The Mat␣2 (␣2) protein is a transcriptional repressor necessary for the proper expression of cell typespecific genes in Saccharomyces cerevisiae. Like many transcription factors, ␣2 is rapidly degraded in vivo by the ubiquitin-proteasome pathway. At least two different ubiquitin-dependent pathways target ␣2 for destruction, one of which recognizes the well-characterized Deg1 degradation determinant near the N terminus of the protein. Here we report that the ␣2 corepressors Tup1 and Ssn6 modify the in vivo degradation rate of ␣2. Tup1 modulates the metabolic stability of ␣2 by directly binding to the Deg1-containing region of the protein. TUP1 overexpression specifically stabilizes Deg1-containing proteins but not other substrates of the same ubiquitination enzymes that recognize Deg1. Point mutations in both ␣2 and Tup1 that compromise the ␣2-Tup1 binding interaction disrupt the ability of Tup1 to stabilize Deg1 proteins. The physical association between Tup1 and ␣2 competes with the ubiquitination machinery for access to the Deg1 signal. Finally, we observe that overproduction of both Tup1 and Ssn6, but not either alone, strongly stabilizes the endogenous ␣2 protein. From these results, we propose that the fraction of ␣2 found in active regulatory complexes with Tup1 and Ssn6 is spared from rapid proteolytic destruction and is stabilized relative to the uncomplexed pool of the protein.The determination of different cell types in Saccharomyces cerevisiae provides a simple model for understanding the transcriptional regulatory mechanisms that specify distinct cellular identities (10,11,17). Haploid yeast cells exist as either of two types, a or ␣, that can conjugate with each other to produce a third kind of cell, the a/␣ diploid. These three cell types are phenotypically distinct because of the expression of cell typespecific genes: cells of the ␣ type exclusively activate ␣-specific genes, while the a-specific genes are transcribed only in a cells. In addition, a set of haploid-specific genes are expressed in both a and ␣ cells but are repressed in a/␣ diploids. These unique patterns of gene expression are regulated by a small number of transcription factors that function in various combinations to create this complex transcriptional circuit (7). For example, the a-specific genes are activated in a cells by the DNA-binding protein Mcm1 but are strongly repressed in ␣ and a/␣ cells through the combinatorial action of Mcm1 and the homeodomain protein Mat␣2 (␣2). The binding of these two proteins to sequences in the upstream region of a-specific genes tethers the Tup1-Ssn6 general repression complex in the vicinity of a-specific gene promoters (19,20), where it potently represses transcription by a variety of mechanisms (35). This repression complex is recruited to target promoters by ␣2 through several distinct protein-protein interactions: Tup1 binds to the N-terminal domain of ␣2, while Ssn6 directly contacts the homeodomain in the C terminus of the protein (22,34,36).Although ␣2 directs the extremely robust an...