Molecular cloning of Drosophila HCF reveals proteolytic processing and self-association of the encoded protein.
HCF-1 functions as a coactivator for herpes simplex virus VP16 and a number of mammalian transcription factors. Mature HCF-1 is composed of two subunits generated by proteolytic cleavage of a larger precursor at six centrally-located HCF(PRO) repeats. The resulting N- and C-terminal subunits remain tightly associated via two complementary pairs of self-association domains: termed SAS1N-SAS1C and SAS2N-SAS2C. Additional HCF proteins have been identified in mammals (HCF-2) and Caenorhabditis elegans (CeHCF). Both contain well-conserved SAS1 domains but do not undergo proteolytic processing. Thus, the significance of the cleavage and self-association of HCF-1 remains enigmatic. Here, we describe the isolation of the Drosophila HCF homologue (dHCF) using a genetic screen based on conservation of the SAS1 interaction. The N-terminal beta-propeller domain of dHCF supports VP16-induced complex formation and is more similar to mammalian HCF-1 than other homologues. We show that full-length dHCF expressed in Drosophila cells undergoes proteolytic cleavage giving rise to tightly associated N- and C-terminal subunits. As with HCF-1, the SAS1N and SAS1C elements of dHCF are separated by a large central region, however, this sequence lacks obvious homology to the HCF(PRO) repeats required for HCF-1 cleavage. The conservation of HCF processing in insect cells argues that formation of separate N- and C-terminal subunits is important for HCF function.
Mahajan
Shahana S
SS
Department of Microbiology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016, USA.
Johnson
Kristina M
KM
Wilson
Angus C
AC
D000375:Aging / Q000502:physiology; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000302:isolation & purification; D004330:Drosophila / Q000235:genetics*; D017405:Herpes Simplex Virus Protein Vmw65 / Q000502:physiology; D051863:Host Cell Factor C1; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D014157:Transcription Factors; D014158:Transcription, Genetic
D018076:DNA, Complementary; D017405:Herpes Simplex Virus Protein Vmw65; D051863:Host Cell Factor C1; D011506:Proteins; D014157:Transcription Factors; D010447:Peptide Hydrolases