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

Amino terminal protein sequencing data and cDNA deduced primary structure had established that LBR is synthesized without a cleavable amino terminal signal sequence . Its integration into the membrane therefore is likely to proceed by its transmembrane segments serving as internal signal and stop transfer sequences (Blobel, 1980; Friedlander and Blobel, 1985) leaving the 205 residue long hydrophilic domain untranslocated on the cytoplasmic side of the ER membrane. In vitro expressed LBR could be integrated into rough microsomal membranes (Fig. 2) . In the absence of membranes ( -M E M ) , both LBR (p58) as well as a secretory protein, preprolactin (pPL), remained in the supernatant (S) fraction, whereas in the presence of membrane (+MEM) most p58 and processed prolactin (PL) cosedimented with the membrane in the pellet (P) fraction (Fig. 2 A) . LBR (p58) was integrated into membranes as it continued to sediment in the pellet fraction after In vitro translation in a reticulocyte lysate system containing p58 mRNA (p55) or pPL mRNA (C) was performed in the presence of microsomal membranes. The reactions were extracted with 0.1 M Na2CO3, pH 11.5, or 6 M urea, and soluble proteins (5) were separated from membrane integrated proteins (P) by centrifugation as described in Materials and Methods. Protein samples in A and B were processed as described in Materials and Methods, resolved on 12.5% SDS-PAGE and detected by autoradiography, pPL and PL indicate the position of preprolactin and mature prolactin, respectively. COS-1 cells transfected with p58FL A and B or mock transfected (C), were fixed and permeabilized 40 h (,4 and C) or 24 h (B) after transfection and processed for indirect immunofluorescence microscopy. (.4) Double staining was performed with mouse anti-HA tag mAbs (panel/) and rabbit anti-SSRa anti-peptide antibodies (panel 2). Secondary antibodies were anti-mouse IgG-FITC and anti-rabbit IgG-Texas red. Panel 1 was photographed with a fluorescein filter and panel 2 shows the same cell photographed with a rhodamine filter. (B) Cells were stained with rabbit anti-p58 antibodies, followed by anti-rabbit IgG-FITC and photographed with a fluorescein filter (panel/5 or phase contrast (panel 2). (C) Cells were stained with autoimmune serum that recognizes the endogenous COS cell LBR, followed by anti-human IgG-FITC. Bar, 10 t~m. treatment with NazCO3 at pH 11.5 or 6 M urea, both of which extracted the translocated (processed) secretory protein (Fig. 2 B) .