Journal: The Journal of Cell Biology
Article Title: PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration
doi: 10.1083/jcb.202206008
Figure Lengend Snippet: E-Syt1 interacts through its C2D-C2E domain with PERK. (A) Representative immunoblot for IP3R3, GFP, PERK, CNX, VDAC1, and CYTC from total lysate and mito crude fractions of DKO HeLa cells transiently transfected with eGFP-E-Syt1. (B and C) Representative immunoblot (B) for E-Syt1 and GFP in WT HeLa cells transiently transfected with eGFP and DKO HeLa cells transiently transfected with eGFP, eGFP-E-Syt1, eGFP-E-Syt1-ΔDE, eGFP-E-Syt1-ΔSMP, eGFP-E-Syt1-ΔCDE; quantification (C) of E-Syt1 expression levels normalized on ACTIN (loading control) in WT, DKO + E-Syt1, DKO + E-Syt1-ΔDE, DKO + E-Syt1-ΔSMP, or DKO + E-Syt1-ΔCDE HeLa cells. The values plotted are the mean ± SEM from three biological replicates using one-way ANOVA, with Tukey’s test for multiple comparisons. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 mutants transfected and E-Syt1 signals for E-Syt1 endogenous and eGFP-E-Syt1 mutants transfected. (D) Abundance of GFP pulled-down normalized on abundance of GFP in mito crude fraction in DKO HeLa cells transiently transfected with eGFP-tagged E-Syt1, E-Syt1-ΔCDE,E-Syt1-ΔSMP, and E-Syt1-ΔDE. The values plotted are the mean ± SEM from four biological replicates analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. (E) Representative immunoblot for GFP and PERK showing PERK-E-Syt1 interaction from total lysate in HEK293-T cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1, E-Syt1-ΔSMP, E-Syt1-ΔDE, and E-Syt1-ΔCDE. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 mutants pulled down. (F) Abundance of GFP pulled-down normalized on abundance of GFP in total cell lysates in HEK293-T cells transiently transfected with eGFP-tagged E-Syt1, E-Syt1-ΔSMP, E-Syt1-ΔDE, and E-Syt1-ΔCDE. The values plotted are the mean ± SEM from biological replicates analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons . (G–I) Quantification of PERK interaction normalized on E-Syt1-ΔSMP (G), E-Syt1-ΔDE (H), or E-Syt1-ΔCDE (I) eGFP-pulled down and relative to control condition (eGFP-E-Syt1). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (J) Representative images from eGFP-E-Syt1 full length or eGFP-E-Syt1-ΔDE transiently co-transfected with STIM1-mCherry in PERK +/+ and PERK −/− MEFs cells. Scale bar, 10 µm. (K) Colocalization analysis of eGFP-E-Syt1/eGFP-E-Syt1-ΔDE and STIM1-mCherry in PERK +/+ and PERK −/− MEFs cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates ( n = 30, n = 29, n = 30, and n = 27 for PERK +/+ +E-Syt1-GFP, PERK +/+ +E-Syt1ΔDE, PERK −/− + E-Syt1-GFP, and PERK −/− + E-Syt1ΔDE, respectively) analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons. **, P < 0.01; ****, P < 0.0001; and NS = not significant. Source data are available for this figure: .
Article Snippet: Mouse monoclonal anti ACTIN, Sigma-Aldrich, A5441; Mouse monoclonal anti c-myc, Sigma-Aldrich, M4439; Rabbit polyclonal anti Calnexin, Enzo, ADI-SPA-865-F; Mouse monoclonal anti CYTC, BD Bioscience, 556433; Rabbit DyLight 680, Thermo Fisher Scientific, 35569; Mouse DyLight 680 Thermo Fisher Scientific, 35519; Mouse DyLight 800 Thermo Fisher Scientific, 35521; Rabbit DyLight 800 Thermo Fisher Scientific, 35571; Mouse monoclonal anti eIF2α, Cell Signaling, 2103S; Rabbit polyclonal anti-E-Syt1, Sigma-Aldrich, HPA016858; Rabbit polyclonal anti-GFP, Cell Signaling, 2555S; Mouse monoclonal anti-GFP,Life technologies, A11122; HRP Mouse Bioké, Cell Signaling, 7076; HRP Rabbit Bioké, Cell Signaling, 7074; Mouse monoclonal anti IP3R3, BD Bioscience, 610312; Rabbit polyclonal anti-PERK, Cell signaling, 3192S; Rabbit polyclonal anti PERK, Cell signaling, 5683S; Rabbit monoclonal anti Phospho-eIF2α (Ser51), Cell signaling, 3597S; Rabbit polyclonal anti PDI Genetex, GTX30716; Mouse monoclonal anti PSD, Santa Cruz, sc-390070; Rabbit polyclonal anti, PSS1 (B-5), Santa Cruz, sc-515376; Rabbit polyclonal anti PSS2, Sigma-Aldrich, SAB1303408; Rabbit polyclonal VDAC1, Cell Signaling, 4866S; Rabbit polyclonal VDAC1, Abcam, ab15895; Veriblot antibody Abcam, ab131366.
Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation
Cell Signaling Technology Inc is a verified supplier 
Cell Signaling Technology Inc manufactures this product 
![PERK modulates the proximity and the mitochondrial Ca 2+ uptake at the EMCS but E-Syt1 does not. (A) Representative immunoblot for PERK, PSD, PSS1, PSS2 in shCTR and shPERK HeLa cells and quantification of PSD, PSS1, PSS2, and PERK, normalized on ACTIN (loading control) and relative to control condition (shCTR). The values shown are the mean ± SEM from three biological replicates analyzed using one sample t test. (B) Abundance of GFP-E-Syt1 pulled-down normalized on abundance of GFP-E-Syt1 in total lysate in HEK293-T cells transiently co-transfected with GFP-E-Syt1 and myc-tagged PERK full-length (FL) or myc-tagged PERK kinase dead mutant (PERK K618A ). The values plotted are the mean ± SEM from three biological replicates analyzed using unpaired Student’s t test. (C) Representative immunoblot for PERK, E-Syt1, CNX, and CYTC from total lysates, mito crude, mito pure, and MAMs fractions of PERK +/+ and PERK -/- MEFs cells. Arrows indicate E-Syt1, PERK, and a non-specific band. (D and E) Quantification of E-Syt1 levels at MAMs (D) and in the total lysate (E) normalized on CNX levels and relative to control condition (PERK +/+ ). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (F and G) Representative images (F) in situ PLA in shCTR and shPERK HeLa cells and quantification (G) of number of dots corresponding to IP3R3-VDAC1 interaction per nucleus and relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. Scale bar, 10 µm. (H and I) Representative images (H) in situ PLA in shCTR and shE-Syt1 HeLa cells and quantification (I) of number of dots corresponding to IP3R3-VDAC1 interaction per nucleus and relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. Scale bar, 10 µm. (J and K) Representative images (J) from EM analysis in shCTR and shE-Syt1 HeLa cells transfected with HRP-KDEL-myc and quantifications (K) of % mitochondria surface engaged into ER–mitochondria contact sites width below 30 nm (mitochondria occupancy %); mitochondrial average perimeter, number of mitochondria normalized on total mitochondrial perimeter and average number of mitochondria. The values plotted are the mean ± SEM ( n = 10 cells) analyzed using unpaired Student’s t test. Scale bar, 500 nm. (L and M) Representative traces (L) of mitochondrial calcium uptake after ER calcium depletion (ATP 100 µM) in shCTR and shPERK transiently transfected with mitochondrial Aequorin WT (mtAeqWT); quantification (M) of the mitochondrial peak of [Ca 2+ ]. The values plotted are the mean ± SEM from four biological replicates ( n = 22) analyzed using unpaired Student’s t test. Arrow indicates the addition of ATP. (N and O) Representative traces (N) of mitochondrial Ca 2+ uptake after ER calcium depletion (ATP 100 µM) in shCTR and shE-Syt1 transiently transfected with mtAeqWT; quantification (O) of the mitochondrial peak of [Ca 2+ ]. The values plotted are the mean ± SEM from three biological replicates ( n = 19 and n = 18 for shCTR and shE-Syt1, respectively) analyzed using unpaired Student’s t test. Arrow indicates the addition of ATP. *, P < 0.05; **, P <0.01; ***, P <0.001; and NS = not significant. Source data are available for this figure: .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8969/pmc09998969/pmc09998969__JCB_202206008_FigS2.jpg)
