Abstract
Employing avian erythrocytes, we have previously isolated a multimeric complex consisting of the lamin B receptor (LBR, or p58), the nuclear lamins, an LBR-specific kinase, a 34-kDa protein, and an 18-kDa polypeptide termed p18. As the LBR kinase and the 34-kDa component have been recently characterized, we now proceed in the characterization of p18. We show here that p18 is an integral membrane protein specific to the erythrocyte nuclear envelope which binds to LBR and B-type lamins. NH2-terminal sequencing indicates that p18 is distinct from other nuclear envelope components, but has similarity to the mitochondrial isoquinoline-binding protein. In situ analysis by immunoelectron microscopy and examination of digitonin-permeabilized cells by indirect immunofluorescence show that p18, unlike LBR and other lamin-binding proteins, is equally distributed between the inner and outer nuclear membrane. Furthermore, cycloheximide inhibition experiments reveal that the fraction of p18 that resides in the outer nuclear membrane does not represent nascent chains en route to the inner nuclear membrane, but rather material in equilibrium with the p18 that partitions with the inner nuclear membrane. The paradigm of p18 suggests that transmembrane complexes formed by the nuclear lamins and LBR provide potential docking sites for integral membrane proteins of the nuclear envelope that equilibrate between the rough endoplasmic reticulum and the inner nuclear membrane.
Highlights
Few integral membrane proteins of the nuclear envelope have been characterized so far (for reviews, see Gerace and Foisner (1994) and Georgatos et al (1994))
We show here that p18 is an integral membrane protein specific to the erythrocyte nuclear envelope which binds to LBR and B-type lamins
The co-isolation of p18 and LBR in a native complex (Simos and Georgatos, 1992) and the direct binding of p18 to LBR and B-type lamins in vitro indicate that these proteins form a transmembrane assembly at the level of the inner nuclear membrane
Summary
Cell Fractionation and Chemical Extraction—Turkey erythrocyte nuclear envelopes and plasma membranes were isolated as described previously (Georgatos and Blobel, 1987a). Samples of urea-extracted nuclear envelopes were analyzed by SDS-PAGE, the proteins transferred to ProBlott membranes, and the 18-kDa band was excised from the blot. Both isolation methods yielded the same results. The incubation mixture was diluted to 0.5 ml with the addition of 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM PMSF, 1% Triton X-100, and 1 mg/ml fish skin gelatin, and LBR was precipitated with affinity-purified aR1 antibodies (7 g) in the presence or absence of the antigenic peptide R1 (100 g). The mixture was incubated for 2 h at room temperature before LBR or the lamins were immunoprecipitated with affinity-purified aR1 or aLI antibodies and protein A-Sepharose. For immunoelectron microscopy on ultrathin frozen sections, erythrocyte ghosts or hypotonically swelled erythrocytes were fixed overnight in 8% paraformaldehyde in 250 mM Hepes, pH 7.4, and processed as described previously by Griffiths et al (1984)
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