Cellular function and pathological role of ATP13A2 and related P-type transport ATPases in Parkinson's disease and other neurological disorders

Mutations in ATP13A2 lead to Kufor-Rakeb syndrome, a parkinsonism with dementia. ATP13A2 belongs to the P-type transport ATPases, a large family of primary active transporters that exert vital cellular functions. However, the cellular function and transported substrate of ATP13A2 remain unknown. To discuss the role of ATP13A2 in neurodegeneration, we first provide a short description of the architecture and transport mechanism of P-type transport ATPases. Then, we briefly highlight key P-type ATPases involved in neuronal disorders such as the copper transporters ATP7A (Menkes disease), ATP7B (Wilson disease), the Na+/K+-ATPases ATP1A2 (familial hemiplegic migraine) and ATP1A3 (rapid-onset dystonia parkinsonism). Finally, we review the recent literature of ATP13A2 and discuss ATP13A2’s putative cellular function in the light of what is known concerning the functions of other, better-studied P-type ATPases. We critically review the available data concerning the role of ATP13A2 in heavy metal transport and propose a possible alternative hypothesis that ATP13A2 might be a flippase. As a flippase, ATP13A2 may transport an organic molecule, such as a lipid or a peptide, from one membrane leaflet to the other. A flippase might control local lipid dynamics during vesicle formation and membrane fusion events

The P5A ATPase Spf1p is stimulated by phosphatidylinositol 4-phosphate and influences cellular sterol homeostasis

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in severe ER stress. However, the function of these ubiquitous membrane proteins, which belong to the P-type ATPase superfamily, is unknown. We purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and observed that the ATP hydrolytic activity of the protein is stimulated by phosphatidylinositol 4-phosphate (PI4P). Furthermore, SPF1 exhibited negative genetic interactions with SAC1, encoding a PI4P phosphatase, and with OSH1 to OSH6, encoding Osh proteins, which, when energized by a PI4P gradient, drive export of sterols and lipids from the ER. Deletion of SPF1 resulted in increased sensitivity to inhibitors of sterol production, a marked change in the ergosterol/lanosterol ratio, accumulation of sterols in the plasma membrane, and cytosolic accumulation of lipid bodies. We propose that Spf1p maintains cellular sterol homeostasis by influencing the PI4P-induced and Osh-mediated export of sterols from the ER

Structural and biochemical insights into mammalian cobalt-substituted methionine sulfoxide reductase B1 using UV-visible spectroscopy and high-resolution NMR spectroscopy

Prior to this study it was discovered that MsrB1 from Mus musculus expressed in Escherichia Coli binds cobalt(II) (hereafter cobalt) in cobalt-supplemented growth media, and it had further been demonstrated that the His-tag was not responsible for this metal uptake. The aim of this study was to investigate the effects of cobalt on the growth of E. Coli in culture, characterize the metal-uptake and metal-binding site of cobalt-substituted MsrB1 by UV-visible spectroscopy and to gain structural information about the protein by high resolution NMR spectroscopy.The effects of cobalt on growth of E. Coli were studied by growing cultures in Lysogeny broth (LB) and minimal (M9)-media supplemented with different concentrations of cobaltdichloride (CoCl2) and monitoring culture growth by optical density (OD) measurements. Growth rates were found to decrease with increasing concentrations of CoCl2. To study the cobalt-uptake of MsrB1, the protein was recombinantly expressed in E. Coli in different cobalt-supplemented growth media, and the purified protein analyzed by UV-vis spectroscopy. Cobalt-uptake was demonstrated in all cases by characteristic absorption peaks owing to the cobalt-ligand complex, and the wavelengths of these peaks matched those of a tetrahedral four-coordinated cobalt-Cys complex. It was argued that these four Cys residues should be the same that constitute the zinc(II)-binding site of MsrB1, indicating that cobalt simply replaces zinc as a structural metal ion in cobalt-substituted MsrB1 (Co-MsrB1). It was argued that the intracellular concentration of cobalt in E. Coli should be significantly higher than zinc, and that this together with similar ionic radii for cobalt and zinc leads to the formation of Co-MsrB1. MsrB1 expressed in nickel(II)-supplemented LB did not lead to formation of Ni-MsrB1, which was argued to result from nickel not being released directly into the cytosol in E. Coli. Co-MsrB1 was produced by zinc-starvation of E. Coli followed by expressing the protein in zinc-free minimal medium supplemented with CoCl2. To investigate if pH-titratable groups could be detected, the protein was dialyzed against buffers with pH 4.9-11.5 and the molar extinction coefficient was found from UV-vis absorption spectra in the different pH. Two titration curves were observed, but assignment of the titrations to specific residues could not be made. Further, Co- and Zn-MsrB1 was dialyzed against buffers with metal chelating agents to remove the metal ions from the two proteins. Cobalt was successfully removed at pH 5.0 and 5.5, while removal of zinc from Zn-MsrB1 was not detected, demonstrating that zinc is more tightly bound to the Cys-ligands than cobalt.To study the ratio of formation of Co-MsrB1 and the native zinc-form Zn-MsrB1 in CoCl2-supplemented growth media, the molar extinction coefficient of Co-MsrB1 was determined, and the concentration of Co-MsrB1 in purified protein samples from protein expression in different growth media was determined. The Co-MsrB1:Zn-MsrB1 ratio was found to be 0.2 in M9 medium supplemented with 10 µM CoCl2, 0.1 in LB supplemented with 50 µM CoCl2 and 0.03 in LB supplemented with 10 µM CoCl2. From 2D- and 3D-NMR experiments on 13C- and 15N-enriched Co-MsrB1, a 70 % backbone assignment and 50 % side chain-assignment was accomplished using Computer Aided Resonance Assignment (CARA). The four structural Cys-residues of the native protein was not found, while the other three Cys-residues of MsrB1 were assigned, confirming that the same Cys-residues are responsible for coordination of cobalt and zinc in MsrB1. Many strongly shifted signals were observed in 1D 1H spectra of Co-MsrB1, some as far upfield as 350 ppm and downfield as -80 ppm, and it was argued that most of the unassigned residues should be found outside the spectral width of the 2D and 3D-NMR spectra.To gain further structural information about Co-MsrB1, pseudocontact shifts (PCSs) were determined for the assigned HN-atoms of Co-MsrB1 by using the published chemical shifts of for Zn-MsrB1. The PCSs were analyzed by AnisoFit using three conformers and the mean conformer of the published Zn-MsrB1 structure, and the best correlations between observed and calculated PCSs were found for conformer 3. The PCSs were plotted against their hypothetical distance to cobalt using the structure of Zn-MsrB1, and a very good PCS-distance proportionality was found, indicating that Co-MsrB1 and Zn-MsrB1 have the same overall fold and structure. The AnisoFit calculations and PCSs of the N-terminus suggested that the N-terminus spends significant time in the proximity of the metal-binding site, and it was argued that this proximity ensures high catalytic efficiency due to the short distance between the catalytic and resolving Cys-residues

Towards defining the substrate of orphan P5A-ATPases

P-type ATPases are ubiquitous ion and lipid pumps found in cellular membranes. P5A-ATPases constitute a poorly characterized subfamily of P-type ATPases present in all eukaryotic organisms but for which a transported substrate remains to be identified.publisher: Elsevier articletitle: Towards defining the substrate of orphan P5A-ATPases journaltitle: Biochimica et Biophysica Acta (BBA) - General Subjects articlelink: http://dx.doi.org/10.1016/j.bbagen.2014.05.008 content_type: article copyright: Copyright © 2014 Elsevier B.V. All rights reserved.status: publishe

Parkinson disease related ATP13A2 evolved early in animal evolution

<div><p>Several human P5-type transport ATPases are implicated in neurological disorders, but little is known about their physiological function and properties. Here, we investigated the relationship between the five mammalian P5 isoforms ATP13A1-5 in a comparative study. We demonstrated that ATP13A1-4 isoforms undergo autophosphorylation, which is a hallmark P-type ATPase property that is required for substrate transport. A phylogenetic analysis of P5 sequences revealed that ATP13A1 represents clade P5A, which is highly conserved between fungi and animals with one member in each investigated species. The ATP13A2-5 isoforms belong to clade P5B and diversified from one isoform in fungi and primitive animals to a maximum of four in mammals by successive gene duplication events in vertebrate evolution. We revealed that ATP13A1 localizes in the endoplasmic reticulum (ER) and experimentally demonstrate that ATP13A1 likely contains 12 transmembrane helices. Conversely, ATP13A2-5 isoforms reside in overlapping compartments of the endosomal system and likely contain 10 transmembrane helices, similar to what was demonstrated earlier for ATP13A2. <i>ATP13A1</i> complemented a deletion of the yeast P5A ATPase <i>SPF1</i>, while none of <i>ATP13A2-5</i> could complement either the loss of <i>SPF1</i> or that of the single P5B ATPase <i>YPK9</i> in yeast. Thus, ATP13A1 carries out a basic ER function similar to its yeast counterpart Spf1p that plays a role in ER related processes like protein folding and processing. ATP13A2-5 isoforms diversified in mammals and are expressed in the endosomal system where they may have evolved novel complementary or partially redundant functions. While most P5-type ATPases are widely expressed, some P5B-type ATPases (ATP13A4 and ATP13A5) display a more limited tissue distribution in the brain and epithelial glandular cells, where they may exert specialized functions. At least some P5B isoforms are of vital importance for the nervous system, since ATP13A2 and ATP13A4 are linked to respectively Parkinson disease and autism spectrum disorders.</p></div

Genetic complementation in yeast with mammalian P5-type ATPases.

<p>The genome of <i>S</i>. <i>cerevisiae</i> contains a single P5A ATPase gene (<i>SPF1</i>) and a single P5B ATPase gene (<i>YPK9</i>). <b>A.</b> Gene knockout of <i>SPF1</i> results in increased sensitivity to caffeine which can be rescued by expression of untagged Spf1p and mATP13A1 respectively, but not by the untagged catalytically dead SPF1 D487N or by untagged mammalian ATP13A2-5. (e.v.–empty vector) <b>B.</b> Deletion of <i>ypk9</i>^<i>-</i> results in increased sensitivity to MnCl₂, which can be rescued by expression of untagged Ypk9p, but not by the untagged and catalytically dead Ypk9p D781N. None of the untagged mammalian P5 ATPase genes showed rescue of <i>ypk9</i>^<i>-</i> (not shown). <b>C.</b> ATP13A1 complements the caffeine phenotype of the <i>spf1</i>^<i>-</i> knockout strain, whereas the catalytically dead mutant D530N fails to complement. <b>D.</b> Expression of Ypk9p and catalytically dead Ypk9p D781N proteins was confirmed by both N-terminal GFP fusion constructs. Ypk9p tagged with GFP show vacuolar localization. <b>E.</b> Expression of mammalian ATP13A1-5 proteins was similarly confirmed by N- and C-terminal GFP fusion constructs. N- or C-terminal positioning of GFP showed no apparent difference in localization patterns. ATP13A1 shows ER localization and is absent in vacuoles, ATP13A2 and ATP13A4 show vacuolar localization, ATP13A3 and ATP13A5 show localization to cytosolic spots which could represent a pre-vacuolar compartment or early endosomes. Scale bars represent 2,5 μm.</p

P5-type ATPase phylogenetic tree.

<p>Simplified view of the phylogenetic tree calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193228#pone.0193228.g001" target="_blank">Fig 1</a>. The P5A group includes only ATP13A1-like sequences (grey), while the P5B group includes subclades belonging to the ATP13A2 (blue), P5B_inv (purple, invertebrate P5B), ATP13A3 (yellow), ATP13A4 (red) and ATP13A5 (green) clades. Sequences from cnidaria, placozoa and ctenophore are marked in white dots. Invertebrate sequences are marked in yellow dots. Sequences from hemichordates and echinoderms are marked with light blue dots and sequences from higher vertebrates are marked in progressively darker shaded blue dots. Yeast Spf1p and Ypk9p are marked with red dots. The yellow numbers indicate the three major P5B gene duplications in vertebrate evolution as discussed in the main text.</p

ATP13A2-4 localize to the endo-/lysosomal system.

<p>HeLa cells were transiently co-transfected with hATP13A2, hATP13A3, mATP13A4 or mATP13A5 (N- or C-terminal mCherry-tag, N-mCh and C-mCh, respectively). Scale bar represents 20 μm. <b>A-C.</b> Co-localization of hATP13A2 (N-terminal mCherry-tag) with various GFP-labeled markers, such as the early endosomal marker RAB5 (A), the late endo-/lysosomal marker RAB7 (B) and RAB11, a marker for the recycling endosomes (C). <b>D-F.</b> Immunocytochemistry of cells overexpressing N- or C-terminal mCherry labeled hATP13A2 with the endogenous markers of late endosomes (CD63, E-F) and lysosomes (lamp2, D). <b>G-I.</b> Co-localization of hATP13A3 with GFP-labeled RAB5 (G), RAB7 (H) and RAB11 (I). <b>J-L.</b> Immunocytochemistry of cells overexpressing N- or C-terminal mCherry labeled hATP13A3 with the endogenous markers of late endo-/lysosomes lamp1 (K, L) and of lysosomes lamp2 (J). <b>M-O.</b> Co-localization of hATP13A4 with GFP-labeled RAB5 (M), RAB7 (N) and RAB11 (O). <b>P-R.</b> Immunocytochemistry of cells overexpressing N- or C-terminal mCherry labeled hATP13A4 with the endogenous markers of late endosomes (CD63, Q-R) and lysosomes (lamp2, P). <b>S.</b> mATP13A5 overexpression is too weak for fluorescence microscopy. When a fluorescent signal is observed, an endosomal-like pattern was seen in 80% of the observations (two left panels), whereas in other images a reticular pattern was observed (right panel). <b>T-W.</b> Bar graphs depict Pearson’s coefficients (PCC) of ATP13A2-5 isoforms with various endosomal markers.</p