Evidence that intramolecular interactions are involved in masking the activation domain of transcriptional activator Leu3p.
The Leu3 protein of Saccharomyces cerevisiae regulates the expression of genes involved in branched chain amino acid biosynthesis and in ammonia assimilation. It is modulated by alpha-isopropylmalate, an intermediate in leucine biosynthesis. In the presence of alpha-isopropylmalate, Leu3p is a transcriptional activator. In the absence of the signal molecule, the activation domain is masked, and Leu3p acts as a repressor. The recent discovery that Leu3p retains its regulatory properties when expressed in mammalian cells (Guo, H., and Kohlhaw, G. B. (1996) FEBS Lett. 390, 191-195) suggests that masking and unmasking of the activation domain occur without the participation of auxiliary proteins. Here we present experimental support for this notion and address the mechanism of masking. We show that modulation of Leu3p is exceedingly sensitive to mutations in the activation domain. An activation domain double mutant (D872N/D874N; designated Leu3-dd) was constructed that has the characteristics of a permanently masked activator. Using separately expressed segments containing either the DNA binding domain-middle region or the activation domain of wild type Leu3p (or Leu3-dd) in a modified yeast two-hybrid system, we provide direct evidence for alpha-isopropylmalate-dependent interaction between these segments. Finally, we use the phenotype of Leu3-dd-containing cells (slow growth in the absence of added leucine) to select for suppressor mutations that map to the middle region of Leu3-dd. The properties of nine such suppressors further support the idea that masking is an intramolecular process and suggest a means for mapping the surface involved in masking.
Wang
D
D
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Hu
Y
Y
Zheng
F
F
Zhou
K
K
Kohlhaw
G B
GB
D000595:Amino Acid Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D005656:Fungal Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry* / Q000502:physiology
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C063667:LEU3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators