Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in vivo and in vitro interactions with the p48 subunit of chicken chromatin assembly factor-1.
We cloned cDNA encoding chicken cytoplasmic histone acetyltransferase-1, chHAT-1, comprising 408 amino acids including a putative initiation Met. It exhibits 80.4% identity to the human homolog and possesses a typical leucine zipper motif. The glutathione S:-transferase (GST) pull-down assay, involving truncated and missense mutants of the chicken chromatin assembly factor-1 (chCAF-1)p48, revealed not only that a region (comprising amino acids 376-405 of chCAF-1p48 and containing the seventh WD dipeptide motif) binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. The GST pull-down assay, involving truncated and missense chHAT-1 mutants, established that a region, comprising amino acids 380-408 of chHAT-1 and containing the leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. In addition, mutation of each of four Leu residues in the leucine zipper motif prevents the in vitro interaction. The yeast two-hybrid assay revealed that all four Leu residues within the leucine zipper motif of chHAT-1 are necessary for its in vivo interaction with chCAF-1p48. These results indicate not only that the proper leucine zipper motif of chHAT-1 is essential for its interaction with chCAF-1p48, but also that the propeller structure of chCAF-1p48 expected to act as a platform for protein-protein interactions may not be necessary for this interaction of chHAT-1.
Ahmad
A
A
Department of Biochemistry, Miyazaki Medical College, 5200, Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Nagamatsu
N
N
Kouriki
H
H
Takami
Y
Y
Nakayama
T
T
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002645:Chickens; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D051548:Histone Acetyltransferases; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D009154:Mutation; D020125:Mutation, Missense; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D000123:Acetyltransferases; D051548:Histone Acetyltransferases; C110880:histone acetyltransferase type B complex; D005982:Glutathione Transferase; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases