Figure 1.

Cloning scheme for the preparation of the pIN-G construct and the transmembrane topology of the pIN-G reporter protein. 1A. A fragment encoding EGFP, obtained by AgeI/SalI digestion of pEGFP-C1, was ligated into an XmaI/SalI digested pDisplay backbone and the entire protein coding region PCR-amplified and ligated into a host backbone generated through NheI/KpnI digestion of pEGFP-C1 to retain the 3' multiple cloning site but exclude the EGFP coding region. The final pIN-G construct, with the appropriate reading frame throughout, was generated via a subsequent round of PCR. See Methods for further details. 1B. Following expression and cleavage of the Igk- chain leader sequence, the pIN-G protein is proposed to adopt a transmembrane orientation where HA and cMyc epitope tags and GFP have an extracellular disposition at the cell surface (or intra-lumenal in organelles or vesicular trafficking intermediates).