Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway

The c-myc epitope tagged and N-glycosylated form of ERGIC-53 designated GM is shown in Fig. 1 . The DNA sequence coding for the c-myc epitope EQKLISEEDL was introduced after the signal sequence at the amino acid position 31 of ERGIC-53 by PCR-based splicing . The PCR amplified DNA was cloned back to the ERGIC-53 cDNA (Schindler et al., 1993) via the Sacll site at position 116 leading to P53cmyc. In a second reaction aspartic acid at position 61 of wild-type ERGIC-53 was mutated to asparagine by PCR mutagenesis leading to a NGT N-glycosylation consensus site. The PCR-amplifled DNA was ligated to the ERGIC-53 cDNA via the BglI restriction site at position 269. This construct was termed P53G1. The relegated DNA was treated with Klenow and cloned into the EcoRV site of Bluescript SK vector (Stratagene, La Jolla, CA). Both constructs were cloned into pECE to test for expression in COS cells. To create the final GM construct the two mutations were combined by ligating the cmyc tagged 5'-end of P53cmyc via the SacI site at position 116 to P53G1. For expression in COS cells GM was cloned as a SalI/XbaI fragment into the pECE expression vector (Ellis et al., 1986) . All mutations were confirmed by DNA-sequencing (Sequenase 2.0; US Biochemical Co., Cleveland, OH).