The mechanism of transmembrane glucose transport in yeast: Evidence for phosphorylation, associated with transport

ARCHIVES

OF

BIOCHEMISTRY

AND

The Mechanism Evidence

130, 244-252 (1969)

BIOPHYSICS

of Transmembrane for

Phosphorylation,

Glucose

Transport

Associated

with

in Yeast:

Transport’

J. VAN STEVENINCK Laboratory

for Medical Received

Chemistry,

September

Wassenaarseweg

18, 1968; accepted

62, Leiden, November

The Netherlands 2, 1968

Glucose transport in Saccharomyces cerevisiae was studied, utilizing iodoacetatepoisoned cells to block fermentation. An initial, rapid, active uptake of about 5 rmoles of glucose per gram yeast could be observed under these conditions. The actively transported glucose could not be recovered from the cells as free glucose. It was found that the transported sugar had been phosphorylated. The intracellular ATP concentration appeared to be far too low to account for this sugar phosphorylation via the hexokinase reaction. ATP synthesis during the experiments could be excluded with a high degree of probability. It appeared that an intracellular conversion of the transported, phosphorylated sugar takes place in iodoacetate-poisoned yeast. The product (or products) of this conversion could not yet be identified. Unlike sugar phosphates, the unknown compound is released again into the medium. Under certain experimental conditions glucose taken up from the medium was phosphorylated by commercial bakers’ yeast, whereas free glucose, present inside the cells (liberated from storage carbohydrates) was not phosphorylated. It is shown that the most obvious interpretation of these phenomena is a phosphorylation of glucose associated with transport, with polyphosphates as phosphate donor. This interpretation tallies with a hypothesis on glucose transport, postulated in previous papers. These experimental results are compared with data on sugar transport in various cells, published in recent literature.

(S-E-phosphate-C)

In previous papers (l-4) evidence has been presented for the existence of two sugar transport systems in yeast: A passive, carrier-mediated facilitated diffusion and an active, metabolically linked transport mechanism. The active mechanism is operative in e.g. glucose transport. Experimental results indicate a permease-carrier transport system in which a phosphorylating reaction takes place, with polyphosphate as phosphate donor. The previously proposed model can be summarized as follows: S + E + (SE) (SE) + C + (KPO,), (S-E-phosphate-C)

(S-phosphate-C)

(KPO&r

i This work was aided by a U.S. Public Research Grant (no. R05-TWOO159).

+ E

(3)

(S = substrate; E = permease; C = carrier; (KPO,),

= polyphosphate).

The glucose-phosphate-carrier complex moves across the membrane. This model implies the use of one high energy phosphate bond during transport of each glucose molecule, corresponding to 50 % of the chemical energy gain of anaerobic metabolism. This fact makes it likely without more ado, that the chemical energy spent in the transport step is conserved some way or other. This aspect was not studied in previous papers, but a few possibilities were suggested: (1) If the free energy of the polyphosphate bond is retained in the sub-

(1)

= +

e

(2) Health 244

GLUCOSE

TRANSPORT

strate-phosphate-carrier complex, free glucose may be released at the inside of the membrane, associated with resynthesis of polyphosphate (5). (2) Glucose-phosphate may be released from the carrier at the inside of the membrane, entering further metabolic pathways and by-passing the hexokinase reaction (5). (3) (S-phosphate-C) may represent a carrier-ATP-glucose complex, and after dissociation from the carrier ATP and glucose may be delivered to a hexokinase molecule for phosphorylation (1). In the present communication the intracellular fate of glucose, transported into iodoacetate-poisoned yeast is discussed. These investigations were undertaken in order to obtain further evidence for the proposed phosphorylation during transport. Moreover, this approach could indicate if the chemical energy, spent during transport, is conserved. METHODS Two yeast strains were used in these experiments: commercial bakers yeast (“Koningsgist,” obtained from the Gist en Spiritusfabriek, Delft) and strain Hansen, C.B.S. 1172. The latter yeast strain was grown and harvested as described previously (5). Prior to use the yeast was starved aerobically overnight in distilled water and subsequently washed three times in about 30 vol of distilled water. To avoid complicating metabolic reactions, fermentation was blocked by iodoacetate (1 mM) under strict anaerobic conditions. In most experiments the final yeast concentration was lO’j&, wet weight. Separation of the cells and medium u-as accomplished by Millipore filtration, followed by washing of the cells on the filter with ice-cold water. Yeast extracts for chromatography were obtained either by boiling the yeast in 1 mM iodoacetate solution (3 ml per gram yeast) during 5 min, or by treating the yeast with 80% ethanol (3 ml per gram yeast). Both extraction methods gave the same results. If necessary, these extracts were concentrated in a heated vacuum desiccator at 35” over anhydrous CaCL. Paper chromatography was performed with S and S 2043b paper, utilizing two solvent systems: Ethyl acetate:propanol: water = 20:60:20 (I) and ethyl acetate:acetic ncid:water = 30:30:10 (II). Yeast extracts for ATP determinations were prepared as described by Feldheim et al. (6). ATP was measured by the firefly tail, luciferin-luciferase system, according to the method of Addanki

IN

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et al.

(7). The assay was carried out in a liquid scintillation counter, recording counts between 15 and 27 set after mixing. Polyphosphates were extracted and assayed according to Lohmann and Langen (8). Extraction of other phosphorus-containing substances and their subsequent determination was done by the method of Juni et al. (9). The concentration of glucose G-phosphate in the yeast cells was measured enzymatically, as by Betz and Chance (10). Fermentation was measured with the standard Warburg technique. Further analytical methods utilized were: For glucose, the glucose oxidase method, as modified by Washko and Rice (II), utilizing Glucostat special (Worthington) ; for fructose and fructose phosphates, the method of Dische and Devi (12) and the enzymatic method described by Bergmeyer (13); for orthophosphate: the method of Fiske and Subbarow, as modified by Meyerhof and glucose was measured in Oesper (14) ; ‘%-labeled a liquid scintillation counter, with the liquid scintillator described by Bray (15). Radioautographs of dried paper chromatograms were made by placing them in close contact with a strip of X-ray film in the dark, for about 5 days. Subsequent quantitative analysis was performed by cutting the chromatogram in small strips, guided by the blackening of the radioautograph and measuring the radioactivity of the strips in a liquid scintillation counter. RESULTS

Experiments zcith Strain Hansen, C.B.S. 1172 In preliminary experiments iodoacetatepoisoned yeast was incubated with low [14C]glucose concentrations, for periods up to 4 hr. The suspension was analyzed for radioactivity at intervals. No decrease of radioactivity was observed even after 4 hr, indicating that fermentation was completely blocked. Control experiments with the Warburg technique gave the same results. Iodoacetate-poisoned yeast shows a small, rapid uptake of glucose during the first few seconds after addition of substrate. As shown previously, this rapid uptake is accounted for by the active transport mechanism (1). The glucose taken up by this rapid transport component appeared to be not reactive with glucose oxidase in yeast extracts, indicating that it does not occur inside the cells as free glucose. Experiments to determine the chemical form of the rapidly transported glucose were performed by paper chromatog-

246

VAN STEVENINCK TABLE

I -- sokent front 1

Strip no. 4 5 6

of yeast-cell-extract FIG. 1. Radioautographs chromatograms, with solvent systems I and II. The yeast had been incubated with [‘YJglucose in the presence of iodoacetate during 15 min. A: Radioautograph of reference chromatogram with [14C]glucose and [14C]fructose(a) and [14C]glucose 6-phosphate and [14C]fructose 6-phosphate (b), run in the presence of nonlabeled yeast extract. With both solvent systems fructose and glucose migrate to the same position (a), as do glucose Sphosphate and fructose 6-phosphate (b). B: Radioautograph of yeast-extract chromatogram, showing radioactive spots corresponding to hexose phosphate and the unidentified compound(s) ‘Cc*” At the location of free hexoses (a), no distinct spot could be detected. l-10: strips, as measured in the liquid scintillation counter.

raphy of yeast-cell extracts, after very short incubation periods with small amounts of l*C-labeled glucose (3 pmoles glucose per gram yeast). Adding glucose, mixing, and transferring the suspension to the Millipore funnel took about 7 set; washing the cells free of medium took another 20 sec. So yeast extraction started about 30 see after addition of glucose. With the solvent systems I and II the radioactivity is recovered at location b, Fig. 1. With increasing glucose concentrations it appeared that the maximal amount of glucose taken up via the rapid transport component and recovered at location b is about 5 pmoles per gram of yeast. With higher glucose concentrations in the medium a gradual increase of uptake is observed. As shown in a previous paper, this additional uptake in iodoacetate-poisoned yeast is accounted for by a relatively slow

I

CHROMATOGRAPHIC DISTRIBUTION OF INTRACELLULAR RADIOACTIVITY IN IODOACETATE-POISONED YEAST HANSEN C.B.S. 1172, AFTER VARYING INCUBATION PERIODS WITH [W]GLUCOSES

I

Solvent system I 30 set

5 min

30min

10set

0.2 0.1 0.1 92.3 0.1 0.0 4.7 0.0 0.0. 2.8

0.0

1

0.0

0.1

2 3 4 5 6 7 8 9

0.1 0.3 4.2 0.5 0.3 3.2 0.1 0.3 91.1

0.4 1.1 20.8 2.2 0.4 3.6 0.2 1.0 70.2

10

TSolvent system II .-

-

1.2 2.3 0.8 2.8 1.1 1.1 80.2 6.3 4.2

I-

I

5 min

IOmin

0.2 2.1 22.3 1.2 3.0 1.0 2.4 60.4 3.4 4.1

0.3 4.2 72.8 2.1 4.1 1.1 0.9 8.3 2.8 3.4

0 Thestrip numbers correspond to the numbers in Fig. 1. The results are given for both solvent systems and are expressed in percentage of the total intracellular radioactivity.

facilitated diffusion, after exhaustion of the active transport system (1). The sugar taken up in excess of the 5 pmoles per gram moves on the chromatogram to the position characteristic for free hexoses (Fig. la), and moreover, reacts with glucose oxidase. To simplify the conditions, limited amounts of glucose were used in all further experiments (up to 6 pmoles per gram of yeast), so that virtually all the glucose taken up is accounted for by the rapid, active transport component. If longer incubation periods (up to 30 min) with limiting concentrations of glucose are allowed before filtering off the yeast, the total amount of glucose taken up by the cells does not increase, as could be shown by analysis of the medium with glucose oxidase reagent. Apparently the maximal uptake is reached already within a few seconds. At glucose concentrations below 6 pmoles per gram yeast this maximal uptake is always 7045% of the total amount of added glucose. With increasing incubation periods, however, an increasing percentage of the intracellular radioactivity is recovered on the chromatogram in a position with a RF value much higher than glucose (Fig. lc).

Incubation time 5 min

30 set

Glucose, pg/ml (glucose oxidase) Total radioactivit) Glucose-radioactivity (spot a) Spot b, radioactivity Spot c, radioactivity

100 22,800 22,800 0 0

18 4,200 4,150 0 50

19 -5,510 4,050 0 1,460

30 min 18 11,590 4,270 0 7,320

n Initial glucose concentration: lOO~g/ml, corresponding to 22,800 counts/min/ml; yeast concentration: corresponding to glucose 10% ; iodoacetate concentration : 1 mM; temperature : 25”. The radioactivity and to spots b and c was measured after paper chromatography of the medium with solvent system I. Radioactivity is expressed in counts/min/ml.

After an incubation period of 30 min, over 90% of the intracellular radioactivity has shifted from position b to position c. This reflects an intracellular conversion, still taking place in iodoacetate-poisoned yeast (Table I). The total amount of radioactivity taken up by the poisoned cells decreases gradually from 30 set to 30 min after adding [14C]glucose, with a concomitant increase of the radioactivity in the medium. The 14Clabeled material, apparently leaking out of the cells in this period, was not glucose, as shown by the quantitatively unchanged glucose oxidase reaction in the medium during that interval. Chromatography of the medium revealed besides glucose a distinct spot, corresponding exactly with spot c in the yeast extracts, with both solvent systems. No traces of radioactivity were found below the glucose spot (Table II). Apparently, glucose taken up by the rapid transport component in iodoacetate-poisoned yeast is recovered primarily in cellular extracts in an altered form, remaining at the starting point of paper chromatograms with solvent system I. It could be shown that this altered glucose is a mixture of fructose 6phosphate and glucose 6-phosphate: 1. Glucose 6-phosphate, fructose 6-phosphate and the 14C-labeled compound behave identically in paper chromatography with both solvent systems, I and II (Fig. 1). 2. Pretreatment of the yeast extracts with alkaline or acid phosphatase at appropriate pH (3 mg enzyme per milliliter extract) prior to chromatography, shifts the radioactivity to the location of free hexoses (a, in Fig. 1).

This shift is not observed if the phosphatase is first inactivated by heating to TO”, during 30 min. 3. Enzymatic analysis of cellular extracts revealed that only traces of glucose 6-phosphate and fructose 6-phosphate are present in iodoacetate-poisoned yeast. No free fructose could be detected enzymatically. The ketose reaction of Dische and Devi (12) is only slightly positive. After addition of glucose there is a small increase in the intracellular glucose 6-phosphate concentration, accounting for only 2-10% of t’he glucose taken up, with a concomitant sharp increase in the fructose 6-phosphate concentration, accounting for SO-90 % of the glucose taken up (Table III). The reaction of Dische and Devi (12) increases accordingly. Ko free fructose could be detected. 4. After pretreatment of the extracts with phosphatase, the sugar phosphates could no longer be detected. Enzymatic analysis proved a quantitative conversion into free glucose and free fructose. 5. Paper chromatography of yeast extracts followed by elution of small strips of the chromatogram showed that the sugar phosphates are located at the radioactive spot b in normal yeast extracts. After pretreatment of the extract with phosphatase, no sugar phosphates could be detect’ed at location b, but the enzymatic reactions of glucose and fructose at location a reflected the conversion into free sugar. 6. With the extraction method of Juni et al. (9) all radioactivity is recovered as glucose 6-phosphate and fructose A-phos-

248

VAN TABLE

STEVENINCK

III

GLUCOSE UPTAKE, GLUCOSE~-PHOSPHATE, FRUCTOSE ~-PHOSPHATE AND COMPOUND “c" CONCENTRATION IN IODOACETATE-POISONED HANSEN YEAST C.B.S.1172, AFTER VARYING PERIODS OF INCUBATION WITH ~~~~~~~~~~~~~ Incubation time 0 ___Glucose, taken up Glucose B-phosphate Fructose 6-phosphate Compound “c”

30 set

5 min ____

30

min

0.00

4.62

4.75

4.74

0.11 0.32

0.26 4.51

0.22 3.61

0.17 0.40

0.00

0.01

0.96

4.00

n Experimental conditions: See legend to Table II. All concentrations are expressed in glucose equivalents &moles) per gram yeast. Glucose uptake was calculated from sugar disappearance from the medium. Sugar phosphates were measured in the yeast extracts. The total concentraand in the tion of compound “c, ” intracellular medium, was determined by paper chromatography and subsequent measurement of radioactivity.

phate in the “soluble organic phosphate” fraction. This could be shown by paper chromatography, followed by elution and enzymatic analysis. This fraction is essentially free of orthophosphate. After treatment of this fraction with phosphatase the radioactivity is enzymatically recovered as free glucose and free fructose, with a concomitant, stoichiometric release of orthophosphate. So it can be concluded that the glucose taken up by the rapid transport component in iodoacetate-poisoned yeast is initially recovered inside the cells mainly as fructose 6-phosphate and to a much lesser extent as glucose 6-phosphate. After longer incubation periods the sugar phosphate concentrations decrease again, with a concomitant, stoichiometric increase of the compound(s), migrating to position c in paper chromatography (Table III). No attempts were made yet to identify the compound “c.” According to the hypothesis on glucose transport discussed in the introduction, this initial glucose phosphorylation is associated with transport and does not occur inside the cell, following transport of free glucose. Intracellular phosphorylation via the hexo-

FFIG. 2. ATP concentration in iodoacetatepoisoned yeast (Hansen C.B.S. 1172), as a function of time. Iodoacetate (1 mnn) was added at zero time. The dotted lines represent the amount of glucose phosphorylated by the yeast, if glucose is added to the suspension after varying incubation periods of the yeast with iodoacetate.

kinase reaction would require one ATP molecule for each glucose molecule phosphorylated. The ATP concentration in iodoacetate-poisoned yeast decreases gradually from an initial value of about 1.2 pmoles to less than 0.1 pmole per gram of yeast, after 30 min. The amount of glucose taken up and phosphorylated by the yeast however, is about 5 pmoles per gram, irrespective of whether the glucose is added only 2 or 60 min after the iodoacetate. Apparently intracellular ATP can only account for from 20 to less than 2% of the amount of glucose phosphorylated (Fig. 2). To exclude any residual metabolic ATP synthesis during the experiments, fermentation of poisoned yeast was studied carefully with the Warburg technique, utilizing high yeast concentrations (up to 300 mg per vessel). No traces of metabolism could be detected. Another possibility would be intracellular ATP synthesis out of the cellular polyphosphate pool. Enzymes, catalyzing the reaction: ADP

+

(KPO,),

=

ATP

+

(KP0&-1

have been described in various micro-organisms (16-18). It appeared, however, that the polyphosphate concentration in poisoned yeast remains constant (about 60 req per gram yeast) during at least 3 hr, both with

GLUCOSE

TRANSPORT

and without addition of an excess of glucose to the medium. This definitely rules out any appreciable synthesis of ATP from polyphosphates. If, via the above equation, 5 pmoles of ATP could be formed from polyphosphates in a few seconds (as needed to account for the observed glucose phosphorylation), the cellular polyphosphate pool would be exhausted within a few minutes. Moreover, the residual phosphorylation capacity of iodoacetate-poisoned yeast would account for 60 pmoles of glucose, instead of the observed 5 pmoles. A peculiar phenomenon was the small decrease of the amount of polyphosphate extracted from the cells in presence of iodoacetate. This decrease was found in all experiments and was always 4-6 peq P per gram yeast, both with and without glucose added to the medium. Complete analysis of cellular phosphorus-containing compounds according to Juni et al. (9) revealed a similar small decrease of the polyphosphate fraction after addition of iodoacetate. About 15 % of this decrease is recovered in the orthophosphate fraction, 15 % in the “soluble organic phosphate” fraction and 70 % in the residual, “unidentified” fraction. On the other hand, if glucose is added to the poisoned yeast, about 60% of the polyphosphate decrease is recovered in the “soluble organic phosphate” fraction, in which sugar phosphates are included. Although the experimental error in this analysis is high (standard error of 5--S% in each fraction), this indicates that this relatively small polyphosphate fraction (as distinct from the major polyphosphate pool) is involved in glucose phosphorylation, in agreement with the hypothesis. Experiments

with

“Koningsgist”

yeast

“Koningsgist” yeast ferments glucose with the same velocity as strain Hansen C.B.S. 1172. Kinetic analysis revealed that the K,,, values for glucose transport, calculated as described before (3), are the same for both yeast strains, within the experimental error (5.1-5.3 112~). If Koningsgist yeast is poisoned with iodoacetate, and glucose added within 2 min, exactly the same phenomena as described for strain Hansen

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YEAST

could be observed. After longer preincubation periods with iodoacetate however, the situation is different. Koningsgist yeast shows a phenomenon, not observed in strain Hansen. If the former yeast strain is poisoned with iodoacetate, storage carbohydrates are degraded to free glucose, which is transported to the medium. The glucose concentration in the medium equals the intracellular concentration as measured with glucose oxidase, and increases during about 3 hr. After 3 hr the glucose concentration in the medium and in the cells remains constant and amounts to 1.0-1.4 pmoles glucose per milliliter, in a 10% yeast suspension. It can be expected that under these circumstances phosphorylation, by the hexokinase reaction, of the free glucose liberated inside the cells, will cause a complete ATP depletion. This was confirmed experimentally: After about 6 min incubation with iodoacetate no ATP could be detected in the cells. If extra glucose was added to the medium after preincubation with iodoacetate during 3 hr, no chemically measurable uptake of glucose was observed in this yeast, as could be expected. If, however, a very small amount of [14C]glucose was added to the medium, it appeared that traces of glucose were still taken up by the cells (O.OOl0.005 pmoles per gram in 60 set). Analysis of cellular extracts showed that again at least 90 % of the transported glucose is recovered inside the cells as a phosphorylated product, notwithstanding the presence of much larger amounts of free glucose inside the cells (0.50.7 pmoles per gram yeast). DISCUSSION

The results described in this paper strongly support the hypothesis of glucose phosphorylation, associated with transport. The experimental evidence can be summarized as follows. Iodoacetate-poisoned Hansen, C.B.S. 1172 can take up about 5 pmoles glucose per gram yeast via the rapid, active-transport component. This glucose can be recovered from the cells as sugar phosphate. It is very unlikely that phosphorylation takes place inside the cell, following transport of free glucose. The amount of available ATP for the hexokinase reaction

250

VAN

STEVENINCK

can only account for the phosphorylation of about 20 % of the transported glucose after short preincubations with iodoacetate, to less than 2 % after more prolonged exposure to this poison (Fig. 2). A residual capacity of metabolic ATP synthesis or of ATP formation from the cellular polyphosphate pool during the experiments could be excluded. Moreover, if any residual ATP synthesis took place in iodoacetate-poisoned yeast (either metabolic or out of polyphosphates), it is diflicult to assume that about 5 pmoles of ATP would be synthesized in the first few seconds after glucose addition (irrespective of the length of the preceding incubation period with iodoacetate), and nothing more in the next 30 min. Another possibility that should be considered is an eventual exchange reaction between free glucose and e.g. phosphates of glucose and fructose, present in the cells. The intracellular concentrations of glucose 6-phosphate and fructose B-phosphate appeared to be far too low, however, to account for the observed [14C]glucose phosphorylation. Moreover, the phosphorylated [‘“Clglucose is added quantitatively to the phosphates of glucose and fructose, already present in the cell (Table III). Consequently, as neither the hexokinase-ATP nor a possible exchange reaction can account for the observed glucose phosphorylation, this phosphorylation must be associated with transport. Further support for the hypothesis is the small decrease of the total polyphosphate concentration during glucose transport, described in a previous paper (5). Attempts to demonstrate a limited decrease of polyphosphate during glucose transport under the present experimental conditions were only partly successful. The decrease that should be expected is relatively small, as only a minor fraction of the total cellular polyphosphates is involved in transport phosphorylation (5). Further, as shown above, iodoacetate interferes with complete extraction of polyphosphates: About 5 peq of polyphosphate can no longer be identified as such in poisoned yeast. It is tempting to assume that this is exactly the fraction involved in transport phosphoryla-

tion; the numerical equality of both fractions favours this assumption. This is in agreement with the observation that iodoacetate also interferes with ion-binding to intact yeast cells (1, 5). The metal-ion-binding groups of intact yeast cells have been identified as polyphosphates, involved in transport phosphorylation (1, 5). How iodoacetate interferes with this ion binding and with complete polyphosphate extraction is still obscure. Finally, the analysis according to the scheme of Juni et al. (9), as described above, indicates the involvement of this polyphosphate fraction in glucose phosphorylation. The experiments with iodoacetatepoisoned Koningsgist yeast showed that with respect to phosphorylation the yeast cell discriminates strictly between intra- and extracellular glucose. Under the experimental conditions described above, relatively large amounts of free glucose are present inside the cells, associated with an exhaustion of the ATP-hexokinase system. Nevertheless, trace amounts of exogenous [‘“Clglucose are still transported into the cells and phosphorylated almost completely. This means that the hexokinase-ATP system cannot be involved in the phosphorylation of this exogenously added substrate. The amount of radioactivity taken up by these cells in 1 min corresponds to O.OOl0.005 pmole glucose per gram yeast. If this labeled glucose were to be transported into the cells as free glucose, it would be diluted 100-500 times with unlabeled glucose, as the intracellular free glucose concentration of these cells is virtually constant at a level of 0.5-0.6 pmole per gram, as pointed out in the Results section. Consequently, any residual capacity of the ATP-hexokinase system would phosphorylate only a minor fraction of the transported, labeled glucose. In fact, however, at least 90 % of the labeled glucose taken up by the cells is phosphorylated. Apparently, this phosphorylation is associated with the transport mechanism, as distinct from the intracellular hexokinaseATP system. If the incubation with labeled glucose is continued for longer periods in the experiments with Koningsgist, a slow labeling of

GLUCOSE

TRANSPORT

the intracellular free glucose also takes place, apparently via the facilitated diffusion system described previously (1). After 1 hr, the specific activity of intracellular glucose is about, g of the specific activity of the extracellular glucose. So this equilibration takes place very slowly under these circumstances and can be neglected in the short incubation experiments described above. It should be emphasized that in cont’rol experiments no differences were observed between sugar transport in Koningsgist yeast and in yeast strain Hansen C.B.S. 1172. The only obvious difference concerns the breakdown of storage carbohydrates in Koningsgist yeast after iodoacetate poisoning, which is not found in strain Hansen. Apparently an intracellular conversion of the accumulated sugar phosphates t:lkes place in iodoacetate-poisoned yeast. The product of this reaction is recovered at position “c” on chromatograms (Fig. 1) and is released to the medium. It is possible that spot ‘(c” represents several products with identical RF values. Preliminary experiments indicated that the unknon-n compound dots not contain phosphorus; a concomitant dephosphorylation must have taken place. In further experiments it will be attempted to identify the compound(s) ‘ic.” The hypothesis implies the presence of polyphosphate outside the protoplasmic membrane. Experimental evidence supporting this assumption has been presented in a previous paper (5). Recent studies utilizing a quite different experimental approach, also indicate a superficial location of a fraction of the cellular polyphosphates. Weimberg and Orton (19) noted the apparent location of a fraction of the polyphosphate pool outside the permeability barrier. Experimental results obtained with other techniques led Souzu (20, 21) to a similar conclusion. If this hypothesis on glucose transport is compared with data from recent literature, it is notable that it agrees with the permeasecarrier model of transport, suggested by Kepes (22). Absolute proof of sugar phosphorylation associated with transmembrane transport in any cell is still lacking. An increasing number of phenomena is described in recent literature however, suggesting such

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transport-associated phosphorylation in various cells. Saha and Coe (23) suggest a glucose transport in Ehrlich ascites tumor cells, coordinated with substrate phosphorylation. The possibility of a phosphorylation involved in p-glucoside transport in yeast was mentioned by Kaplan and Tacreiter (24). Hengstenberg et al. (25, 26) demonstrated an intimate relationship between phosphorylation and membrane passage of various sugars in Staphylococcus aweus. Recent experiments on 2-deoxy-&glucose transport in y-east strongly suggest phosphorylation of this sugar, directly associated with transport (27). Finally, Kundig et al. (28) and Simoni et al. (“9) showed that a phosphotransferase system is an essential component of the glycoside permease system in Escherichia coli. ACKNOWLEDGMENTS The author is greatly indebted to Miss J. A. A. de Jong for her skilled technical assistance, and to Dr. A. Rothst’ein for critical and illuminating discussions concerning this paper. REFERENCES J., AND ROTHSTEIN, A., J. Gen. Physiol. 49, 235 (1965). ROTHSTICIN, A., AND VAN STEVENINCK, J., Ann. N.Y. Acad. Sci. 137,606 (196(i). VAN STEVENINCIC, J., AND DAWSON, E. C., Biochim. Biophys. Acta 150, 47 (1968). VAN STEVENINCIC, J., Biochim. Biophys. Acta 160, 424 (19G8). V.~N STEVENINCK, J., AND BOOIJ, H. L., J. Gen. Physiol. 48,43 (1964). FELDHEIM, M. E., AUGUSTIN, H. W., AND E., Biochem. 2. 344, 238 (1966). HOFMANN, ADDANKI, S., SOTOS, J. F., AND REARICK, P. D., Anal. Biochem. 14, 261 (1966). LOHMANN, K., AXD L.~NGEN, P., Biochem. Z. 328, 1 (1957). JUNI, E., KAMEN, Nl. D., REINER, J. M., AND SPIEGELMAN, S., Arch. Biochem. Biophys. 18,387 (1948). BETZ, k, AND CHANCE, B., Arch. Biochem. Riophys. 109, 585 (1965). WASHKO, hl. E., AND Rrcb;, E. W., Clin. Chem. ‘7, 542 (1961). DISCHE, Z., ASD DI~:vI, A., Biochim. Biophys. Acfa 39, 140 (1960). BERGMEYISR, II. U., “Methods of Enzymatic Analysis,” Academic Press, 1965, p. 134 and p. 156.

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23. SAHA, J., AND COE, E. L., Biochem. Biophys. Res. Comm. 26, 441 (1967). 24. KAPLAN, J. G., AND TACREITER, W., J. Gen. Physiol. 60,Q (1966). 25. HENGSTENBERG, W., EGAN, J. B., AND MORSE, M. L., Proc. Natl. Acad. Sci. 68,274 (1967). 26. HENGSTENBERG, W., EGAN, J. B., AND MORSE, M. L., J. Biol. Chem. 243, 1881 (1968). 27. VAN STEVENINCK, J., Biochim. Biophys. Acta 163,386 (1968). 28. KUNDIG, W., KUNDIG, F. D., ANDERSON, B., AND ROSEMAN, S., J. Biol. Chem. 241, 3243 (1966). 29. SIMONI, R. D., LEVINTHAL, M., KUNDIG, F. D., KUNDIG, W., ANDERSON, B., HARTMAN, P. E., AND ROSEMAN, S., Proc. Natl. Acad. Sci. 68, 1963 (1967).