Membrane integration of non-albino wild-type (wt) and mutant ( Snowflake ) G518R sequences. (a) Schematic representation of the engineered leader peptidase (Lep) model protein. Wild-type Lep has two transmembrane (TM) helices (H1 and H2) and a large C-terminal luminal domain (P2). It inserts into rough microsomal (RM) membranes in an Nt/Ct ER luminal orientation. SLC45A2 TM12 domains (TM12) wild-type and G518R mutant were inserted in the P2 domain flanked by two glycosylation acceptor sites (G1 and G2). If the inserted sequence integrates into the membrane, only the G1 site is glycosylated (left), whereas both G1 and G2 sites are glycosylated for the sequences that do not integrate into the membrane (right). (b) Plasmids encoding the constructs were transcribed and translated in vitro in the absence (−) and presence (+) of RM membranes. Non-glycosylated protein bands are indicated by a white dot; singly or doubly glycosylated proteins are indicated by one or two black dots, respectively. (c) SLC45A2 TM12 sequence in each construct and ΔGapp values predicted using the ΔG Prediction Server (http://dgpred.cbr.su.se/) and deduced from the data in panel b are shown.