Y3H analysis of HIV-1 Nef and AP-2 α–σ2 hemicomplexes with mutations of residues revealed in the crystal structure. (A) Diagram of the plasmids used in Y3H analysis. NL4-3 Nef or mouse Tyrosinase cytosolic tail was cloned into MCS1 of pBridge and expressed as a GAL4BD fusion protein. AP-2 σ2 or AP-1 σ1 was cloned into MCS2 of pBridge and expressed without Met. AP-2 α or AP-3 δ was cloned into MCS of pGADT7 and expressed as a GAL4AD fusion protein. (B–D). The indicated combinations of double transformants were plated in media lacking Leu, Trp, Met and His (−HIS),−HIS with 3-AT (1 mM or 5 mM) or Leu, Trp and Met (+HIS). mTyr, mouse Tyrosinase cytosolic domain.