The first membrane spanning region of the lamin B receptor is sufficient for sorting to the inner nuclear membrane

To detect chick LBR expressed in COS cells, an epitopetagged chick LBR eDNA was constructed (see Materials and Methods) that contained a nine amino acid epitope of the influenza virus hemagglutinin (HA) inserted after Ser 29 of p58. The epitope-tagged eDNA was cloned into an SV-40 expression vector and the construct (p58FL) was introduced into COS cells by calcium phosphate-mediated transfection. Localization of the protein was monitored by immunofluo-rescence microscopy using anti-HA rnAbs. As shown in Fig. 3 A, 1, the protein was localized predominantly at the nuclear rim. In contrast, when the same cell was stained with antibodies to an endogenous integral membrane protein of the ER, known as signal sequence receptor o~ (SSRa) (Wiedmann et al., 1987) , there was nuclear rim staining and diffuse cytoplasmic staining characteristic for an ER protein (Fig. 3 A, 2) . As the ER and the nuclear envelope are one continuous membrane system that is accessible by lateral diffusion to all membrane proteins targeted to and integrated into the ER, the exclusive nuclear rim staining of HA-tagged p58 can be considered to represent correct sorting to the inner nuclear membrane. If the HA-tagged p58 were specifically sorted to the outer nuclear membrane it should also be located in the ER, and like SSRo~, should yield cytoplasmic staining which it did not.