Transport-associated Phosphorylation Research Articles

Investigation of transport-associated phosphorylation of sugar in yeast mutants (snf3) lacking high-affinity glucose transport and in a mutant (fdp1) showing deficient regulation of initial sugar metabolism

Pool-labeling experiments with 2-deoxyglucose in derepressed cells of the yeastSaccharomyces cerevisiae confirmed the previously reported results pointing to the possible existence of transport-associated phosphorylation of sugar. In yeast mutants containing a disruption or an inactivating point mutation in thesnf3 gene, which codes for the high-affinity glucose carrier, no evidence for transport-associated phosphorylation of 2-deoxyglucose was observed. If transport-associated phosphorylation in yeast exists, it is apparently not mediated by the low-affinity glucose carrier. Mediation by the high-affinity carrier would fit with the known requirement of an active kinase for high-affinity sugar transport. A mixed type of uptake in cells having both carriers would explain many of the problems associated with the 2-deoxyglucose pool-labeling experiments. Since mutants that have only low-affinity glucose transport are not deficient in the glucose-induced RAS-mediated cAMP signal, transport-associated phosphorylation of glucose is not required for or involved in the induction of the signal. The yeastfdp mutant, which dies on media containing fermentable sugars because of overaccumulation of sugar phosphates, also did not show any evidence for the existence of transport-associated phosphorylation. The same was true for the double mutantfdp snf3. The latter also showed the typicalfdp phenotype, indicative that the lethality on media containing fermentable sugar is owing to aberrant regulation of low-affinity transport. The high protein kinase activity in thefdp mutant does not appear to be responsible for the absence of evidence for transport-associated phosphorylation, because another mutant with high protein kinase activity, thebcy mutant, displayed normal transport behavior.

Mechanism of transport-associated phosphorylation of 2-deoxy- d-glucose in the yeast Kluyveromyces marxianus: characterization of the phosphoryl source

The mechanism of 2-deoxy- d-glucose transport via the glucose/fructose carrier in Kluyveromyces marxianus has been studied by comparing the concentrations and specific activities of polyphosphate, orthophosphate, ATP and sugar phosphate in cells, pulse-labeled with [ 32P]orthophosphate. During aerobic 2-deoxyglucose uptake, phosphate accumulates in the sugar phosphate pool, whereas both the orthophosphate- and the polyphosphate pool diminish. Comparison of the specific activities suggests that ATP is the primary phosphoryl donor. ATP is recycled at the expense of orthophosphate. Polyphosphate appears to replenish the orthophosphate pool and therefore has, at least mainly, an indirect role in sugar phosphorylation. Reduction of the ATP concentration results in a decrease of the 2-deoxyglucose transport rate. The uptake velocity can be increased again by raising the ATP level. These results corroborate the conclusion that ATP donates the phosphoryl group to the sugar.

37] Sugar transport in normal and mutant yeast cells

This chapter focuses on the sugar transport in normal and mutant yeast cell. Most studies of sugar transport in yeasts have been carried out with baker's yeast, Saccharomyces cerevisiae , which exhibits a variety of sugar transport processes––namely, facilitated diffusion, active transport, and, possibly, group translocation by transport-associated phosphorylation. The high- and low-affinity processes for monosaccharide uptake in S. cerevisiae may differ with respect to mechanism as well as kinetics. The low-affinity process is dearly due to a constitutive, facilitated diffusion mechanism; however, the high-affinity process may be mediated by a group translocation mechanism––namely, a transport-associated phosphorylation. Transport is customarily studied in washed cells suspended in distilled water or buffered salt solutions. Radiolabeled sugars are added to the cell suspensions, and samples are filtered and washed on glass microfiber or membrane filters. Glass microfiber filters are less expensive and allow much higher filtration rates than membrane filters.

Studies on the mechanism of the glucose-induced cAMP signal in glycolysis and glucose repression mutants of the yeast Saccharomyces cerevisiae.

When glucose is added to cells of the yeast Saccharomyces cerevisiae grown on non-fermentable carbon sources, a cAMP signal is induced which triggers a protein phosphorylation cascade. Addition of glucose or fructose to cells of a phosphoglucose isomerase mutant also induced the cAMP signal indicating that metabolization of the sugar beyond the sugar phosphate step is not necessary. Glucose 6-phosphate might stimulate the triggering reaction since induction with fructose shows a significant delay. Experiments with double and triple mutants in hexokinase 1, hexokinase 2 or glucokinase indicated that the presence of one of the three kinases was both necessary and enough for induction of the cAMP signal by glucose and the presence of one of the two hexokinases necessary and enough for induction by fructose. The product of the kinase reaction itself however does not appear to be the trigger of the reaction: when the increase in the level of glucose 6-phosphate and fructose 6-phosphate was measured as a function of time after addition of different glucose concentrations, no correlation was observed with the increase in the cAMP level. From the dependence of the cAMP increase on the external concentration of glucose, a rough estimate was obtained of the Km of the triggering reaction: about 25 mM. This value clearly fits with the Km of the low-affinity glucose carrier (about 20 mM) and differs by at least an order of magnitude from the Km values of the high-affinity glucose carrier and the three kinases. The present results situate the primary triggering reaction at the level of transport-associated phosphorylation. The main (= low-affinity) glucose carrier appears to be the receptor while association of the corresponding kinase is needed for induction of the signal. Since it is known that the presence of the kinases influences the characteristics of sugar transport, no definite conclusion can be given on whether the necessity of the kinases reflects the need for a certain type of transport or the need for phosphorylation of the sugar. The increase in the level of fructose 1,6-bisphosphate, on the other hand, correlated very well with the cAMP increase. However, it clearly lagged behind the cAMP increase, confirming the previously suggested importance of the cAMP signal for the stimulation of glycolytic flux at the level of phosphofructokinase 1.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of ATP on sugar uptake mediated by the constitutive glucose carrier of Saccharomyces cerevisiae

The glucose carrier of Saccharomyces cerevisiae transports the phosphorylatable sugars glucose, mannose, fructose and 2-deoxy- d-glucose (2-dGlc) and the non-phosphorylatable sugar 6-deoxy- d-glucose (6-dGlc). Reduction of the ATP concentration by, for example, incubating cells with antimycin A, results in a decrease in uptake of 2-dGlc and fructose. These uptake velocities can be increased again by raising the ATP level. These results establish a role of ATP in sugar transport. Transport of glucose and mannose is less affected by changes in the ATP concentration than 2-dGlc and fructose uptake, while the 6-dGlc transport is independent of the amount of ATP in the cells. Also, reduction of the kinase activity by incubation with xylose diminished transport of 2-dGlc and fructose, while the uptake of glucose and mannose remained unchanged. It is discussed that these results are due to transport-associated phosphorylation with ATP as substrate and the hexokinases and the glucokinase as phosphorylating enzymes.

Transport-associated phosphorylation of 2-deoxyglucose in rat adipocytes

The relationship between ATP levels and 2-deoxyglucose uptake was investigated. When the concentration in the medium lies between 1 and 10 mM 2-deoxyglucose uptake causes a marked decrease in ATP level. This could partly be explained by an inhibiting effect of 2-deoxyglucose and 2-deoxyglucose 6-phosphate on ATP synthesis in the mitochondria. A good correlation between the various ATP levels induced by 2,4-dinitrophenol and the rate of uptake of 5 μM and 0.5 mM (but not 5 mM) 2-deoxyglucose was observed. The addition of glucose and 2-deoxyglucose to cells incubated in the presence of trace amounts of 2-deoxy-[1- 14C]glucose induced marked changes in the uptake of the tracer that were associated with a rapid decline in ATP level. It appeared that the phosphorylation of 2-deoxyglucose is an important step in the uptake of the sugar. It is hypothesized that the processes of transport and phosphorylation of 2-deoxyglucose are coupled in rat adipocytes.

The involvement of high-energy phosphate in 2-deoxy- d-glucose transport in Kluyveromyces marxianus

Under aerobic conditions 2-deoxy- d-glucose was accumulated in Kluyveromyces marxianus mainly in a phosphorylated form. During sugar uptake both ATP, polyphosphate and orthophosphate levels decreased. Under anaerobic conditions considerably less sugar was taken up. The intracellular free sugar concentration did not exceed the medium concentration, whereas sugar phosphorylation leveled off at about 3 μmol/g yeast. In response to anaerobic 2-deoxy- d-glucose uptake only ATP and polyphosphate appeared to decrease. Within the experimental error sugar phosphorylation was counterbalanced by the polyphosphate decrease. Pulse labeling experiments revealed transport-associated phosphorylation under these anaerobic conditions, Further, kinetic studies on permeabilized cells showed that cytoplasmic ATP could not be the phosphoryl donor in this transport-associated phosphorylation. These results confirm and extend previous observations, indicating that polyphosphate plays a crucial role in 2-deoxy- d-glucose transport in Kluyveromyces marxianus.

Facilitated diffusion of 6-deoxy-D-glucose in bakers' yeast: evidence against phosphorylation-associated transport of glucose.

6-Deoxy-D-glucose, a structural homomorph of D-glucose which lacks a hydroxyl group at carbon 6 and thus cannot be phosphorylated, is transported by Saccharomyces cerevisiae via a facilitated diffusion system with affinity equivalent to that shown with D-glucose. This finding supports the facilitated diffusion mechanism for glucose transport and contradicts theories of transport-associated phosphorylation which hold that sugar phosphorylation is necessary for high-affinity operation of the glucose carrier.

Active transport of l-sorbose and 2-deoxy- d-galactose in Saccharomyces fragilis

Sorbose and 2-deoxy- d-galactose are taken up in Saccharomyces fragilis by an active transport mechanism, as indicated by the energy requirement of the process and the accumulation of free sugar against the concentration gradient. There are no indications for transport-associated phosphorylation as mechanism of energy coupling with these two sugars. The measured sugar-proton cotransport and the influx inhibition by uncouplers suggest a chemiosmotic coupling mechanism. Thus there are at least two different active transport mechanisms operative in Saccharomyces fragilis: transport-associated phosphorylation in the case of 2-deoxy- d-galactose and chemiosmotic coupling in the case of sorbose and 2-deoxy- d-galactose. The difference between the two mechanisms are discussed. Uncouplers do not stimulate downhill sorbose transport in energy-depleted cells and evoke an almost complete inhibition of efflux and of exchange transport. The differences between this sugar-proton cotransport system and similar systems in bacteria and Chlorella are discussed.

Transport of 2-deoxy- d-galactose in Saccharomyces fragilis

2-Deoxy-D-galactose (dGal) transport in Saccharomyces fragilis is characterized by energy requirement and accumulation of the free sugar against a concentration gradient, indicating active transport. Besides free sugar dGal-1-phosphate, UDP-dGal and a trehalose-like derivative were found inside the cells. The accumulation of the phosphorylated derivatives was balanced by a concomitant decrease of ATP, orthophosphate and polyphosphates. With pulse labeling experiments it could be shown that the free sugar is transported into the cells. This conclusion was supported by several other experimental results, e.g. the lack of correlation between the sugar transport parameters and the dGal phosphorylation capacity, and the countertransport of free dGal evoked by galactose in the medium. The typical differences between this active transport mechanism and the transport-associated phosphorylation system, described previously, are discussed.

Transport-associated phosphorylation of 2-deoxy- d-glucose in Saccharomyces fragilis

2-Deoxy- d-glucose transport and metabolism was studied in Saccharomyces fragilis. Inside the cells four phosphorylated and three non-phosphorylated derivatives were found and identified. Accumulation of phosphorylated 2-deoxyglucose derivatives was balanced by a concomitant decrease of cellular ATP, orthophosphate and polyphosphates. The free sugar was concentrated against a concentration gradient, contradicting facilitated diffusion. Pulse labeling experiments revealed transport-associated phosphorylation. Theoretical considerations and analysis of the effects of iodoacetate showed that an intracellular hexokinase activity was not involved in 2-deoxyglucose phosphorylation, although this sugar is a good substrate for the enzyme in in vitro experiments.

Transport and transport-associated phosphorylation of galactose in Saccharomyces cerevisiae

The galactose transport mechanism in three strains of Saccharomyces cerevisiae was investigated in some detail, both in glucose-grown cells and in galactose-induced cells. In glucose-grown yeast the uptake of galactose and 2-deoxy- d-galactose could be characterized as carrier-mediated facilitated diffusion. Competition and counter-transport studies showed that these sugars are taken up via the same carrier as glucose and sorbose. After induction the transport system has changed from a low affinity to a high affinity system, as reflected by a decrease of the K m by two orders of magnitude. The uphill transport of 2-deoxy- d-galactose after induction indicated that this high affinity system is an active transport mechanism. Competition and countertransport studies showed that this active transport takes place via the same carrier as the facilitated diffusion in glucose-grown cells. In the yeast strain NCYC 240 the induction of the active transport system occurred before the induction of galactose metabolism. During this phase of the induction process galactose was partly phosphorylated, whereas the free sugar concentration in the cells increased to about 50 times the medium concentration. Radioactive pulsing experiments during this interval showed transport-associated phosphorylation of galactose, as demonstrated previously for some other carbohydrates in yeast.

The transport mechanism of -methylglucoside in yeast evidence for transport-associated phosphorylation.

1. α-Methylglucoside transport in Saccharomyces cerevisiae Strain NCYC 240 was studied in uninduced and in maltose-induced cells. It appeared that active transport occurred in induced cells, apparently via the maltose permease. 2. α-Methylglucoside taken up by induced cells was recovered in cellular extracts partly as the free sugar, partly as a phosphate ester. The ratio free α-methylglucoside/α-methylglucoside phosphate in the cells increased during incubation in the course of time. 3. During de-adaptation the α-methylglucoside-phosphorylating capacity of the cells decreased linearly with the maltose and α-methylglucoside transport capacity. 4. With 14C-labeled α-methylglucoside pulsing experiments, it appeared that shortly after addition of the radioactive pulsing dose the specific activity of the intracellular α-methylglucoside phosphate fraction exceeded the specific activity of the free fraction. This demonstrates that the phosphate ester is the precursor of the intracellular free α-methylglucoside. 5. The experimental results indicated transport-associated phosphorylation of α-methylglucoside, with subsequent release of α-methylglucoside phosphate at the inner face of the membrane. The enzyme catalyzing α-methylglucoside phosphorylation is apparently an integral part of the active transport system.

Evidence for a Functional Change in the Plasma Membrane of Murine Sarcoma Virus-infected Mouse Embryo Cells: TRANSPORT AND TRANSPORT-ASSOCIATED PHOSPHORYLATION OF 14C-2-DEOXY-d-GLUCOSE

Abstract Enhancement of sugar uptake occurs in murine sarcoma virus-infected mouse cells at the time of transformation. Direct analysis of the sugar transport and transport-associated phosphorylation system of mouse cells infected with the Harvey strain of MSV was performed with 2-deoxy-d-glucose-14C. It was shown that 2-deoxy-d-glucose-14C was taken up at various concentrations (10-2 to 10-6 m) at a greater rate and was transported with a lower Km (21.0 to 23.3 x 10-4 m for uninfected and 3.5 to 5.0 x 10-4 m for infected cells) and higher Vmax (18.2 to 19.2 nmoles per mg of protein per min at 37° for uninfected and 52.3 to 83.5 nmoles per mg of protein per min for infected cells) in the infected cells. The transported substrate was found mainly as a phosphorylated product, 2-deoxy-d-glucose 6-phosphate. The increased phosphorylation of 2-deoxy-d-glucose-14C by the infected cells was obtained only with intact cells, but not with cell homogenates, indicating that this phosphorylation is not due to increased intracellular hexokinase activity but rather based on a membrane-bound transport system. These data support the hypothesis that alterations in a plasma membrane sugar transport (penetration plus phosphorylation) system occurred during cellular transformation by murine sarcoma virus.

Transport and transport-associated phosphorylation of 2-deoxy- d-glucose in yeast

1. 1. 2-Deoxy- d-glucose taken up by yeast cells was recovered inside the cells partly as free sugar, partly as 2-deoxy- d-glucose-6-phosphate. 2. 2. At low deoxyglucose concentrations in the medium there was an uphill transport of the free sugar. Concentration ratios of 20–30 could be reached, indicating an active transport mechanism. 3. 3. The accumulation of doexyglucose phosphate was counterbalanced quantitatively by a simultaneous decrease of cellular ATP, orthophosphate and polyphosphate. Intracellular hydrolysis of accumulated deoxyglucose phosphate could be demonstrated. 4. 4. With a [ 14C]deoxyglucose pulsing technique it could be shown that deoxyglucose phosphate behaved as the precursor of intracellular free deoxyglucose. This indicates that there is not a transport of free deoxyglucose into the cells followed by intracellular phosphorylation by the hexokinase-ATP system, but that phosphorylation of deoxyglucose is associated with transport (followed by partial intracellular hydrolysis). 5. 5. The apparent analogy between the active sugar transport system in yeast and the phosphotransferase-dependent sugar transport in bacteria, described in recent literature, is discussed.