The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal

To determine if the amino-terminal domain of LBR can function as a nuclear envelope targeting signal for an integral membrane protein, it was attached to the transmembrane segment of a type II protein of the ER/plasma membrane. COS-7 cells were transfected with plasmid LMBR-CHL that codes for a chimeric protein containing the amino-terminal domain of LBR fused to the amino-terminal side of the transmembrane segment of chicken hepatic lectin. In cells transfected with this plasmid, nuclear rim fluorescence without ER labeling was observed consistent with an inner nuclear membrane localization (Fig. 5 a) . No appreciable ER fluorescence was observed even 72 h after transfection. Hence, the amino-terminal domain of LBR can function as a nuclear envelope targeting signal and direct an integral membrane protein synthesized on the ER to the inner nuclear membrane. To confirm this hypothesis, the majority of the aminoterminal domain was deleted from full-length LBR and replaced by c~-globin. The expressed chimeric protein was detected in the ER and nuclear envelope of transfected cells (Fig. 5 b) . An exclusive fluorescence labeling of the nuclear envelope, which was seen with LBR and the LBR-chicken hepatic lectin chimeric protein, was never observed even when cells were examined only 12 h after transfection. Fluorescence labeling of the ER and nuclear envelope does not exclude the possibility that some of the expressed protein is in the inner nuclear membrane or nuclear pore membranes; however, a large portion of the ot-globin-LBR chimeric protein is retained in the ER. Therefore, even if some of the ex- Figure 5 . The amino-terminal domain of LBR is a nuclear envelope targeting signal for integral membrane proteins. Top panels show immunofluorescence photomicrographs of COS-7 cells transfected with plasmids LMBR-CHL (a) and AG-LMBR (b) that express chimeric proteins with and without the LBR amino-terminal domain (see text and Fig. 2) . Antibodies against LBR were used in a and antibodies against c~-globin in b. Bottom panels show DAPI fluorescence for the same microscopic fields as shown above. In a the nucleus of an untransfected cell that is not recognized by anti-LBR antibodies can be identified by DAPI fluorescence. Bars, 4/an. pressed chimeric protein reaches the inner nuclear membrane, its targeting signal is weaker than that of the two proteins that contain the amino-terminal domain of LBR.