The LxxLL motifs are critical for LZIP activation function.
(A) Wild-type and mutant versions of
Gal4-LZIPN92 were cotransfected into BHK21 cells together
with the p5xGal-E1B-luc reporter (500 ng), and luciferase activity was
measured after 40 h. (B) Yeast two-hybrid
interaction assay to monitor association between the β-propeller
domain of HCF-1 and wild-type or mutant LZIPN92 fragments.
A yeast GAL1-LacZ reporter strain was transformed with expression
plasmids encoding the wild-type or P134S mutant HCF-1 β-propeller
domain (residues 1–380) fused to the LexA DNA-binding domain together
with plasmids expressing wild-type, LxxLL 1KO, LxxLL 2KO, LxxLL1/2KO,
and LxxLLHBMKO versions of LZIPN92 fused to the B42
activation domain. Strains were grown on 5-bromo-4-chloro-3-indolyl
β-D-galactoside indicator plates. For each
combination, five independent transformants were patched.