High‐affinity Glucose Uptake in Saccharomyces cerevisiae is not Dependent on the Presence of Glucose‐Phosphorylating Enzymes

Abstract

Glucose uptake in Saccharomyces cerevisiae is believed to consist of two kinetically distinguishable components, the affinity of which is modulated during growth on glucose. It has been reported that triple hexose-kinase deletion mutants do not exhibit high-affinity glucose uptake. This raises the question of whether and how high-affinity glucose uptake is related to the presence of glucose-phosphorylating enzymes. In this study the kinetics of glucose uptake in both wild-type cells and cells of hexose-kinase deletion mutants, grown on either glycerol or galactose, were determined using a rapid-uptake method. In wild-type cells glucose uptake measured over either 5 s or 200 ms exhibited high affinity. In contrast, in cells of hexose-kinase deletion mutants the apparent affinity of glucose uptake was dependent on the time scale during which uptake was measured. Measurements on the 5-s scale showed apparent low-affinity uptake whereas measurements on the 200-ms scale showed high-affinity uptake. The affinity and maximal rate of the latter were comparable to those in wild-type cells. Using a simple model for a symmetrical facilitator, it was possible to simulate the experimentally determined relation between apparent affinity and the time scale used. The results suggest that high-affinity glucose transport is not necessarily dependent on the presence of glucose-phosphorylating enzymes. Apparent low-affinity uptake kinetics can arise as a consequence of an insufficient rate of removal of intracellular free glucose by phosphorylation. This study underlines the need to differentiate between influences of the translocator and of metabolism on the apparent kinetics of sugar uptake in yeast.

Metabolic Signals Trigger Glucose-Induced Inactivation of Maltose Permease in Saccharomyces

“…The hexokinases (and glucokinase) appear to function as glucose sensors in glucose-induced inactivation of maltose permease because of their pivotal role in controlling the metabolic flux through the initial steps of glycolysis. This control is achieved indirectly by regulating the rate of facilitated glucose transport as a result of regulating the rate of glucose phosphorylation (43).…”

“…HXK1, HXK2, and GLK1 encode Saccharomyces cerevisiae hexokinase 1, hexokinase 2, and glucokinase, respectively (reviewed in reference 25). These enzymes catalyze the first step in glycolysis and reportedly are involved in high-affinity glucose transport (1,2,43). HXK2 is an upstream negative regulator of the glucose repression pathway (reviewed in reference 25).…”

Strategies to determine the extent of control exerted by glucose transport on glycolytic flux in the yeast Saccharomyces bayanus

“…The zero trans-influx rate of glucose, however, does not represent the actual activity of glucose transport under steady-state conditions. Glucose transport is connected to the rest of metabolism by intermediary pools of metabolites, most obviously the product of the glucose transport step, intracellular glucose (Walsh et al, 1994a ;Smits et al, 1996). Glucose transport can be stimulated or inhibited by various metabolites, the concentrations of which are determined by the total set of kinetic parameters of metabolism.…”

Glucose sensing and signalling properties in Saccharomyces cerevisiae require the presence of at least two members of the glucose transporter family

“…The initial rate of glucose uptake was assayed over 5 s as described previously (16,17). The 5-s method was employed since in these mutants there is no limitation to be expected by hexose-kinase activity (15,17). Where tested, 0.2-s uptake measurements (15,17) yielded activities identical to those determined by 5-s measurements.…”

“…The 5-s method was employed since in these mutants there is no limitation to be expected by hexose-kinase activity (15,17). Where tested, 0.2-s uptake measurements (15,17) yielded activities identical to those determined by 5-s measurements. Analysis of kinetic data has been described previously (16).…”