Protection against Mitochondrial and Metal Toxicity Depends on Functional Lipid Binding Sites in ATP13A2

Figure 1

Mutations in the key lipid binding sites of ATP13A2 inhibit activity but not subcellular targeting. (a) Multiple positively charged residues in previously identified lipid binding sites [14] (blue, underlined) were substituted for Ala. LBS3 overlaps with an alternative splicing site rendering an insertion of five additional residues in splice variant 1 of ATP13A2. Ma, membrane-associated region. (b) Expression levels of ATP13A2 were analyzed by immunoblotting with a primary anti-ATP13A2 antibody in comparison to GAPDH as a loading control. Breakdown of LBS mutant proteins by the proteasome was assessed via treatment with the proteasome inhibitor MG-132 (100 μM) for 6 h. The same gel for the expression of ATP13A2 between cell lines has been provided at both low and high intensities. OE, overexpression. (c) Autophosphorylation assay (EP) on microsomes of ATP13A2 WT or LBS mutant expressing SHSY5Y cells. At the top, the same gel was depicted twice at different exposure times. Below, equal protein loading on the SDS-PAGE gel was confirmed by Coomassie staining. We consider that the double bands visible in LBS2 and LBS3 at longer exposure time are background levels while the actual ATP13A2 related EP band (visible in LBS1 and WT-OE) is located in between, but closer to the double band. Quantification of ATP13A2 expression ((b) ATP13A2/GAPDH) and autophosphorylation levels ((c) EP/Coomassie) are depicted. (d) Expression and localization of endogenous (FLUC), WT-OE, LBS1, LBS2, LBS3, or LBS1.2.3 ATP13A2 were confirmed by colocalization experiments with the lysosomal marker LAMP-1 and captured using Olympus IX73 fluorescent microscope. Representative enlarged images of colocalization for all cell lines have been provided as image inserts. Data are representative of 3 independent experiments. Scale bars represent 10 μM.

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