Human EndoU localizes to the ER and controls ER morphology. (A) Schematic of the structures of XendoU, human EndoU2, and EndoU-short. The locations of conserved catalytic residues and putative signal sequence/transmembrane domains are indicated. Residue 112 in XendoU is where the homology between the X. laevis and human proteins begins; the N termini are relatively unconserved. (B) Quantitative RT-PCR showing effective knockdown of EndoU with esiRNAs directed against the coding region and 3′ UTR. (C) HeLa cells were cotransfected with EndoU2-GFP and mCherry-Sec61β, and analyzed by fluorescence microscopy. (D) HeLa cells were cotransfected with EndoU-short-GFP and mCherry-Sec61β and analyzed as in C. (E) Cells treated with control esiRNAs (left) or esiRNAs against the coding region (middle) or 3′ UTR (right) of EndoU were transfected with mCherry-Sec61β and analyzed by fluorescent microscopy. (F) Quantification of the percentage of cells exhibiting expanded ER sheets in control, EndoU coding, and EndoU 3′ UTR RNAi. Error bars indicate standard deviation. At least 200 cells were scored and the experiment was performed in triplicate. (G) HeLa cells were treated with control or EndoU 3′ UTR esiRNAs, then transfected with wild-type or catalytically dead EndoU2-GFP or EndoU-short-GFP (resistant to RNAi, lacking the 3′ UTR) and mCherry Sec61β. The percentage of cells exhibiting expanded ER sheets is quantified. Error bars indicate standard deviation. At least 200 cells from three replicates of each condition were scored. Inset panels in C and D are magnified 3×. Bars: (main panels) 10 µm; (insets) 2 µm.