Evidence That Intramolecular Interactions Are Involved in Masking the Activation Domain of Transcriptional Activator Leu3p

Abstract

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 ␣-isopropylmalate, an intermediate in leucine biosynthesis. In the presence of ␣-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 ␣-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.

Yeast Transcriptional Regulator Leu3p

“…The finding that Leu3p exhibited perfectly normal regulatory behavior when expressed in mammalian cells (13) was consistent with the notion that masking of the activation domain in the absence of ␣-IPM and its unmasking in the presence of ␣-IPM did not require extraneous factors but were intramolecular events. This idea was supported by a recent analysis of the mechanism of masking (10). Permutation of the AD revealed the presence of two types of residues as follows: those that, when mutated, appear to loosen the masking interactions (e.g.…”

“…Transfer of the suppressor mutations to wild type Leu3p caused constitutivity, i.e. such mutants were active irrespective of the presence or absence of ␣-IPM, a result consistent with an involvement of the affected residues in masking (10). In the present work, nine individual constitutivity-causing residues are identified.…”

“…Site-directed Mutagenesis of the Leu3p Middle Region-A previous effort to identify suppressors of the Leu3-dd phenotype had yielded multiple site suppressors that mapped to a region bordered by residues 472 and 765 (10). To find out which, if any, particular residue was responsible for suppression, PCR methodology (18) was used to generate all of the single mutations that made up a given multiple mutation suppressor.…”

“…D872N and D874N). When the latter two aspartates were simultaneously changed to asparagines, the resulting mutant (designated Leu3-dd) was permanently masked (10). Leu3-dd, like all Leu3p mutants with an impaired AD, caused repression of LEU2 expression and severely reduced cell growth in the absence of leucine.…”

“…The AD had early on been found to be located within the C-terminal 30 residues and to function when fused directly to a DNA binding domain (9). A recent study showed that as few as 9 of the 30 C-terminal residues suffice to allow substantial transcriptional activation (10). This core AD contains three acidic and four hydrophobic residues.…”