Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn2 +efflux and protects against toxicity

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2011; 108(2):858-63. DOI: 10.1073/pnas.1013642108
Source: PubMed


P-type ATPases transport a wide array of ions, regulate diverse cellular processes, and are implicated in a number of human diseases. However, mechanisms that increase ion transport by these ubiquitous proteins are not known. SPCA1 is a P-type pump that transports Mn(2+) from the cytosol into the Golgi. We developed an intra-Golgi Mn(2+) sensor and used it to screen for mutations introduced in SPCA1, on the basis of its predicted structure, which could increase its Mn(2+) pumping activity. Remarkably, a point mutation (Q747A) predicted to increase the size of its ion permeation cavity enhanced the sensor response and a compensatory mutation restoring the cavity to its original size abolished this effect. In vivo and in vitro Mn(2+) transport assays confirmed the hyperactivity of SPCA1-Q747A. Furthermore, increasing Golgi Mn(2+) transport by expression of SPCA1-Q747A increased cell viability upon Mn(2+) exposure, supporting the therapeutic potential of increased Mn(2+) uptake by the Golgi in the management of Mn(2+)-induced neurotoxicity.

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    • "Isolation of Golgi membrane fractions by sucrose gradient flotation Preparation of Golgi membranes performed with the methods described previously ( Balch et al . 1984 ; Mukhopadhyay and Linstedt 2011 ) with some modifications . Panc1 - bC2GnT - M ( c - Myc ) and LNCaP cells from ten - twelve 75 cm² cell culture flasks were harvested by PBS containing 0 . "
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    ABSTRACT: The Golgi apparatus undergoes morphological changes under stress or malignant transformation, but the precise mechanisms are not known. We recently show that non-muscle myosin IIA (NMIIA) binds to the cytoplasmic tail of Core 2 N-acetylglucosaminyltransferase-M (C2GnT-M) and transports it to the endoplasmic reticulum for recycling. Here, we report that Golgi fragmentation induced by Brefeldin A (BFA) or β-COP knockdown in Panc1-bC2GnT-M (c-Myc) cells is accompanied by increased association of NMIIA with C2GnT-M and its degradation by proteasomes. Golgi fragmentation is prevented by inhibition or knockdown of NMIIA. Using multiple approaches, we have shown that the speed of BFA-induced Golgi fragmentation is positively correlated with the levels of this enzyme in the Golgi. The observation is reproduced in LNCaP cells which express high levels of two endogenous glycosyltransferases - C2GnT-L and β-galactoside α2,3sialyltransferase 1. NMIIA is found to form complexes with these two enzymes but not Golgi matrix proteins. Knockdown of both enzymes or prevention of Golgi glycosyltransferases from exiting the endoplasmic reticulum reduced Golgi-associated NMIIA and decreased the BFA-induced fragmentation. Interestingly, the fragmented Golgi detected in colon cancer HT-29 cells can be restored to a compact morphology after inhibition or knockdown of NMIIA. The Golgi disorganization induced by microtubule or actin destructive agent is NMIIA-independent and does not affect the levels of glycosyltransferases. We conclude that NMIIA interacts with Golgi residential but not matrix proteins, and this interaction is responsible for Golgi fragmentation induced by β-COP knockdown or BFA treatment. This is a novel non-enzymatic function of Golgi glycosyltransferases.
    Full-text · Article · Feb 2013 · Glycobiology
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    • "This suggests its participation in the management of Mn 2+ -induced neurotoxicity . This is also supported by in vivo studies reporting that brain areas with high SPCA1 expression also show enhanced Mn 2+ accumulation upon continuous systemic MnCl 2 infusion in mice (Sepulveda et al. 2012) and by the observation that a gain-of-function mutation of SPCA1, which specifically enhances Golgi Mn 2+ transport, improves survival of Mn 2+ -exposed cells (Mukhopadhyay and Linstedt 2011). "
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    ABSTRACT: Excess Mn(2+) in humans causes a neurological disorder known as manganism, which shares symptoms with Parkinson's disease. However, the cellular mechanisms underlying Mn(2+) -neurotoxicity and the involvement of Mn(2+) -transporters in cellular homeostasis and repair are poorly understood and require further investigation. In this work, we have analyzed the effect of Mn(2+) on neurons and glia from mice in primary cultures. Mn(2+) overload compromised survival of both cell types, specifically affecting cellular integrity and Golgi organization, where the secretory pathway Ca(2+) /Mn(2+) -ATPase is localized. This ATP-driven Mn(2+) transporter might take part in Mn(2+) accumulation/detoxification at low loads of Mn(2+) , but its ATPase activity is inhibited at high concentration of Mn(2+) . Glial cells appear to be significantly more resistant to this toxicity than neurons and their presence in cocultures provided some protection to neurons against degeneration induced by Mn(2+) . Interestingly, the Mn(2+) toxicity was partially reversed upon Mn(2+) removal by wash out or by the addition of EDTA as a chelating agent, in particular in glial cells. These studies provide data on Mn(2+) neurotoxicity and may contribute to explore new therapeutic approaches for reducing Mn(2+) poisoning.
    Preview · Article · Jul 2012 · Journal of Neurochemistry
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    • "As plasmid controls, a pEGFP-N1 plasmid was obtained from Clontech (Mountain View, CA, USA) and a pcDNA3.1 plasmid was obtained from Invitrogen (Basel, Switzerland). A plasmid expressing HA-tagged human SPCA1 was kindly provided by Dr. Adam Linstedt (Carnegie Mellon University, Pittsburgh, PA) [29]. Expression plasmids containing human EGFP-LC3B (plasmid #11546; [30]), rat LAMP1-RFP (plasmid #1817, [31]), human RFP-Rab5A (plasmid #14437, [32]), human GFP-Rab7A (plasmid #12605; [33]), human GFP-Rab9A (plasmid #12663; [33]) and human GFP-Rab11A (plasmid #12674; [33]) were obtained from Addgene. "
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    ABSTRACT: Mutations in the ATP13A2 gene (PARK9) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS), a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s) by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and heterozygous missense mutations in ATP13A2 associated with early-onset forms of parkinsonism.
    Full-text · Article · Jun 2012 · PLoS ONE
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