PLD3 has phospholipase D activity in isolated lysosomes.

A. Overexpression of PLD3 does not produce increased phospholipase D activity in total lysate of NSC cells compared to untransfected NSC total lysate. N = 4 biological/assay replicates. B. Lysosomes isolated following transfection of PLD3 via iron dextran nanoparticles and magnetic affinity columns display enrichment for lysosomal marker LAMP2 and expected lack of cytoskeletal marker tubulin. Lysosomal fraction is enriched with PLD3, further indicating that PLD3 is a lysosomal protein. C. Lysosomal fractionation demonstrates that overexpressed wild-type PLD3 ( P ) is appropriately trafficked to the lysosomal fraction. Similarly, introduction of the AD-associated PLD3 mutation V232M ( V ) or a mutation in the HKD domain, K418R ( K ), did not impact routing to the lysosome (representative blot shown), indicated by Western Blot. Samples isolated via lysosomal isolation detailed in the Methods section. D . Data derived from lysosomes isolated from transfected NSC cells, each condition containing n = 4 biological/assay replicates per condition and n>16 sample replicates per condition. Phospholipase D (PLD) activity measured by the Amplex Red PLD assay with negative (control lysosomes) and positive (cabbage PLD) controls in each assay run. Lysosomes expressing PLD3 display significant increase in PLD activity compared to control lysosomes, which is similar in magnitude to the PLD activity seen with purified cabbage PLD. Upon cotransfection of siRNA against PLD3 alongside PLD3 transfection, these lysosomes display no PLD activity above the control condition. PLD3-K418R transfected lysosomes, which contain a mutated lysine residue in its putative HKD domain, have significantly reduced PLD activity compared to wild-type PLD3 transfected lysosomes. Furthermore, human AD-risk associated variant PLD3-V232M displays no significant PLD activity, and displays a significant reduction in PLD activity compared to PLD3. Data represented as mean +/- SEM. Data analyzed via one-way ANOVA. ****p<0.0001 following Bonferroni ...