Fig. 2. Extracellular secretion of epimorphin is dependent on the SNARE/TM domain. (A) Schematic of chimeric and mutant constructs. 3-Hlx, triple-helical N-terminal domain; SNARE, SNARE domain; TM, transmembrane domain. Epimorphin (Epm) is shown in red, syntaxin 1A is shown in blue. (B) Cell surface presentation of epimorphin (TE), syntaxin 1A (TS) and the domain-swapped molecules (TES, TSE), but not Epm lacking the SNARE/TM domain (TES) or the cytoplasmic β-actin protein. PT67 cells transiently transfected with indicated constructs were labeled with membrane-impermeant biotinylation reagents. Cell lysates were incubated with streptavidin-agarose beads and bound (Ex) or unbound (In) fractions were probed with antibodies against β-actin and the T7 tag. (C) Transfection with TE or TSE causes secretion of a soluble form of the molecules into the medium (sup), whereas transfection with TS, TES, or TES does not. Loaded amount of supernatant was five times higher than that of cell lysate. (D) Proteomic analysis of secreted 30 kDa epimorphin. Supernatant of TE transfectant was collected, the 30 kDa soluble epimorphin was retrieved with anti-T7-coated beads and separated by SDS-PAGE. Protein amount from cells of one culture dish (10-cm diameter) was visible with Coomassie Brilliant Blue staining. The protein band was isolated and tryptic fragments were analyzed by mass spectrometry (MS-MS). Identified sequences are underlined. The most C-terminal peptide identified ends with glutamic acid (E), indicating the cleavage site.