Fig. 4

Insertion of hydrophobic regions of BB0173 into membranes using Lep as model protein. a The HR sequence in each construct is shown together with the predicted G apparent value, which was estimated using the ∆G prediction algorithm available on the Internet (http://dgpred.cbr.su.se/). Glycosylation acceptor site is shown in bold. b Schematic representation of the Lep construct used to report insertion of hydrophobic regions of BB0173 into endoplasmic reticulum membranes. The TM segment under investigation (HR-tested) was introduced into the P2 domain of Lep, flanked by two artificial glycosylation acceptor sites (G1 and G2). Recognition of the tested sequence as a TM domain by the translocon machinery results in the location of only G1 in the luminal side of the ER membrane, preventing G2 glycosylation (left). The Lep chimera will be doubly glycosylated when the sequence being tested is translocated into the lumen of the microsomes (right). c In vitro translation in the presence of membranes of the different Lep constructs. Constructs containing HR1 (residues 7 to 25; lanes 1–3), HR2 (residues 55 to 77; lanes 4–6), HR3 (residues 163 to 185; lanes 7–9) and HR4 (residues 310 to 328; lines 10–12) were translated in the presence (+) and absence (−) of rough microsomes (RM) and proteinase K (PK). Bands of non- glycosylated proteins are indicated by a white dot; singly and doubly glycosylated proteins are indicated by one and two black dots, respectively. In the case of Lep-HR3 construct a triply glycosylated band was observed (lane 8) due to the presence of an acceptor NGS site (residues 169–171) within the (translocated) hydrophobic region. The protected doubly-glycosylated H2/HR3/P2 fragment is indicated by an arrowhead. Control HRs were used to verify sequence translocation (translocated; lanes 13–15)