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

ArticleinProceedings of the National Academy of Sciences 108(2):858-63 · January 2011with10 Reads
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.
    • "To date, three separate transporters/pumps have been found to be capable of mediating Mn efflux: The solute carriers, ferroportin and SLC30A10, and the P-type ATPase secretory pathway Ca-ATPase 1 (SPCA1)515253545556. While there is clear evidence supporting the ability of both ferroportin and SPCA1 to mediate Mn efflux and detoxification in cell culture [51,53,55,56], their roles in mediating Mn detoxification at the whole organism level remains to be clarified. In contrast, SLC30A10 appears to play a fundamental role in maintaining cellular Mn levels and protecting against Mn toxicity at the whole organism level. "
    [Show abstract] [Hide abstract] ABSTRACT: Manganese (Mn) is an essential trace element necessary for physiological processes that support development, growth and neuronal function. Secondary to elevated exposure or decreased excretion, Mn accumulates in the basal ganglia region of the brain and may cause a parkinsonian-like syndrome, referred to as manganism. The present review discusses the advances made in understanding the essentiality and neurotoxicity of Mn. We review occupational Mn-induced parkinsonism and the dynamic modes of Mn transport in biological systems, as well as the detection and pharmacokinetic modeling of Mn trafficking. In addition, we review some of the shared similarities, pathologic and clinical distinctions between Mn-induced parkinsonism and Parkinson's disease. Where possible, we review the influence of Mn toxicity on dopamine, gamma aminobutyric acid (GABA), and glutamate neurotransmitter levels and function. We conclude with a survey of the preventive and treatment strategies for manganism and idiopathic Parkinson's disease (PD).
    Full-text · Article · Jul 2015
    • "Recent studies in mammalian cells indicate that the maintenance of Mn homeostasis is more complex than simply a matter of compartmentalization of Mn. A human Mn sensing glycoprotein (GPP130) has been found to induce vesicle trafficking from Golgi to multivesicular vesicles in response to elevated Golgi Mn concentrations as a way to regulate Mn extrusion from cells [57, 58]. A similar mechanism remains yet to be identified in plants. "
    [Show abstract] [Hide abstract] ABSTRACT: Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species.
    Full-text · Article · Dec 2014
    • "For example, it could be a negative regulator of components involved in reducing cytoplasmic Mn. One mechanism of Mn control involves its sequestration in the Golgi, followed by secretion (Culotta et al., 2005; Mukhopadhyay and Linstedt, 2011 ). When extracellular Mn is low, activities such as SPCA1-mediated movement of Mn into the Golgi may be dampened to maintain basal levels of Mn in the cytoplasm. "
    [Show abstract] [Hide abstract] ABSTRACT: Manganese (Mn) protects cells against lethal doses of purified Shiga toxin by causing the degradation of the cycling transmembrane protein GPP130, which the toxin uses as a trafficking receptor. Mn-induced GPP130 down-regulation, in addition to being a potential therapeutic approach against Shiga toxicosis, is a model for the study of metal regulated protein sorting. Significantly, however, the mechanism by which Mn regulates GPP130 trafficking is unknown. Here we show that a transferable trafficking determinant within GPP130 bound Mn and that Mn binding induced GPP130 oligomerization in the Golgi. Alanine substitutions blocking Mn binding abrogated both oligomerization of GPP130 and GPP130 sorting from the Golgi to lysosomes. Further, oligomerization was sufficient because forced aggregation, using a drug-controlled polymerization domain, redirected GPP130 to lysosomes in the absence of Mn. These experiments reveal metal-induced oligomerization as a Golgi sorting mechanism for a clinically relevant receptor for Shiga toxin.
    Article · Jul 2014
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