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Molecular and Cellular Biology, June 2000, p. 4199-4209, Vol. 20, No. 12
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Two Regulators of Ste12p Inhibit Pheromone-Responsive Transcription by Separate Mechanisms

K. Amy Olson, Chris Nelson, Georgia Tai, Wesley Hung, Carl Yong, Caroline Astell, and Ivan Sadowski*

Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada

Received 25 October 1999/Returned for modification 22 December 1999/Accepted 13 March 2000

The yeast Saccharomyces cerevisiae transcription factor Ste12p is responsible for activating genes in response to MAP kinase cascades controlling mating and filamentous growth. Ste12p is negatively regulated by two inhibitor proteins, Dig1p (also called Rst1p) and Dig2p (also called Rst2p). The expression of a C-terminal Ste12p fragment (residues 216 to 688) [Ste12p(216-688)] from a GAL promoter causes FUS1 induction in a strain expressing wild-type STE12, suggesting that this region can cause the activation of endogenous Ste12p. Residues 262 to 594 are sufficient to cause STE12-dependent FUS1 induction when overexpressed, and this region of Ste12p was found to bind Dig1p but not Dig2p in yeast extracts. In contrast, recombinant glutathione S-transferase-Dig2p binds to the Ste12p DNA-binding domain (DBD). Expression of DIG2, but not DIG1, from a GAL promoter inhibits transcriptional activation by an Ste12p DBD-VP16 fusion. Furthermore, disruption of dig1, but not dig2, causes elevated transcriptional activation by a LexA-Ste12p(216-688) fusion. Ste12p has multiple regions within the C terminus (flanking residue 474) that can promote multimerization in vitro, and we demonstrate that these interactions can contribute to the activation of endogenous Ste12p by overproduced C-terminal fragments. These results demonstrate that Dig1p and Dig2p do not function by redundant mechanisms but rather inhibit pheromone-responsive transcription through interactions with separate regions of Ste12p.

* Corresponding author. Mailing address: Biochemistry and Molecular Biology, 2146 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada. Phone: (604) 822-4524. Fax: (604) 822-5227. E-mail: sadowski{at}interchange.ubc.ca.

Molecular and Cellular Biology, June 2000, p. 4199-4209, Vol. 20, No. 12
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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