Molecular Cloning and Yeast Two-Hybrid Assays

The cloning of the tail of APP (residues 649–695), full-length human μ4, and all the other yeast two-hybrid (Y2H) constructs, and the cloning of the C-terminal domain of μ4 for expression in E. coli was described previously [18] (link). To generate a construct for mammalian expression, full-length human μ4 was obtained by PCR amplification and cloned into the EcoRI and SalI sites of pEGFP-N1 (BD BiosciencesClontech, Mountain View, CA) including a stop-codon before the GFP coding sequence. The cloning of APP-GFP carrying the double mutation F615P/D664A was described previously [34] (link). Single amino acid substitutions were introduced using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Y2H assays were performed as previously described [30] . A set of pCI-neo (Promega) constructs encoding hemagglutinin (HA)-epitope tagged wild-type or mutants of human μ4 was a generous gift of J. Bonifacino and R. Mattera (Cell Biology and Metabolism Program, NICHD, NIH). The nucleotide sequences of all recombinant constructs were confirmed by dideoxy sequencing.