After cDNA microinjection, FP-17 remains in the ER, wheras FP-22 is exported by a COPII mechanism and travels through the Golgi complex to the plasma membrane. (a) Schematic representation of the two constructs. The N-terminal monomeric EGFP, Cerulean or Venus (green), are followed, in sequence, by the following: a linker (red) containing the myc epitope, a repeated Gly-Ser sequence, and residues 94–106 of rabbit cyt b5; the TMD (yellow) of cyt b5 (17 residues) or a modified version thereof extended by 5 residues (ILAAV); and the seven-residue C-terminal polar peptide (blue) of rabbit cyt b5. The N-terminal FP is exposed to the cytosol (Bulbarelli et al., 2002). (b) CV1 cells were microinjected with the cDNAs coding for Venus-17 and Cerulean-22 (left) or Cerulean-17 and Venus-22 (middle and right). 60 min after microinjection, 30 μg/ml cycloheximide was added. Cells were imaged alive by wide field microscopy for the two FPs at the indicated times after microinjection. The middle and right columns show the same cell, 30 (middle) and 90 (right) min after the addition of cycloheximide. In the bottom row, which shows the merged images for each time point, red and green pseudocolors are attributed to FP-22 and -17, respectively. (c) Exit of FP-22 from the ER is COPII-dependent. CV1 cells were cotransfected with H79G-Sar1 and YFP (as a marker of transfection; left) and 12 h thereafter microinjected with the cDNA coding for FP-22. Cells were fixed 6 h later and imaged by wide-field microscopy. 2D-deconvolved images are shown. The left and middle show the same field of microinjected cells visualized for YFP (left), and Cerulean-22 (middle), which remains in the ER in the H79G-Sar1–expressing cells. The right shows two microinjected cells that were negative for YFP and in which Cerulean-22 is at the cell surface. Bars, 10 μm.