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Fig. 4. The C-terminus of Sec15p directly interacts with Bem1p, and the N-terminus of Bem1p is necessary and sufficient for Sec15p interaction. (A) Schematic diagram of recombinant proteins used in this study. Several proteins that have been reported to interact with specific domains of Bem1p are indicated. SH3, Src-homology 3; PX, Phox homology domain; PB1, Phox and Bem1 domain; 6xHis, 6-histidine tag. (B) The Sec15p C-terminal region can directly interact with Bem1p. The C-terminus of Sec15 (residues 557-910) was fused to GST (GST-Sec15^557-910), purified and immobilized on the resin. Bem1p was fused to a 6xHis tag and purified from baterial lysates. These proteins were used in an in vitro binding assay as described in the Materials and Methods. The panel on the left shows purified recombinant GST-Sec15^557-910 and GST constructs resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. Proteins of appropriate molecular masses were observed. The C-terminal fragment of Sec15 immobilized to glutathione-Sepharose beads was incubated with bacterially purified Bem1p. The beads were then washed and the bound proteins were resolved by SDS-PAGE and detected by western blot analysis using anti-Bem1p serum. (C) The interaction with Sec15p requires the N-terminal region of Bem1p. The panel on the left shows N-terminally truncated alleles of Bem1p expressed in bacteria. For binding studies, crude bacterial lysates containing these truncated constructs were incubated with GST-Sec15^557-910 immobilized on glutathione beads. The beads were washed and the bound products were resolved by SDS-PAGE and detected by western blot analysis using anti-His monoclonal antibody. (D) The N-terminal 138 residues are necessary and sufficient for mediating the Sec15p interaction. The crude bacterial lysates containing the minimal 138 amino acids at the N-terminus of Bem1 were incubated with GST-Sec15^557-910 on glutathione beads and the binding assay was performed in similar fashion to panel C.

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