C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya

To determine whether proteins in different subcellular compartments can be expressed by the C3P3-G1 technology, cells were plated and transfected as described above with plasmids encoding the green EGFP or the red RFP fluorescent proteins fused in-frame with mitochondrial, nuclear or endosomal subcellular signals and imaged by direct fluorescence (Supplementary Table S1 ). The human transferrin receptor type I (hTFR1) N-terminal signal localizes the fusion protein to the surface of vesicles of the endosomes (14) . The mitochondrial presequence of S. cerevisiae cytochrome c oxidase subunit 4 (COX4) targets fusion proteins to the inner membrane of the mitochondrial matrix (15) . The eNLS is a strong nuclear signal from SV40 for nuclear subcellular localization (16) . CHO-K1 cells were co-transfected with pCMV-NP868R-(G 4 S) 2 -K1ERNAP(R551S) and the plasmids encoding the different fluorescent protein under control of the K1E promoter. For comparison with standard nuclear expression system, cells were also transfected with plasmids containing the same coding sequences in pCMV-Script backbone. To confirm the localization to the endosomal and mitochondrial compartments of the RFP fluorescent proteins expressed by the C3P3-G1 system, we have investigated their colocalization with EGFP tagged with the human LDLR and rat OCT signal peptides expressed with pCMV-Script plasmids, respectively. Transfected cells were fixed in 4% paraformaldehyde for 20 min at room temperature, then washed with 50 mM glycine solution in PBS for 10 min and permeabilized for 10 min in 0.1% Triton X-100. Cell nuclei were stained with Hoechst 33342 for 15 min, mounted in the anti-fade Vectashield Mounting Medium (Vector Laboratories), and imaged by direct fluorescence as described above.