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J Biol Chem, Vol. 274, Issue 27, 19017-19024, July 2, 1999

Yeast Transcriptional Regulator Leu3p
SELF-MASKING, SPECIFICITY OF MASKING, AND EVIDENCE FOR REGULATION BY THE INTRACELLULAR LEVEL OF Leu3p

Dake Wang, Feng Zheng, Steen Holmberg^¶, and Gunter B. Kohlhaw

From the  Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 and the ^¶ Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark

Recent work suggests that the masking of the activation domain (AD) of yeast transactivator Leu3p, observed in the absence of the metabolic signal -isopropylmalate, is an intramolecular event. Much of the evidence came from the construction and analysis of a mutant form of Leu3p (Leu3-dd) whose AD is permanently masked (Wang, D., Hu, Y., Zheng, F., Zhou, K., and Kohlhaw, G. B. (1997) J. Biol. Chem. 272, 19383-19392). In a modified two-hybrid experiment, the ADs of both wild type Leu3p and Leu3-dd were shown to interact with the remainder of the Leu3 protein, in an -isopropylmalate-dependent manner. The finding that masking and unmasking proceed apparently normally when full-length Leu3p is expressed in mammalian cells is also consistent with the notion of intramolecular masking. Here we report on the identification of nine missense mutations (all of them suppressors of the Leu3-dd phenotype) that cause permanent unmasking of Leu3p. The nine mutations map to three short segments located within a 140-residue-long region of the C-terminal part of the middle region of Leu3p. These segments may be part of a spatial trap for the AD. We also performed "domain swaps" between Leu3p and Cha4p, a serine/threonine-responsive activator that, like Leu3p, belongs to the family of Zn(II)₂Cys₆ proteins. We show that AD masking and response to the appropriate metabolic signal only occur when a given AD remains attached to its own middle region; middle region swapping results in constitutively active proteins. Finally, we show that the extent to which Leu3p regulates reporter gene expression depends on the intracellular concentration of Leu3p. The possible physiological significance of this observation is discussed in light of the known regulation of Leu3p by Gcn4p.

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