The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

The Parkinson-associated human P5B-ATPase ATP13A2 protects against the iron-induced cytotoxicity

AbstractP-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1–P5. The ion transported by the P5-ATPases is not known. Five genes named ATP13A1–ATP13A5 that belong to the P5-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor–Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA).Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage

The N- or C-terminal cytoplasmic regions of P4-ATPases determine their cellular localization

Mammalian P4-ATPases specifically localize to the plasma membrane and the membranes of intracellular compartments. P4-ATPases contain 10 transmembrane domains, and their N- and C-terminal (NT and CT) regions face the cytoplasm. Among the ATP10 and ATP11 proteins of P4-ATPases, ATP10A, ATP10D, ATP11A, and ATP11C localize to the plasma membrane, while ATP10B and ATP11B localize to late endosomes and early/recycling endosomes, respectively. We previously showed that the NT region of ATP9B is critical for its localization to the Golgi apparatus, while the CT regions of ATP11C isoforms are critical for Ca2+-dependent endocytosis or polarized localization at the plasma membrane. Here, we conducted a comprehensive analysis of chimeric proteins and found that the NT region of ATP10 proteins and the CT region of ATP11 proteins are responsible for their specific subcellular localization. Importantly, the ATP10B NT and the ATP11B CT regions were found to harbor a trafficking and/or targeting signal that allows these P4-ATPases to localize to late endosomes and early/recycling endosomes, respectively. Moreover, dileucine residues in the NT region of ATP10B were required for its trafficking to endosomal compartments. These results suggest that the NT and CT sequences of P4-ATPases play a key role in their intracellular trafficking

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

Highly exposed segment of the Spf1p P5AATPase near transmembrane M5 detected by limited proteolysis

The yeast Spf1p protein is a primary transporter that belongs to group 5 of the large family of P-ATPases. Loss of Spf1p function produces ER stress with alterations of metal ion and sterol homeostasis and protein folding, glycosylation and membrane insertion. The amino acid sequence of Spf1p shows the characteristic P-ATPase domains A, N, and P and the transmembrane segments M1-M10. In addition, Spf1p exhibits unique structures at its N-terminus (N-T region), including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (D region). Here we used limited proteolysis to examine the structure of Spf1p. A short exposure of Spf1p to trypsin or proteinase K resulted in the cleavage at the N and C terminal regions of the protein and abrogated the formation of the catalytic phosphoenzyme and the ATPase activity. In contrast, limited proteolysis of Spf1p with chymotrypsin generated a large N-terminal fragment containing most of the M4-M5 cytosolic loop, and a minor fragment containing the C-terminal region. If lipids were present during chymotryptic proteolysis, phosphoenzyme formation and ATPase activity were preserved. ATP slowed Spf1p proteolysis without detectable changes of the generated fragments. The analysis of the proteolytic peptides by mass spectrometry and Edman degradation indicated that the preferential chymotryptic site was localized near the cytosolic end of M5. The susceptibility to proteolysis suggests an unexpected exposure of this region of Spf1p that may be an intrinsic feature of P5A-ATPases.Fil: Petrovich, Guido Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Corradi, Gerardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Pavan, Carlos Humberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Noli Truant, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Adamo, Hugo Pedro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Novel ATP13A2 (PARK9) homozygous mutation in a family with marked phenotype variability

Mutations in the ATP13A2 (PARK9) and FBXO7 (PARK15) genes are linked to different forms of autosomal recessive juvenile-onset neurodegenerative diseases with overlapping phenotypes, including levodopa-responsive parkinsonism, pyramidal disturbances, cognitive decline, and supranuclear gaze disturbance. However, the associated genotypes and phenotypes are poorly characterized due to the small number of patients described. Here, we report clinical, instrumental, and genetic findings in an Italian family with novel PARK9 and PARK15 mutations. The proband developed a severe progressive phenotype including juvenile-onset parkinsonism, pyramidal disturbances, cognitive decline, and oculomotor abnormalities. On the contrary, his brother only shows mild abnormalities (pyramidal, cognitive, and oculomotor) on the neurological examination at the age of 31 years. These two brothers both carry a novel homozygous PARK9 missense (p.G877R) and a novel heterozygous PARK15 mutation (p.R481C). The PARK9 mutation replaces a crucial residue for the ATPase activity, and is therefore most likely a loss-of-function mutation and disease-causing in homozygous state. The pathogenic significance of the PARK15 single heterozygous mutation remains unclear. In both sibs, DaTSCAN single photon emission computed tomography showed marked nigrostriatal dopaminergic defects, and transcranial magnetic stimulation detected prolonged central motor conduction time. MRI, including T2*-weighted imaging, detected no evidence of brain iron accumulation. This family, the third reported with homozygous PARK9 mutations and the first with mutations in two genes for atypical juvenile parkinsonism, illustrates that PARK9-linked disease might display wide intra-familial clinical variability and milder phenotypes, suggesting the existence of strong, still unknown, modifiers

ATPase reaction cycle of P4-ATPases affects their transport from the endoplasmic reticulum

P4‐ATPases belonging to the P‐type ATPase superfamily mediate active transport of phospholipids across cellular membranes. Most P4‐ATPases, except ATP9A and ATP9B proteins, form heteromeric complexes with CDC50 proteins, which are required for transport of P4‐ATPases from the endoplasmic reticulum (ER) to their final destinations. P‐type ATPases form autophosphorylated intermediates during the ATPase reaction cycle. However, the association of the catalytic cycle of P4‐ATPases with their transport from the ER and their cellular localization has not been studied. Here, we show that transport of ATP9 and ATP11 proteins as well as that of ATP10A from the ER depends on the ATPase catalytic cycle, suggesting that conformational changes in P4‐ATPases during the catalytic cycle are crucial for their transport from the ER

Characterising ATP13A3 biological function and its role in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare but devastating disease characterised by the progressive remodelling of the small pulmonary vessels. Although the causes may vary, similar pathobiological features are shared among different forms of PAH, with endothelial dysfunction, the hyperproliferation of smooth muscle cells and mesenchymal cells in the vascular wall, as well as inflammation contributing to this process. Despite the availability of licensed therapies, 5-year survival for patients remains less than 60%. To uncover additional fundamental mechanisms of PAH pathobiology, our group undertook whole genome sequencing in 1038 idiopathic or heritable PAH patients and identified disease-associated heterozygous mutations in ATP13A3, a P5B-type ATPase. P5 ATPases, namely ATP13A1-5, are putative transmembrane proteins that generate and maintain significant chemical gradients across biological membranes by active transport of cations. However, to date, studies of their functions are sparse. Therefore, the focus of my project was to characterise ATP13A3 in the pulmonary vasculature and to uncover the underlying mechanism of how mutations in this gene contribute to PAH pathogenesis. Although the biological functions of ATP13A3 are elusive, its homologue (CATP-5) in C. elegans was previously reported as a polyamine transporter. Polyamines are a group of naturally existing polycations essential for many cellular processes. Therefore, my working hypothesis was that ATP13A3 loss of function might disrupt the polyamine homeostasis and hence contribute to the abnormal vascular remodelling in PAH. To address this, I first confirmed ATP13A3 as residing in the recycling endosome of endothelial cells. This localisation of ATP13A3 suggested it might be related to polyamine transport since the endocytic pathway was recognised as a crucial route for polyamine trafficking. To expand this finding, I developed molecular tools to knockdown or to overexpress ATP13A3 in vascular cells and analysed how these affected polyamine transport. This led to the discovery of putrescine as the preferred substrate for ATP13A3 and also revealed a basal reduction of other polyamines in endothelial cells. Further investigation highlighted an essential role of ATP13A3 in pulmonary vasculature with its deficiency restrained cell growth and predisposed endothelial cells to apoptosis. To validate these findings, blood outgrowth endothelial cells (BOECs) were isolated from a PAH patient bearing an LK726X ATP13A3 frameshift mutation (LK726X). Intriguingly, ATP13A3 causing ATP13A3 reduction, impaired polyamine uptake and induced apoptosis in BOECs. This provides the first evidence for the pathogenic effects of a PAH associated ATP13A3 mutation. In addition, using a lentiviral overexpression system, I also demonstrated impairments of other PAH- associated mutations on ATP13A3 mediated polyamine uptake, reinforcing the involvement of ATP13A3 in PAH pathobiology. To explore whether ATP13A3 mutations can cause PAH, our laboratory has generated a mouse model harbouring a human ATP13A3 mutation (P452Lfs) via the MRC Harwell Gene editing service. My colleague, Dr Ekaterina Legchenko and I have discovered that the Atp13a3P452Lfs/P452Lfs mice spontaneously develop PAH-like hemodynamic changes at 6-months of age. Preliminary data also revealed lower Atp13a3 level and reduced polyamine content in the lungs of the mutant mice, suggesting that Atp13a3 frameshift mutation (P452L) may contribute to PAH pathogenesis via the disruption of polyamine homeostasis in vivo. In conclusion, my work characterises for the first time that ATP13A3 is a polyamine transporter in vascular cells with putrescine as its preferable transport substrate. ATP13A3 mutations identified from PAH patients are highly likely to impair polyamine homeostasis and contribute to PAH progression. These findings open up a new field for investigating PAH pathobiology and also highlight the potential role of rebalancing polyamine homeostasis in PAH treatment