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

Ru-pSg The starting point for the cloning was a plasmid, pETgc/S (a giR from Dr. Danny Schnell), which contained the DNA sequence encoding the mature small subunit of ribulose-l,5-bispbosphate carboxylase (RuBP-Case) cloned into the prokaryotic expression vector pETgc (Rosenberg et ai., 1987) . pETgc/S was digested with XbaI, which cuts 5' to the gene, blunt ended with DNA polymerase, Klenow fragment, and digested with Kpni, a unique site within the RuBPCase gene. This fragment (containing the first 76 codons of the mature subunit of RuBPCase) was used to replace the 5'untranslated region and codons I through 45 and p58AN (including the HA tag) by cloning into the large fragment ofp58AN produced by digestion with EcoRI, blunt end formation with DNA polymerase, Klenow fragment and digestion with KpnI. Ru-p58 contains p58 amino acids 190-637. 13~a/-TML pCHllO (Pharmacia Fine Chemicals), a ~-galactosidase expression plasmid, was digested with ScaI and EcoRI and the resulting large fragment (containing the 5' half of the ampicillin gene, the SV-40 early prorooter, and the gene encoding a lac Z fusion protein), was llgated to the SeaI-EcoRI fragment of pSVX containing the 3' half of the ampicillin gene and the polylinker. The resulting plasmid contained the iac Z fusion (minus the last 16 amino acids from the carboxy end) fused to the polylinker in pSVX. Next, a fragment containing codons 201 through 246 was amplified from p58ATM2-8 by PCR using 5' PCR primer, 5'-GAGAATTCGGTG-CAAGATTCGCGACC-3' (EcoRI site is underlined) and 3' PCR primer, 5'-GTCCACTCTAGAGGATCC-3' (XbaI site is underlined). The resulting fragment was digested with EeoRI and XbaI and cloned into the EeoRI and XbaI sites of the polylinker in the pCH110-derived vector, 3' to and in frame with the lac Z gene. BgaI-TM1 contains p58 amino acids 201-246.