP13A2 were probed with anti-V5 antibody to PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39942