A three component switch consisting of Gal4p, a transcriptional
activator, Gal80p, an antagonist of Gal4p, and Gal3p, an antagonist of
Gal80p, provides galactose-specific regulation of GAL genes in S.
cerevisiae . In absence of galactose Gal80p's association with Gal4p
inhibits Gal4p's transcriptional activator function. In presence of
galactose, Gal3p interacts with Gal80p and releases Gal80p's inhibition
of Gal4p. Just how the Gal3p-Gal80p interaction alters the Gal80p-Gal4p
interaction is unknown although Leuther and Johnston (Science 256
1333-1335) have presented evidence for nondissociation of Gal4p and
Gal80p in presence of galactose. Here we present in vivo . 2-hybrid data showing that in presence of galactose Gal3p destabilizes the
interaction between Gal4AD(aa768-881) and Gal80p. In separate in
vitro . experiments we quantify the binding of Gal80p to
Gal4AD(aa768-881) in the presence and absence of Gal3p, ATP, and
galactose. We find that Gal3p acts in a galactose and ATP dependent
manner to markedly destabilize the Gal4AD/Gal80 complex. In other
experiments utilizing co-immunoprecipitation of Gal80p and full length
Gal4p(aa1-881) we observe less Gal80p/Gal4p complex in presence of Gal3p
and galactose and ATP. We hypothesize that in response to galactose (and
ATP) Gal3p destabilizes binding of Gal80p to the C-terminal end (aa768-881) of Gal4p resulting in the displacement of Gal80p to a second lower
affinity site on Gal4p.
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