Fig. 3.

Insertion of viroporin 2B hydrophobic regions 1 (HR1) and 2 (HR2) into microsomal membranes using Lep as a model protein. (A) Schematic representation of the leader peptidase (Lep) construct used to report insertion into the ER membrane of 2B HR1 and HR2. The HR under study is inserted into the P2 domain of Lep flanked by two artificial glycosylation acceptor sites (G1 and G2). The recognition of the HR by the translocon machinery as a TM domain locates only G1 in the luminal side of the ER membrane, preventing G2 glycosylation. The Lep chimera will be doubly glycosylated when the HR being tested is translocated into the lumen of the microsomes. (B) In vitro translation in the presence of membranes of the different Lep constructs. Constructs containing HR1 (residues 35 to 55; lanes 1 and 2) and HR2 (residues 61 to 81; lanes 3 and 4) were transcribed and translated in the presence of membranes. Control HRs were used to verify sequence translocation (trans.; lanes 5 and 6) and membrane integration (inser.; lanes 7 and 8). Bands of nonglycosylated protein are indicated by a white dot; singly and doubly glycosylated proteins are indicated by one and two black dots, respectively. (C) The HR sequence in each construct is shown together with the predicted ΔG apparent value, which was estimated using the ΔG prediction algorithm available on the Internet (http://dgpred.cbr.su.se/). Lysine residues in HR1 are shown in boldface. (D) In vitro translation of HR1-derived mutants at lysine 46.