Figure 4. PERK mediates E-Syt1... | Rockefeller University Press

Figure 4.

$PERK mediates E-Syt1 localization at the EMCS through E-Syt1 C2D-C2E domain. (A) Representative immunoblot for eGFP and PERK showing PERK-E-Syt1 interaction from mito crude fractions in DKO HeLa cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 pulled down. (B) Schematic representation of the E-Syt1 mutants anchored at the ER membrane used in this study; E-Syt1 full length (FL), E-Syt1-ΔSMP, E-Syt1-ΔDE, and E-Syt1-ΔCDE. (C) Representative immunoblot for eGFP and PERK showing PERK-E-Syt1 interaction from mito crude fractions in DKO HeLa cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1, E-Syt1-ΔCDE, E-Syt1-ΔSMP, and E-Syt1-ΔDE. (D–F) Quantification of PERK interaction normalized on E-Syt1-ΔCDE (D), E-Syt1-ΔSMP (E), or E-Syt1-ΔDE (F) eGFP pulled down and relative to control condition (eGFP-E-Syt1). The values plotted are the mean ± SEM from four biological replicates analyzed using one sample t test. (G) Representative images from eGFP-E-Syt1 full length, eGFP-E-Syt1-ΔDE or eGFP-E-Syt1-ΔSMP transiently transfected and co-stained with MitoTracker Far Red in DKO HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (H) Colocalization analysis of eGFP-E-Syt1/eGFP-E-Syt1-ΔDE/eGFP-E-Syt1-ΔSMP and MitoTracker Far Red in DKO HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates (n = 24, n = 32, and n = 30 for eGFP-E-Syt1, eGFP-E-Syt1-ΔDE, and eGFP-E-Syt1-ΔSMP respectively) analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. (I) Representative images from eGFP-E-Syt1 full length or eGFP-E-Syt1-ΔDE transiently co-transfected with STIM1-mCherry in DKO HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (J) Colocalization analysis of eGFP-E-Syt1/eGFP-E-Syt1-ΔDE and STIM1-mCherry in DKO HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates (n = 28 and n = 25 for eGFP-E-Syt1 and eGFP-E-Syt1-ΔDE, respectively) analyzed using unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; and NS = not significant. Source data are available for this figure: SourceData F4.$

PERK mediates E-Syt1 localization at the EMCS through E-Syt1 C2D-C2E domain. (A) Representative immunoblot for eGFP and PERK showing PERK-E-Syt1 interaction from mito crude fractions in DKO HeLa cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 pulled down. (B) Schematic representation of the E-Syt1 mutants anchored at the ER membrane used in this study; E-Syt1 full length (FL), E-Syt1-ΔSMP, E-Syt1-ΔDE, and E-Syt1-ΔCDE. (C) Representative immunoblot for eGFP and PERK showing PERK-E-Syt1 interaction from mito crude fractions in DKO HeLa cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1, E-Syt1-ΔCDE, E-Syt1-ΔSMP, and E-Syt1-ΔDE. (D–F) Quantification of PERK interaction normalized on E-Syt1-ΔCDE (D), E-Syt1-ΔSMP (E), or E-Syt1-ΔDE (F) eGFP pulled down and relative to control condition (eGFP-E-Syt1). The values plotted are the mean ± SEM from four biological replicates analyzed using one sample t test. (G) Representative images from eGFP-E-Syt1 full length, eGFP-E-Syt1-ΔDE or eGFP-E-Syt1-ΔSMP transiently transfected and co-stained with MitoTracker Far Red in DKO HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (H) Colocalization analysis of eGFP-E-Syt1/eGFP-E-Syt1-ΔDE/eGFP-E-Syt1-ΔSMP and MitoTracker Far Red in DKO HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates (n = 24, n = 32, and n = 30 for eGFP-E-Syt1, eGFP-E-Syt1-ΔDE, and eGFP-E-Syt1-ΔSMP respectively) analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. (I) Representative images from eGFP-E-Syt1 full length or eGFP-E-Syt1-ΔDE transiently co-transfected with STIM1-mCherry in DKO HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (J) Colocalization analysis of eGFP-E-Syt1/eGFP-E-Syt1-ΔDE and STIM1-mCherry in DKO HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates (n = 28 and n = 25 for eGFP-E-Syt1 and eGFP-E-Syt1-ΔDE, respectively) analyzed using unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; and NS = not significant. Source data are available for this figure: SourceData F4.