Fig. 2. The C-terminus of Sec15p physically interacts with Bem1p, whereas the N-terminus of Sec15p is required for the interaction with Sec10p. Cell growth, yeast lysate preparations, GST pull-down and western blotting procedures were as described in the Materials and Methods. (A) Schematic diagram of Sec15p constructs employed in this study. (B) Sec15-Bem1 complexes. Glutathione-Sepharose pull-down products from lysates of cells expressing GST-fusion products of Sec15 (Sec15 GST; NY2559) and Sec15-1 (sec15-1 GST; NY2560), and GST (NY2561), were analyzed by standard SDS-PAGE and western blotting with the indicated antibodies. (C) Glutathione-Sepharose pull-down products from lysates of cells expressing GST-fusion products of the Sec15p constructs described in A were analyzed by standard SDS-PAGE and western blotting with the indicated antibodies. The purified Sec15-GST constructs were resolved on an SDS-PAGE gel and the gel was analyzed by western blotting using anti-GST antibodies. Lysates were loaded to indicate that there were similar amounts of Sec10p present in all lysates although only the full-length Sec15p containing the extreme N-terminal region can pull-down Sec10p. (D) Diagram of the protein-protein interactions of Sec15.