FIGURE 10.

Intracellular localization of GCase DSPIIV variants in Gba1^−/− cells. A, typical examples of immunofluorescence localization of DSPIIV-substituted GCase variants following transient transfections. The DSPIIV (WT) and ESPIIV show co-localization of GCase (FITC) with either Lamp1 (red) or LIMP-2 (purple) (top right), indicating that the retention of charge by Glu³⁹⁹ does not impact localization. The cells are typical for either Asp³⁹⁹ (DSPIIV) or Glu³⁹⁹ (ESPIIV). The DSPAIV (I402A) or DSPIAV (I403A) mutant shows no co-localization with ldLIMP-2 or Lamp1 (bottom left) (i.e. no binding to ldLIMP-2 in the cell and no localization to the lysosome (Lamp1/ldLIMP2)). The black and white images in the bottom right indicate the relative localization of LAMP-1 (lysosomes) and WT GCase (ER/Golgi and lysosomes) in Gba1^−/− fibroblasts for comparison. Sham control is shown in the top left. B, Pearson indices for co-localization of the various mutants in the ER/Golgi (black) or lysosome (hatched). The substitution of Glu for Asp (WT) at 399 (ESPIIV) did not alter the co-localization compared with WT. In comparison, ASPIIV (D399A) and the double mutant, DSPAAV (I402A/I403A) showed little co-localization to the lysosome but significant retention in the ER/Golgi. The single mutants, DSPAIV (I402A) and DSPIAV (I403A), showed partial co-localization to the lysosome but greater retention in the ER/Golgi. Error bars, S.E.