Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose

Abstract

By using a modified technique to measure glucose uptake in Saccharomyces cerevisiae, potential uncertainties have been identified in previous determinations. These previous determinations had led to the proposal that S. cerevisiae contained a constitutive low-affinity glucose transporter and a glucose-repressible high-affinity transporter. We show that, upon transition from glucose-repressed to -derepressed conditions, the maximum rate of glucose transport is constant and only the affinity for glucose changes. We conclude that the transporter or group of transporters is constitutive and that regulation of glucose transport occurs via a factor that modifies the affinity of the transporters and not via the synthesis of different kinetically independent transporters. Such a mechanism could, for instance, be accommodated by the binding of kinases causing a change in affinity for glucose.Until very recently, glucose uptake in Saccharomyces cerevisiae was thought to be mediated by two kinetically distinct mechanisms. A constitutive low-affinity transport system, consisting of a nonconcentrative facilitated diffusion process with a Km of approximately 20 mM (50 mM for fructose), and a kinase-dependent, glucose-repressible high-affinity transport system, also consisting of a facilitated diffusion process with a Km of approximately 1 mM (5 mM for fructose), have been described (3). The diagnostic technique used in the elucidation of these two systems was the biphasic nature of Eadie-Hofstee plots obtained from short-time-scale (5-s) uptake experiments using radiolabelled glucose. Analysis of such plots, however, is complex and requires computer-assisted nonlinear regression. Simple linearization of the two slopes in the biphasic plot can produce poor estimations of the kinetic parameters or, in severe cases, fail to detect all of the systems present (24, 26).The widespread use of these experimental and analytical techniques has led to the description of high-and low-affinity glucose transporters in several Saccharomyces species (7) as well as other yeasts such as Kluyveromyces lactis (23), Kluyveromyces marxianus (9), and Candida wickerhamii (20).More recent genetic studies have identified a number of genes involved in glucose transport in S. cerevisiae. SNF3 (sucrose nonfermenting), a glucose-repressible component of high-affinity glucose transport (4) and HXT1 and HXT2 (hexose transport) are proposed components of high-affinity glucose transport (14,16). Further studies have revealed that HXT3, HXT4, and an unidentified component are likewise involved in high-affinity glucose transport while SNF3 may be involved only in the regulation of high-affinity transport (13). While at least six components have been identified as being involved in the high-affinity uptake, the nature of the lowaffinity component has remained elusive, and this has led to questions regarding the nature of this component. Several groups have suggested that low-affinity uptake may be a consequence of passive diffusion (8,10,11 Strains and growth con...

Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter

“…The results presented here, and other observations, indicate that metabolic reactions after translocation affect the rate of glucose uptake. In some studies v max was the same for both high-and low-affinity transport systems, and for uptake of hexoses transported by different systems (Mark & Romano, 1971;Scarborough, 1970b;Walsh et al, 1994). In Neurospora crassa it was shown that the maximum uptake rate was similar to the capacity of disrupted mycelium to phosphorylate glucose (Scarborough, 1970a).…”

“…K s values determined for growth in both batch and chemostat cultures depend on substrate uptake, metabolism and growth. Many investigations, however, have focused on separation of the characterization of the uptake process from interfering effects of growth and metabolism by determination of initial glucose uptake rates of starved or washed cells (MacCabe et al, 2003;Mark & Romano, 1971;Torres et al, 1996;vanKuyk et al, 2004;Walsh et al, 1994), isolated plasma membrane vesicles (Fuhrmann et al, 1989) or by description of uptake of non-metabolizable glucose analogues (Brown & Romano, 1969;Moore & Devadatham, 1979).…”

Expression and activity of the Hxt7 high‐affinity hexose transporter of Saccharomyces cerevisiae

“…Glucose uptake was assayed as described (Walsh et al, 1994) at five concentrations of D-U-( 14 C)-glucose [1, 2.5, 5, 10 and 25 mM (371,297,148,74 and 30 MBq/mol,respectively)]. The data were fit to the Michaelis-Menten equation using ENZFITTER software (Elsevier-Biosoft).…”

“…At high glucose concentrations yeast exhibits lowaffinity glucose transport, and at low glucose concentrations or in the absence of glucose (e.g. after the diauxic shift of a glucose-grown batch culture) the affinity displayed by yeast for glucose is high (Bisson and Fraenkel, 1984;Walsh et al, 1994). These differences in affinity presumably reflect the substrate affinities of the individual hexose transport proteins synthesized under these conditions (Reifenberger et al, 1997).…”

Control of specific growth rate in Saccharomyces cerevisiae

“…Zero trans-influx rates of sugars were determined in a 5 s assay, according to Walsh et al (1994), at 30 uC with the modification that growth medium was used instead of phosphate buffer. Cold glucose concentrations were verified by using a glucose oxidase assay: glucose oxidase (Roche Diagnostics), peroxidase and ABTS in 0.5 M Tris/HCl were mixed with standard or sample and incubated at 37 uC for 1 h under slow agitation after which the absorbance at 415 nm was measured using a SPECTRAmax PLUS384 Microplate Spectrophotometer (Molecular Devices).…”