FIG. 7. Mapping the transmembrane topography of hBest1 by insertion of TEVP cleavage sites.
A, control experiment in which an efficiently secreted hGH-TEV-myc-HER3EC fusion protein was expressed in transiently transfected 293 cells, and cell homogenates were subject to TEVP cleavage in the presence or absence of 1% Triton X-100. Immunoblotting with an anti-Myc mAb reveals cleavage at the TEVP site and release of the C-terminal myc-HER3EC fragment only in the presence of TEVP and detergent. The diagram illustrates the exclusion of added TEVP (scissors) from the interior of an intact membrane vesicle (left) and the access of TEVP to the vesicle interior in the presence of detergent (right). The vesicle lumen is topologically equivalent to the outside of the cell. B, TEVP cleavage of the indicated hBest1 mutants carrying a TEVP site insertion. Each mutant is tagged at the extreme C terminus with the Rim3F4 epitope, and proteins were visualized by immunoblotting with mAb Rim3F4. In the 1st 5 panels, each mutant hBest1 carries the sequence GGENLYFQGGG inserted inframe after the indicated codon. The TI-10–3Myc mutant (lower right autoradiogram) carries an additional insertion of 3 Myc epitopes N-terminal to the TEVP cleavage site. The cleavage of this mutant by TEVP (scissors) is shown schematically at the lower right. A part of a membrane bilayer (double lines) separates the lumen (upper area) from the cytosol (lower area). Cleavage occurs on the cytosolic face of the membrane. N, N terminus; 3Myc, triple Myc epitope tag. For each mutant, the molecular mass of the C-terminal fragment released by TEVP cleavage (asterisk) is determined by the point of insertion. Bars indicate molecular mass standards (from top to bottom) of 173, 111, 80, 61, 49, 36, and 25 kDa.