Figure 1

Association of DAT with CPE. A, Using the Yeast 2-Hybrid System 3 (Invitrogen), 1.7 × 106 independent clones of a human substantia nigra library (in pGAD10) were screened using DAT-NT, containing the amino terminus up to the first transmembrane domain (TM1) and DAT-CT, containing the entire carboxyl terminus after the 12th transmembrane domain (TM12) as baits. Interacting clones were selected on media lacking Leu, Trp, and His, in the presence of 2.5 mM 3-AT. Of 158 colonies growing under this selection, 35 were positive for lacZ expression. Sequence analysis of these clones showed that two of them represented fragments of carboxypeptidase E (CPE). One clone showing very strong lacZ expression encoded a portion of CPE beginning at amino acid residue 123 of the mature protein. No interaction was observed between CPE construct and Gal4 BD fused to murine p53 or human lamina C or between any of the DAT-BD fusions and Gal4 AD fused to SV40 large T antigen. B, GST fusion protein precipitation assay using the complete C and N terminus of DAT fused to GST. Aliquots containing GST fusion fragments were incubated with rat striatal extracts and analyzed by Western blot using a polyclonal anti-CPE antibody. C, Using an in vitro binding assay, [³⁵S]-CPE probe bound with GST-DAT-CT, but not with GST-DAT-NT or GST alone. D, DAT coimmunoprecipitated with CPE antibodies from rat striatal extracts.