Gal80's linked interactions: a new model for the GAL gene switch.
Vepkhia Pilauri, Cuong Diep, James Hopper
Biochem & Mol Biol, Penn State Univ, 500 University Drive, Hershey, PA, 17033, USA
The GAL gene switch of S. cerevisiae consists of the Gal3, Gal80 and Gal4 proteins. In the absence of galactose, Gal80 inhibits the transcriptional activator Gal4 through its binding to Gal4's activation domain (Gal4AD). This inhibition is relieved by Gal3's binding to Gal80, an event that appears to occur in the cytoplasm. So, Gal80's binding reactions with Gal4 and Gal3 are key mechanistic features of the switch. Here we identify Gal80's binding determinants for Gal4AD and Gal3. Using PCR mutagenesis, gap repair and reverse two-hybrid selections we selected two original classes of Gal80 single aa variants: those impaired in binding with Gal4AD (class I) and those impaired in binding with Gal3 (class II). These classes were cross-evaluated for their association with Gal3 and Gal4AD and for self-association. These analyses yielded eight 4-/3+ and six 3-/4- and one 3-/4+. Five of the six 3-/4- variants were distinguished from all other variants as severely impaired in self-association. We mapped the mutations on a derived YiaK-Gal80 3-D model, taking advantage of fact that both YiaK (2,3-diketogulonate reductase) and Gal80 are members of NAD oxidoreductase family. The two major classes of mutations (I and II) show strikingly separate clustering. The positions, the distinctive clustering of the two classes of mutants and their differential effects on Gal80-self-association suggests a new mechanism underlying Gal80's pivotal role in the Gal3-Gal80-Gal4 transcriptional switch.
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