The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope

After its integration into the ER membrane, gp210 is sorted to the pore membrane, most likely by lateral diffusion in the plane of the membrane. To locate the determinant(s) for sorting, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210 (Fig. 1) ; subcloned them into a transient eukaryotic expression vector (pSVL); injected the DNA directly into the nuclei of subconfluent, unsynchronized 3T3 cells; and monitored localization of the expressed protein by immunofluorescence microscopy using the appropriate antibodies. Typically, 100-200 cells were injected for each experiment and the location of expressed protein was assessed 4-5 h after injection. This time period was experimentally determined to be optimal as it yielded levels of expressed protein that were sufficient for immunofluorescence detection but avoided overexpression and concomitant mislocalization.