Fig. 1. TACE interacts with the PDZ3 domain of SAP97. (A) Interaction analyzed by yeast two-hybrid system. TACE cytoplasmic tail and SAP97 mutant forms are expressed in yeast as fusion proteins with a GAL4 DNA-binding domain and a GAL4 activation domain, respectively (see Materials and Methods) and are schematically represented in the lower part of the figure. (B) Western blot showing that SAP97 mutant forms fused to the GAL4 activation domain are expressed in yeast. Detection was made using anti-SAP97 monoclonal antibody from C. Garner. (C) In vitro pull-down experiment. Upper panel: In vitro synthesized SAP97_DPDZ1-2 and SAP97_DPDZ1-3 (lanes 1 and 2, respectively) were affinity purified with glutathione-agarose bound GST-TACE (lanes 3 and 4, respectively) or with glutathione-agarose bound GST (lanes 5 and 6, respectively). Lower panel: Coomassie staining of GST-TACE cytoplasmic tail (lanes 3 and 4, position indicated by the arrow) and GST (lanes 5 and 6, position indicated by the arrow head).