Abstract
UDP-galactose 4'-epimerase (GALE) catalyzes the final step of the highly conserved Leloir pathway of galactose metabolism. Loss of GALE in humans results in a variant form of the metabolic disorder, galactosemia. Loss of GALE in yeast results in galactose-dependent growth arrest. Although the role of GALE in galactose metabolism has been recognized for decades, the precise relationship between GALE activity and galactose sensitivity has remained unclear. Here we have explored this relationship by asking the following. 1) Is GALE rate-limiting for galactose metabolism in yeast? 2) What is the relationship between GALE activity and galactose-dependent growth arrest in yeast? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? To answer these questions we engineered a strain of yeast in which GALE was doxycycline-repressible and studied these cells under conditions of intermediate GALE expression. Our results demonstrated a smooth linear relationship between galactose metabolism and GALE activity over a range from 0 to approximately 5% but a steep threshold relationship between growth rate in galactose and GALE activity over the same range. The relationship between abnormal accumulation of metabolites and GALE activity was also linear over the range from 0 to approximately 5%, suggesting that if the abnormal accumulation of metabolites underlies galactose-dependent growth-arrest in GALE-impaired yeast, either the impact of individual metabolites must be synergistic and/or the threshold of sensitivity must be very steep. Together these data reveal important points of similarity and contrast between the roles of GALE and galactose-1-phosphate uridylyltransferase in galactose metabolism in yeast and provide a framework for future studies in mammalian systems.
Highlights
Galactose is metabolized in species ranging from Escherichia coli to mammals via a series of reactions collectively known as the Leloir pathway
1) Is galactosemia is epimerase (GALE) ratelimiting for galactose metabolism in otherwise Leloir wild-type yeast and, if so, over what range? 2) What is the relationship between GALE activity and galactose-dependent growth impairment in yeast? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? With this last question we are seeking to know if some galactose metabolites may correlate more closely with the degree of galactose sensitivity than do others, perhaps implicating a role for these compounds in the mechanism of sensitivity
We have addressed three fundamental questions regarding the role of GALE in galactose metabolism and sensitivity in yeast
Summary
6% GALE appeared to be in excess for all of the outcomes measured. Together, these data both quantify the role of GALE and define key similarities and distinctions between GALT and GALE as mediators of galactose metabolism and sensitivity in yeast. With this last question we are seeking to know if some galactose metabolites may correlate more closely with the degree of galactose sensitivity than do others, perhaps implicating a role for these compounds in the mechanism of sensitivity To ask these questions we inserted a doxycycline-regulated promoter just upstream of the GAL10 open reading frame in otherwise Leloir wild-type yeast, rendering the encoded GALE enzyme doxycycline repressible. We grew these cells under conditions of differential drug concentration and followed their growth and metabolic response to galactose exposure.
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