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.

Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae

“…Again maltose should be used as the carbon source in order to avoid inhibitory effects of extracellular D-glucose on the uptake of pentoses. Moreover, it has been shown before that hexo-/glucokinase deficient yeast mutants accumulate high intracellular levels of D-glucose when incubated with maltose [27,28]. …”

A history of research on yeasts 11. The study of solute transport: the first 90 years, simple and facilitated diffusion1

“…In particular, Karel van Dam and his colleagues, at the E.C. Slater Institute of the University of Amsterdam, showed that assaying the initial rates of glucose uptake on a sub‐s time scale, as opposed to a 5 s scale, had a marked effect on estimates of the apparent kinetic parameters of the glucose carrier171. Figure 21 demonstrates explicitly that the high‐affinity mode of glucose uptake ( K m 2–3 m M ), which appeared to be lacking in the triple‐kinase mutant's behaviour when the assays were based on 5 s samples, was clearly manifest when the sample time was reduced to 0.2 s, by means of a quench flow apparatus.…”

“… Kinetics of glucose uptake by wild‐type and a triple kinase deletion mutant of Saccharomyces cerevisiae grown on glycerol. Uptake was measured (A) after 5 s, (B) after 200 ms. Reproduced from171 …”

“…The Dutch workers concluded in their 1996 paper that ‘high‐affinity transport is not dependent on the presence of the hexose kinases’ [171, p. 446]. This was on the grounds that both the respective values of K m and V max for glucose uptake, by wild‐type and a triple kinase deletion mutant, are very similar (Figure 21), provided the ultra‐fast kinetic techniques are employed171. Should one then conclude further that previous indications of a functional connexion between the carrier mechanism and the kinases are all due to imperfect rate measurements?…”

“…Should one then conclude further that previous indications of a functional connexion between the carrier mechanism and the kinases are all due to imperfect rate measurements? van Dam and his colleagues appear to caution against that view, citing the behaviour of 6‐deoxyglucose, a sugar which yeasts do not metabolize171. As Bisson and Fraenkel reported in 1983, a relatively high‐affinity mode of uptake of 6‐deoxyglucose was evident in a wild‐type strain but was lacking in the triple kinase mutant, unless one of these kinases had been reintroduced by means of an appropriate plasmid32.…”