Regulation of the galactose pathway in Saccharomyces cerevisiae : Induction of uridyl transferase mRNA and dependency on GAL4 gene function

InSaccharomyces cerevisiae , utilization of galactose requires four inducible enzyme activities. Three of these activities (galactose-1-phosphate uridyl transferase, EC 2.7.7.10; uridine diphosphogalactose 4-epimerase, EC 5.1.3.2; and galactokinase, EC 2.7.1.6) are specified by three tightly linked genes (GAL7, GAL10 , andGAL1 , respectively) on chromosome II, whereas the fourth, galactose transport, is specified by a gene (GAL2 ) located on chromosome XII. Although classic genetic analysis has revealed both positive and negative regulatory genes that coordinately affect the appearance of all four enzyme activities, neither the basic events leading to the appearance of enzyme activities nor the roles of the regulatory genes have yet been determined. Regulation of inducible enzyme activity could be mediated by events related to transcription, translation, or enzyme activation. For the purpose of studying galactose pathway induction and its regulation, we have developed an immunoprecipitation assay that enables us to detect theGAL7 specified uridyl transferase polypeptide in yeast extracts and among the polypeptides synthesized in an RNA-dependentin vitro translation system. Use of this immunoprecipitation assay in conjunction within vivo labeling experiments demonstrates the presence of [3 H]leucine-labeled transferase in extracts prepared from cells grown in galactose but not from cells grown in glucose. This galactose-specific induction of transferase polypeptide is mediated by thede novo appearance of a functional mRNA species whose synthetic capacity is detectable by the combination ofin vitro translation and immunoprecipitation. The appearance of functional transferase mRNA depends on wild-type expression of the positive regulatory gene,GAL4 . Cells carrying a nonsense (amber) mutation in theGAL4 gene fail to produce the transferase mRNA, whereas a nonsense suppressor of theGAL4 amber mutant regains the galactose-specific mRNA response. Our results establish that the induction of theGAL7 specified uridyl transferase activity is mediated byde novo appearance of a functional mRNA and that this galactose-specific response is dependent on a wild-typeGAL4 gene product.