Fig. 4.

Insertion of HR1 into microsomal membranes using the Lep′ construct. (A) Schematic representation of the Lep-derived construct (Lep′). In this Lep′ construct (∼40 kDa; the nonglycosylated form), HR1 replaces the H2 domain from Lep. The glycosylation acceptor site (G2′) located in the beginning of the P2 domain will be modified only if HR1 inserts into the membrane, while the G1′ site, embedded in an extended N-terminal sequence of 24 amino acids, is always glycosylated. (B) In vitro translation in the presence of membranes. Bands of nonglycosylated protein are indicated by a white dot; singly and doubly glycosylated proteins are indicated by one and two black dots, respectively. The protected glycosylated HR1/P2 fragment (∼36.5 kDa) is indicated by a black triangle.