The bZIP protein LZIP interacts specifically with HCF. (A) Yeast two-hybrid assays showing specificity of the HCF–LZIP interaction. Growth of the yeast GAL1–HIS3 reporter strain transformed with both a Gal4 DNA-binding domain (DBD) fusion expression plasmid and a Gal4 activation domain (AD) fusion expression plasmid is shown. The identity of the fusion constructs is indicated in the diagram. Growth with histidine (+histidine) demonstrates that the expression plasmids are not deleterious to yeast growth, and growth without (−histidine) demonstrates activation of the GAL1–HIS3 reporter gene. The yeast were grown for 7 days at 30°C in both the presence and absence of histidine. (B) Amino acid sequence comparison of human LZIP (hLZIP) with mouse LZIP (mLZIP; Burbelo et al. 1994) and two regions of the LZIP-related Drosophila protein BBF-2/dCREB-A (Abel et al. 1992; Smolik et al. 1992, labeled dBBF-2). Sequence identities to hLZIP are indicated by dots in the comparison. The DNA-binding basic region and dimerization–interface leucine zipper are bracketed, with an asterisk above the seven hydrophobic residues that make up the extended leucine zipper of LZIP. The location of an additional 25 amino acids introduced by an alternate splice variant of mouse LZIP (Burbelo et al. 1994) is indicated (▾). The AD fusion points of the original two-hybrid screen isolates are indicated by the arrows above the top line. (HBM) HCF-binding motif. (C) Association of endogenous HCF and LZIP from human cells. Human 293 whole-cell nuclear extracts were precipitated with the nonspecific 12CA5 influenza hemagglutinin monoclonal antibody (lane 1) or the M2 anti-HCF monoclonal antibody (lane 2), and immunoblots of the precipitates were probed with the C7 LZIP antipeptide antibody.

 LZIP and VP16 recognize HCF similarly. Yeast two-hybrid interactions between mutated HCF_N380 and LZIP (top) and VP16 (bottom). The DBD ⋅ HCF_KEL 1–6 mutants (see diagram) contain individual proline-to-alanine substitutions as follows: KEL1, P30A; KEL2, P79A; KEL3, P134A; KEL4, P197A; KEL5, P252A; KEL6, P319A. The yeast were grown for 7 days at 30°C in both the presence (left) and absence (right) of histidine. A control with wild-type DBD ⋅ HCF_N380 with AD ⋅ LZIP and AD ⋅ VP16 grown in parallel showed the same growth rate as the cells grown in sectors A and E.

 The HCF tsBN67 cell-cycle arrest mutation disrupts interaction with LZIP. Yeast two-hybrid interaction assays with wild-type HCF_N380 (DBD ⋅ HCF_N380, top panels) and tsBN67 HCF_N380 (DBD ⋅ HCF_N380P134S, bottom panels) were performed as indicated in the diagram. The yeast were grown for 5 days at 30°C in both the presence (left) and absence (right) of histidine.

 Mammalian LZIP, Drosophila BBF-2, and VP16 share a small HCF-binding motif. (A) HCF-binding motif sequence alignment. The VP16 sequences are derived from homologs of VP16 from bovine herpesvirus (GenBank accession no. Z54206; BTIF), equine herpesvirus (GenBank accession no. L16590), and varicella-zoster virus (GenBank accession no. X04370; ORF 10). Dots indicate sequence identities with the HSV VP16 (viral) and hLZIP (cellular) sequences shown. The position of the carboxy-terminal residue within the parental sequence is indicated to the right. The HCF-binding motif is boxed and a consensus is shown at the bottom. (D/E) Either aspartic or glutamic acid; (X) nonconserved variable residue. (B) HCF association with wild-type and mutant LZIP and VP16. Epitope-tagged wild-type or nine substitution mutants of LZIP (top panels) and VP16 (bottom panels) were transiently expressed in human 293 cells. The mutated residue in each mutant is indicated above each lane. In LZIP, all residues were changed to alanine, except for the threonine, which was changed to asparagine. In VP16, all nonalanine residues were changed to alanine, and the alanine was changed to serine. Expression of each mutant protein is shown by direct immunoblot with the anti-T7 epitope antibody of the 293 cell extract (lanes 1–10). Extracts were precipitated with the N18 HCF antipeptide antibody (lanes 11–20) or the nonspecific anti-N-Oct-3 antibody (lanes 21–30). In each panel, LZIP or VP16 was detected by immunoblot analysis with the anti T7 epitope tag antibody. (C) HCF recognizes the Drosophila BBF-2 HCF-binding motif. T7-epitope-tagged wild-type and mutant LZIP and BBF-2 were transiently expressed in human 293 cells. Position 1 of the HCF-binding motif in LZIP (D to A) and BBF-2 (E to A) were mutated (Mut). 293 cell extracts were immunoprecipitated with the N18 HCF antibody (lanes 1–4) or the nonspecific anti-N-Oct-3 (lanes 5–8) and the precipitates analyzed by immunoblot with the anti-T7 epitope tag antibody. Direct immunoblot with the anti T7 epitope tag antibody showed that for both LZIP and BBF-2 the wild-type and mutant proteins were expressed at the same level, but the BBF-2 proteins were expressed at higher levels than LZIP (data not shown).