Sigma-1 Receptor Chaperone at the ER-Mitochondrion Interface Mediates the Mitochondrion-ER-Nucleus Signaling for Cellular Survival (original) (raw)
Figure 1
IRE1 localizes at the MAM.
(a) The subcellular distribution of ER stress sensors. All endogenous proteins were prepared from wild-type non-stressed CHO cells. P1, nuclear; Mito, mitochondrial; P3, microsomal; Cyt, cytosolic fractions. NucleoP, nucleoporin p62; Complex V, complex V ATP synthase inhibitor; Cyto c, cytochrome c; ERp57, ER thiol-disulfide oxidoreductase p57; ERK, extracellular signal-regulated kinase. Phosphatidylserine (PtSer) synthase activity was measured by the autoradiographic measurement of 14C-PtSer as described [10]. All other proteins were measured by immunoblotting. (b) The subcellular distribution of IRE1 in CHO cells with reduced expression of mitofusin-2 (MFN2) or Sig-1Rs. Control (siCon) or specific siRNAs (siMFN2, siSig-1R) were transfected two days before the membrane fractionation. (c) Confocal microscopic observation of the subcellular distribution of Sig-1Rs and IRE1 in CHO cells. In top panels, endogenous ERp57 and transfected full-length IRE1-V5 were immunostained. Asterisks indicate CHO cells transfected with IRE1-V5 (Note: no V5 immunoreactivity in non-transfected cells, verifying the high selectivity of V5 immunostaining). In bottom panels, GFP targeting mitochondria was expressed by gene transfection. Arrows indicate clusters of IRE1-FLAG apposing mitochondria. Scale = 10 µm. Insets on a 5× magnification.