Figure 1.

Use of biotin tagging to discern MRAP orientation. (A) Schematic representation showing the sequence of the acceptor peptide (AP) for bacterial biotin ligase that catalyzes addition of biotin to Lys. Addition of C-terminal KDEL sequence to BirA targets it to the ER. Circles depict a potential N-linked glycosylation site (Asn-X-Ser/Thr) with or without glycosylation. An AP sequence was added immediately after the initiating Met in AP–MRAP or at the C-terminus in MRAP–AP as detailed in Section “Materials and Methods.” (B) ACTH responses of CHO cells expressing a cAMP-responsive CRE-luciferase reporter, MC2 receptor, and MRAP constructs.