The N-terminal region of OsEro1 is necessary and sufficient for rER membrane localization. (A) Schematic representation of constructs. Blue boxes, OsEro1; red boxes, predicted TMD of OsEro1 (Ala-37 to Ser-55); yellow lines, catalytic active sites of OsEro1 (Cys-134-Cys-139 and Cys-391-Cys-394); green boxes, GFP. (B) Extracts from seeds (10 DAF) expressing each of 3 chimeric genes encoding OsEro1-GFP (lane 1), spGFP-OsEro1ΔN (lane 2), and OsEro1ΔC-GFP (lane 3) were subjected to SDS/PAGE, followed by Western blot analysis with anti-GFP antibody. The predicted molecular mass of OsEro1-GFP, spGFP-OsEro1ΔN, and OsEro1ΔC-GFP are 80, 78, and 34 kDa, respectively. (C–E) Confocal fluorescence images of the subaleurone cells (10 DAF) expressing OsEro1-GFP (C), spGFP-OsEro1ΔN (D), and OsEro1ΔC-GFP (E). PB-I was labeled with Rhodamine. Arrowheads indicate the rER. The fluorescence signals of DsRed-Sec61β are visible in C, but those of DsRed-Sec61β (D) and Cherry-Sec61β (E) are not detectable. (Scale bar, 5 μm.)