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: 9.81). 01/2011; 108(2):858-63. DOI: 10.1073/pnas.1013642108
Source: PubMed

ABSTRACT 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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    • "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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