Uptake of glucose, fructose, and the nonmetabolizable analog 6-deoxyglucose was measured in wild-type Saccharomyces cerevuiae and two mutant strains, one (hxkl hxk2) lacking both hexokinase A (P-I) and B (P-Il) but containing glucokinase (and hence able to grow on glucose but not fructose) and the other (hxkl hxk2 gik) also lacking glucokinase (and not able to grow on glucose either). Uptake of the nonmetabolized substances (i.e., 6-deoxyglucose in all three strains, fructose in the two mutants, and glucose in the triple mutant) reached a plateau at or below the external concentration. The kinetic characteristics of uptake were determined from 5-sec incubations by plotting velocity (V) vs. velocity/substrate concentration (V/S) curves. According to such plots, in the wild-type strain uptake had two components, "high affinity uptake" with Km values of ca. 1 mM for glucose and 6 mM for fructose and "low affinity uptake" with Km values of ca. 20 and 50 mM, respectively. The double kinase mutant showed both components for glucose but only the high Km component for fructose, while the triple kinase mutant showed only high Km uptake for both glucose and fructose. Genetic analysis showed that only in strains lacking both hexokinases (hxkl hxk2) was the low Km system for fructose absent. Low Km uptake was restored to the triple mutant by introduction of the cloned wild-type genes: HXK1 orHXK2, for fructose uptake, and HXKl, HXK2, or GLKl, for glucose uptake. A phosphoglucose isomerase mutant had both low and high Km uptake for glucose. These results indicate the presence of two types of uptake mechanism for glucose and fructose in yeast, the functioning of one of which, the low Km system, is influenced by the cognate kinases.Glucose transport in yeast is not well understood. Studies of specificity have implicated a constitutive membrane carrier for uptake of glucose, fructose, and mannose and their analogs (1-3). The internal concentration of glucose is low during its metabolism, and the problem, as posed by van Steveninck and Rothstein (4), has been whether the low concentration reflects metabolic phosphorylation following entry by facilitated diffusion or a more intimate connection between entry and metabolism-for example, a transient phosphorylation during entry, or even formation of the first metabolic intermediate, hexose-6-phosphate, occurring by an obligatory vectorial phosphorylation (as with the bacterial phosphotransferase system). Several experimental lines of evidence seem to implicate metabolism with transport. (i) Considerations of kinetics do not simply accord with a membrane carrier of fixed properties delivering hexose to the cytoplasm (5, 6). (ii) Metabolic inhibitors, such as iodoacetate, are known to affect apparent affinity for uptake (7) and, in derepressed cells, as measured by fermentation rate, affinity for glucose is decreased by oxygen (8). (iii) Experiments with 2-deoxyglucose have established that its internal free poolin whatever compartment-is preceded by 2-deoxyglucose 6-phosphat...

Glucose uptake mutants have not been previously obtained in Saccharomyces cerevisiae, possibly because there seem to be at least two transport systems, of low and high affinities. We showed that snf3 (sucrose nonfermenting) mutants did not express high-affinity glucose uptake. Furthermore, their growth was completely impaired on low concentrations of glucose in the presence of antimycin A (which blocks respiration).Several genes which complemented the original snJ3 gene were obtained on multicopy plasmids. Some of them, as well as plasmid-carried SNF3 itself, conferred a substantial increase in high-affinity glucose uptake in both snf3 and wild-type hosts. The effects of glucose on the expression of such a plasmid-determined high-affinity uptake resembled those in the wild type. Other genes complementing snJ3 seemed to cause an increase in low-affinity glucose uptake. We suggest that SNF3 may function specifically in high-affinity glucose uptake, which is needed under some conditions of growth on low glucose concentrations. SNF3 itself or the other complementing genes may specify components of the glucose uptake system.The uptake of glucose and fructose in Saccharomyces cerevisiae is mediated by both high (Km = 1 to 2 mM for glucose and 5 to 7 mM for fructose)-and low (Km = 15 to 20 mM for glucose and 50 to 100 mM for fructose)-affinity transport systems (1). Low-affinity uptake appears to occur by constitutive facilitated diffusion. High-affinity uptake, on the other hand, is not expressed in cells grown in the presence of high glucose concentrations (3), and mutant studies revealed that this transport system is under glucose catabolite repression control (L. Bisson, unpublished data).The activity of the high-affinity hexose transport system is dependent upon the activity of glucose-phosphorylating enzymes within the cell. In S. cerevisiae, there are three known enzymes catalyzing the phosphorylation of glucose at the six position: hexokinase A (PI), hexokinase B (Pll), and glucokinase; the hexokinases also catalyze the phosphorylation of fructose (11,12). While triple kinase mutants (hxkl hxk2 glk) lacking all three kinase activities do not display high-affinity uptake of either glucose or fructose (1), a double kinase mutant (hxkl hxk2) lacking both hexokinases A and B but retaining glucokinase lacks high-affinity uptake of fructose but retains high-affinity uptake of glucose (1). Transformation of a triple kinase mutant with a plasmid carrying a gene encoding any one of the kinases (pHXKI, pHXK2, or pGLK) restored high-affinity uptake of glucose; only pHXKI and pHXK2 were able to restore high-affinity uptake of both glucose and fructose (1). High-affinity uptake of a nonphosphorylatable glucose analog, 6-deoxyglucose, was also found to be "kinase dependent," suggesting some role of the kinases in high-affinity uptake other than simple phosphorylation of the substrate (2).Understanding the role of the kinases in kinase-dependent transport as well as the physiological roles of both low-and high-affinity uptake...

A single gene mutant lacking phosphoglucose isomerase (pgi) was selected after ethyl methane sulfonate mutagenesis of Escherichia coli strain K-10. Enzyme assays revealed no pgi activity in the mutant, whereas levels of glucokinase, glucose-6-phosphate dehydrogenase, and gluconate-6-phosphate dehydrogenase were similar in parent and mutant. The amount of glucose released by acid hydrolysis ofthe mutant cells after growth on gluconate was less than 2% that released from parent cells; when grown in the presence of glucose, mutant and parent cells contained the same amount of glucose residues. The mutant grew on glucose one-third as fast as the parent; it also grew much slower than the parent on galactose, maltose, and lactose. On fructose, gluconate, and other carbon sources, growth was almost normal. In both parent and mutant, gluconokinase and gluconate-6-phosphate dehydrase were present during growth on gluconate but not during growth on glucose. Assay and degradation of alanine from protein hydrolysates after growth on glucose-1-'4C and gluconate-1-_4C showed that in the parent strain glucose was metabolized by the glycolytic path and the hexose monophosphate shunt. Gluconate was metabolized by the Entner-Doudoroff path and the hexose monophosphate shunt. The mutant used glucose chiefly by the shunt, but may also have used the Entner-Doudoroff path to a limited extent. MATERIALS AND METHODS Chemicals. Gluconate-1-_4C was from Nuclear-Chicago Corp., Des Plaines, Ill. Glucose-1-14C, Lalanine-U-14C and DL-alanine-1-14C were from New England Nuclear Corp., Boston, Mass. 2-Keto-3deoxygluconate-6-phosphate was a generous gift from W. A. Wood. Sodium gluconate was from Eastman Organic Chemicals, Rochester, N.Y., and contained 1571

Screening of a mutagenized strain carrying a multicopy ENOI-'lacZ fusion plasmid revealed a new mutation affecting most glycolytic enzyme activities in a pattern resembling that caused by gcrl: levels in the range of 10% of wild-type levels on glycerol plus lactate but somewhat higher on glucose. The recessive single nuclear gene mutation, named gcr2-1, was unlinked to gcrl, and GCRI in multiple copies did not restore enzyme levels. GCR2 was obtained by complementation from a YCp5O genomic library; the complemented strain had normal enzyme levels, as did a strain with GCR2 in multiple copies. GCR2 in multiple copies did not suppress geri. A chromosomal gcr2 null mutant was constructed; its pattern of enzyme activities resembled that of the gcr2-1 mutant and, like the gcr2-1 mutant, its growth defect on glucose was only partial (in contrast to the glucose negativity of the gcrl mutant). Northern (RNA) analysis showed that gcr2 and gcrl affect ENO] mRNA levels.The glycolytic pathway is a major metabolic route in Saccharomyces cerevisiae. Most of the S. cerevisiae enzymes, which form a significant fraction of the total protein (11,14), depend on structural genes expressed at high rates. Whether their expression is regulated may be strain dependent, with reports ranging from virtual constitutivity (1, 6) to apparent induction by glucose (24) of at least some of the enzymes. gcrl mutants were recognized as a class of mutants impaired in their growth on glucose and low in their levels of most of the glycolytic enzymes but relatively normal in their levels of other proteins (6,7,21). GCRJ has been cloned and sequenced (1, 17) and may be a positive, trans-acting transcriptional regulator.Most mutations affecting single glycolytic enzymes have been shown to identify the structural gene rather than specific regulatory elements. Thus, with an ENOJ-'lacZ fusion gene for screening, no mutations so far analyzed appeared to affect only ENO] (unpublished data). However, several mutations affecting more than one enzyme were obtained. The present report describes one of them, named gcr2, which resembles gcrl in its effect on the levels of glycolytic enzymes but identifies a different gene, which we have cloned and mutated. MATERLALS AND METHODSStrains and plasmids. The S. cerevisiae strains used are listed in Table 1. Strain 2845 was the wild-type strain used to isolate mutants in this study.Escherichia coli DH5a (F-endAl hsdRJ7 supE44 thi-J recAl gyrA96 relAl A(1acU169 480 dlacZAM15) was used to propagate all plasmids (13, 31a respectively. pGCR8 (21) is a derivative of YEp13 carrying GCRI. All these plasmids also carry LEU2. YEp352 (15), a multicopy shuttle vector carrying URA3, was used for the subcloning of the GCR2 gene. YIp351 (15), an integration type plasmid carrying LEU2, was used to examine the linkage between the cloned gene and the gcr2 mutation. pGCR2 is a GCR2 clone obtained from an S. cerevisiae genomic DNA library in YCp5O. Plasmids pML16-2, pML17-1, pML18-1, and pML19-1 are "dropout" derivatives of pGCR2. Plasmid...

Yeast hexokinase 2 is known to be a phosphoprotein in vivo, prominently labeled from 32P-inorganic phosphate after a shift of cells to medium with low glucose concentration [Vojtek, A. B., & Fraenkel D. G. (1990) Eur. J. Biochem, 190, 371-375]. The principal and perhaps sole site of phosphorylation is now identified as residue serine-15, by observation of a single tryptic peptide difference, its sequencing and size determination by mass spectrometry, and by mutation to alanine, which prevents phosphorylation in vivo. Although protein kinase A was unlikely to accomplish the phosphorylation in vivo, serine-15 does belong to a protein kinase A consensus phosphorylation sequence, and in vitro phosphorylation by protein kinase A at serine-15 could be shown by labeling and by peptide determination. The alanine-15 mutant enzyme was not phosphorylated in vitro.