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, 108 (2), 858-63

Identification of a Gain-Of-Function Mutation in a Golgi P-type ATPase That Enhances Mn2+ Efflux and Protects Against Toxicity

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Identification of a Gain-Of-Function Mutation in a Golgi P-type ATPase That Enhances Mn2+ Efflux and Protects Against Toxicity

Somshuvra Mukhopadhyay et al. Proc Natl Acad Sci U S A.

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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Increased intra-Golgi Mn2+ induces degradation of GPP130. (A) HeLa cells were treated with 500 μM of Mn2+ for the indicated times and imaged to detect GPP130 and giantin. (Scale bar, 10 μm.) (B) Cells treated with control or anti-SPCA1 siRNAs for 48 h were transfected with HA-tagged SPCA1-WT and, after 24 h, were exposed to 500 μM of Mn2+ for 4 h and stained using anti-HA (to detect SPCA1) and anti-GPP130. After Mn2+, GPP130 was degraded in cells transfected with the control siRNA as well as in cells in which SPCA1 was not depleted after knockdown (single asterisk). Cells with no detectable SPCA1 after knockdown did not exhibit Mn2+-induced loss of GPP130 (double asterisk). (Scale bar, 10 μm.) (C) Levels of GPP130, myc-SPCA1-WT, and tubulin in cells treated exactly as described in B were determined by immunoblot. (D) Quantitation after immunoblotting to determine the levels of GPP130 remaining after Mn2+, normalized to tubulin, in cells transfected with control or anti-SPCA1 siRNAs (mean ± SE; n = 3; P < 0.05). (E) Cells treated with anti-SPCA1 siRNA for 48 h were transfected with RNA-resistant HA-tagged SPCA1-WT or mutated constructs. After 24 h they were exposed to 500 μM of Mn2+ for 4 h and imaged to detect SPCA1 using an anti-HA antibody and GPP130. After Mn2+, GPP130 was lost in cells expressing SPCA1-WT but not D350A or G309C (single asterisk). Knockdown cells not transfected with SPCA1-WT did not exhibit a GPP130 Mn2+ response (double asterisk). (Scale bar, 10 μm.) (F) Quantitation of percentage of transfected cells that did not have detectable GPP130 after Mn2+ from E (mean ± SE; n = 100 cells per experiment from three independent experiments; P < 0.05).
Fig. 2.
Fig. 2.
SPCA1-Q747A enhances the GPP130 Mn2+ response. (A) HeLa cells were transfected with HA-tagged SPCA1-WT or mutated constructs and 24 h later imaged to detect HA and GPP130. GPP130 was degraded in cells transfected with SPCA1-Q747A but not WT or the double mutants (single asterisk). In the SPCA1-Q747A transfected culture, GPP130 was clearly detected in untransfected cells (double asterisk). (Scale bar, 10 μm.) (B) Quantitation of percentage of transfected cells without detectable GPP130 (mean ± SE; n = 100 cells per experiment from six independent experiments; P < 0.05). (C) HeLa cells were transfected with HA-tagged SPCA1-WT or Q747A and harvested 24 h posttransfection as described previously (15). Immunoblotting was performed to detect GPP130, HA, and tubulin.
Fig. 3.
Fig. 3.
The Q747A substitution increases the size of the SPCA1 ion permeation cavity. (AC) SPCA1-WT was modeled using the MODBASE server as described in Materials and Methods. The M4 and M6 transmembrane domains are depicted in cartoon (A), surface (B), and ribbon (C) forms using the open source PyMol software. Ion-binding residues, E308, N738, and D742 are depicted in red; residue Q747 is in green; and V313 is in blue. Arrowheads indicate the path used by ions to reach the ion-binding site. (DF). Distances across the ion permeation cavity are shown for the computed structures of SPCA1-WT, SPCA1-Q747A, and SPCA1-Q747A+V314I. Distances between M4 and M6 were measured using the “Measurement Wizard” in PyMol and the structures of the SPCA1 mutants were obtained using the “Mutagenesis Wizard” of PyMol. Blue arrowhead in F shows the rotation of the side chain of V313 after the V314I substitution. Black arrowheads indicate the likely path traversed by ions en route to the ion-binding site. (G) HeLa cells were transfected with HA-SPCA1-Q747A or HA-SPCA1-Q747A+V314I and imaged 24 h later to detect GPP130 and HA. GPP130 was degraded in cells transfected with SPCA1-Q747A but not in those expressing the V314I mutation (single asterisk). Untransfected cells in the SPCA1-Q747A transfected culture exhibited Golgi-localized GPP130 (double asterisk). (Scale bar, 10 μm.) (H) Quantitation of percentage of transfected cells lacking detectable GPP130 from G above (mean ± SE, n = 50 cells from four independent experiments, P < 0.05).
Fig. 4.
Fig. 4.
SPCA1-Q747A is a hyperactive Mn2+ transporter. (A) Golgi membranes were isolated from cells transfected with HA-SPCA1-WT or Q747A as described in Materials and Methods and 2 μg of total protein was immunoblotted to detect giantin, GPP130, and HA. (B) Uptake of 54Mn2+ by isolated Golgi vesicles was performed as described in Materials and Methods. Uptake was normalized to the uptake by untransfected cells (set to 100) and the experiment was replicated three times (mean ± SE; P < 0.05 for the difference in uptake in untransfected cells with and without excess cold Mn2+ and for the difference in uptake between SPCA1-WT and Q747A). (C) Uptake of 54Mn2+ in permeabilized cells was done as described in Materials and Methods. Uptake in absence of cold Mn2+ was normalized to 100 for each experiment (mean ± SE, n = 3, P < 0.05). (D) Uptake of 54Mn2+ on FACS sorted cells expressing either GFP alone or GFP and SPCA1-WT or GFP and Q747A was performed as in C above. Uptake in cells expressing GFP alone (set to 100) was used for normalization (mean ± SE, n = 3, P < 0.05 for the difference between SPCA1-WT and Q747A). (E) Cells were loaded with 54Mn2+ and intracellular radioactivity was measured at various time points. Uptake at 30 min (set to 100) was used for normalization (mean ± SE, n = 3, P < 0.05). (F) Control or BFA-treated cells were loaded with 54Mn2+ for 30 min, washed, and chased for 30 min. Intracellular radioactivity after a 30-min loading was normalized to 100 for control and BFA (mean ± SE, n = 3, P < 0.05 for the difference in intracellular radioactivity retained after the chase in control cultures). (G) Distribution of intracellular and extracellular radioactivity after a 30-min chase in the presence or absence of BFA. (H) Cells were loaded with 54Mn2+ for 30 min at 37 °C, washed, and chased for 30 min at 37 °C or 20 °C. Intracellular radioactivity after a 30-min loading was normalized to 100 (mean ± SE, n = 3, P < 0.05 for the difference in intracellular radioactivity retained between 37 °C and 20 °C groups). (I and J) FACS sorted cells expressing SPCA1-WT or Q747A were loaded with 54Mn2+ for 30 min and subsequently chased for an additional 30 min. Intracellular radioactivity retained and secreted after the 30-min chase was normalized to 50 for WT-expressing cells (mean ± SE, n = 3, P < 0.05 for the difference in radioactivity retained and radioactivity secreted between WT and Q747A).
Fig. 5.
Fig. 5.
Expression of SPCA1-Q747A protects cells from Mn2+ toxicity. (A) HeLa cells were exposed to 1, 2, and 5 mM of Mn2+ for 16 h and viability was determined using the MTT assay. Controls were untreated (0 mM of Mn2+). Samples were normalized using absorption at 570 nm of the controls (set to 100) for each experiment (mean ± SE, n = 3, P < 0.05). (B) FACS sorted cells expressing GFP alone, GFP and SPCA1-WT, or GFP and SPCA1-Q747A were left untreated or exposed to 1 mM of Mn2+ for 16 h and viability was determined using the MTT assay. Viability in cells expressing GFP alone (set to 100) was used for normalization (mean ± SE, n = 3, P < 0.05 for the difference in viability between SPCA1-WT and Q747A). (C) Cells were treated with control or anti-SPCA1 siRNAs for 48 h and then exposed to 0 or 1 mM of Mn2+ for 16 h. Cell viability was assessed using the MTT assay and viability in cells treated with the control siRNA (set to 100) was used for normalization (mean ± SE, n = 3, P < 0.05 for the difference in viability between control and anti-SPCA1 siRNA treated groups). (D) Cells were transfected with SPCA1-WT or Q747A and mCherry for 24 h and then exposed to 1 mM of Mn2+ for 16 h. Propidium iodide staining was performed as described in Materials and Methods. (Scale bar, 10 μm.) (E) Quantitation of percentage of transfected cells positive for propidium iodide from D above (mean ± SE, n = 25 cells from three independent experiments per group, P < 0.05 for the difference between SPCA1-WT and Q747A after Mn2+). (F) Cells were transfected with a GFP marker alone or cotransfected with GFP and SPCA1-WT or Q747A for 24 h and then exposed to 1 mM of Mn2+ for 16 h at 20 °C. Control cultures did not receive Mn2+. Cell viability was then assayed using propidium iodide staining (mean ± SE, n = 25 cells from three independent experiments per group, P > 0.05 for the difference in cell viability between GFP, SPCA1-WT, and Q747A after Mn2+ and P < 0.05 for the difference in cell viability with and without Mn2+ in the GFP group). (G) Schematic showing that Mn2+ detoxification is mediated via transport into the Golgi in mammalian cells. Increasing Golgi Mn2+ uptake reduces cytosolic Mn2+ and protects against Mn2+ toxicity. Decreasing transport of Mn2+ into or out of the Golgi has the opposite effect.

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