Galactose Transport in Saccharomyces cerevisiae - Journal of ...

JOURNAL OF BACTERIOLOGY, Sept. 1970, p. 671-678 Copyright 0 1970 American Society for Microbiology

Vol. 103, No. 3 Printed in U.S.A.

Galactose Transport in Saccharomyces cerevisiae II. Characteristics of Galactose Uptake and Exchange in Galactokinaseless Cells SHOU-CHANG KOU,1 MICHAEL S. CHRISTENSEN, AND VINCENT P. CIRILLO' Department of Biochemistry and the Molecular and Cellular Biology Program, Division of Biological Sciences, State University of New York, Stony Brook, New York 11790

Received for publication 8 June 1970

The characteristics of the inducible galactose system in Saccharomyces cerevisiae were studied by using the nonmetabolized galactose analogues, L-arabinose and D-fucose, and galactokinaseless and transportless mutants. Induced wild-type cells transport L-arabinose by facilitated diffusion. Transportless cells transport neither galactose nor L-arabinose above the noninduced rate, whereas galactokinaseless cells transport galactose L-arabinose and D-fucose by facilitated diffusion. Determination of unidirectional rate of 'C-labeled galactose uptake by preloaded galactokinaseless cells, containing a large unlabeled free-galactose pool, showed that the rate of galactose uptake by facilitated diffusion is greater than the rate of galactose metabolism at similar external galactose concentrations. All investigators agree that, in noninduced cells, galactose is transported by facilitated diffusion as a low-affinity substrate of the glucose carrier (4, 6, 8, 9, 18, 25, 27). However, there is no agreement about the mechanism of galactose transport in induced cells; active transport, facilitated diffusion, and phosphorylative transfer have been proposed by different authors. Galactose transport in galactose-induced bakers' yeast depends on the Ga 2 gene. Douglas and Condie (14) found that haploid cells carrying the recessive mutant gene, ga 2, could only metabolize galactose when the sugar is present at high external concentrations, although the cells are normal for the following galactose pathway enzymes: galactokinase [adenosine triphosphate (ATP), a-D-galactose-1-phosphotransferase (E.C. 2.7.1.6)], transferase [uridine diphosphoglucose (UDPG), a-D-galactose-l-phosphate uridyltransferase (E.C. 2.7.7.12)], and epimerase [UDPG-4-epimerase (E.C. 5.1.3.2)]. Galactokinase, transferase, and epimerase are the products of the structural genes Ga 1, Ga 7, and Ga 10, respectively (14). de Robichon-Szulmajster reported (13) active transport of galactose in transport-positive, galactokinaseless (ga 1, Ga 2) cells of strain CH-1. Cirillo (10), in the first paper of this series, could only find evidence for facilitated diffusion of the galactose analogues, D-fucose and L-arabinose, in a different strain of IPresent address: Microbiology Institute, Rutgers, The State University, New Brunswick, N.J. 08903. 2 Send reprint requests to V.P.C.

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similar genotype. On the basis of the studies with the nonmetabolized analogues, he proposed that the product of the Ga 2 gene is a galactose carrier which transports galactose into the yeast cell by facilitated diffusion, in which it is phosphorylated by intracellular galactokinase. Van Steveninck and his associates (27-30), however, have proposed a phosphorylation-associated transport mechanism for galactose uptake based on differences in the kinetics of galactose uptake by normal and iodoacetate-inhibited, induced, wild-type cells. This paper presents evidence from a study of galactose uptake by galactokinaseless cells, which supports the role of facilitated diffusion in galactose uptake by metabolizing cells. MATERIALS AND METHODS

Yeast strains. Characteristics of various haploid strains of S. cerevisiae used in this study are listed in Table 1. They were kindly provided by Howard C. Douglas of the Department of Microbiology, University of Washington, Seattle, Wash. Media and growth of organism. The yeast cells were grown in induction medium (10, 15) containing 2% peptone (Difco), 1% Yeast Extract (Difco), 0.2% D-glucose, and 0.2% D-galactose as inducer. The sugars and the sugar-free portions of the liquid medium were autoclaved separately at 120 C for 15 min in double strength and mixed aseptically before use. Cells from a 24-hr Sabouraud Dextrose Agar (Difco) slant were used to inoculate 250 ml of liquid medium contained in a 500-ml flask. After 24 hr of incubation on a reciprocal water bath shaker at 30 C, the cells were harvested by centrifugation at

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KUO, CHRISTENSEN, AND CIRILLO

TABLE 1. Yeast strains used in this study Galactose phenotype

Strain

Galactose genotype

GrwhInduciibility

Growth

346-3B Wild type 346-3C ga 1 (galactokinaseless) 346-3D ga 2 (carrierless) 103-lB ga 7 (transferaseless)

+ + -

+ + + +

a Galactose positive only in media of high galactose concentrations (9).

3,000 X g at 4 C. The cells were washed twice by resuspension in, and centrifugation from, 200 ml of glass-distilled water. Washed cells were resuspended in 200 ml of glass-distilled water and shaken at 30 C for 2 to 3 hr to deplete endogenous reserves. Sugar uptake activity. Except where otherwise indicated, 20% yeast suspensions in distilled water (wet weight/volume) were mixed with an equal volume of 1 mm sugar containing 0.1 ,uCi of D-galactose-1-"4C/ ml, 0.5 ,uCi of L-arabinose-1-'4C/ml, or 0.2 ,uCi of D-fucose-6-T/ml and incubated in a water bath maintained at 30 C. The mixture was stirred magnetically; at intervals, 0.1-ml samples were run into 5 ml of ice-cold water standing over 25-mm membrane filters (porosity = 0.45 nm; Millipore Corp., Bedford, Mass.). The cells were concentrated by suction and washed with two 5-ml portions of ice-cold distilled water. For determination of total uptake, the filter and cells were transferred directly to scintillation vials containing 10 ml of Bray's solution (1). The radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer. The apparent concentration gradient of sugar is expressed as the ratio of concentration of sugar in the cell, C i, to that of the medium C0. Cell water is assumed to be 50% of the packed cell volume (7). The C i/Co ratio is only meaningful when the intracellular sugar is not metabolized. Chromatographic separation of L-arabinose or D-galactose accumulation products. Washed cells yvere transferred to 1 ml of absolute ethanol in a boilingwater bath. After evaporation of ethanol, 1 ml of distilled water was added and extraction was continued at room temperature for 2 hr. The cells were removed by filtration and the extract was concentrated under reduced pressure. The concentrated extracts were applied to Whatman no. 1 filter paper and developed by ascending chromatography in the following solvent: 7.5 parts 95% ethanol-3.0 parts 1 M ammonium acetate (adjusted to pH 7.5 with ammonium hydroxide) (24). The dried chromatograms were cut into 1-cm strips and counted in 10 ml of Bray's solution. Galactose-1-phosphate was identified by its chromatographic behavior and its acid lability (24). Determination of galactose-l-phosphate. A 0.5-ml amount of cell-free filtrate without concentration (see above) was passed through microcolumns of Dowex-1X-10 (Cl- form). The columns were washed five times

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with 0.5 ml of distilled water, and the galactose-l-

phosphate which is retained on the column was eluted

with five 0.5-ml portions of 3 N HCI (16). The effluents

were collected directly into scintillation vials. The

hydrochloric acid was removed by placing the scintillation vials in an oven at 80 C overnight before addition of Bray's solution. Exchange-transfer experiments. In a typical experiment, 0.5-ml yeast suspensions (20% wet weight/ volume) were mixed with the same volume of nonradioactive 200 mm galactose for 10 min or 1 hr at 25 C. These preloaded cells were packed by centrifugation, cooled, suspended in 5 ml of ice-cold distilled water, and centrifuged. The supernatant fluid was decanted, and the walls of the centrifuge tube were carefully wiped with a cotton swab. The pellet was kept in an ice-cold water bath until ready for use. At zero time, 5 ml of radioactive 14C-galactose was added at the temperatures noted. At intervals, 0.5 ml of sample was transferred to 5 ml of ice-cold water and washed on a membrane filter. The washed cells were counted in the usual manner. Sources of reagents. D-Galactose-1-14C (specific activity, 10 mCi/mmole), L-arabinose-1-14C (specific activity, 25 mCi/mmole), and D-fucose-6-T (specific activity, 200 mCi/mmole) were purchased from Calbiochem, Los Angeles, California. All other chemicals were reagent-grade. D-Galactose was purified by incubation as a 5% solution with noninduced, wild-type cells for 2 hr at 30 C and then by concentration to a syrup, clarification by charcoal, and recrystallization from alcohol

(32).

RESULTS Uptake of L-arabinose and D-galactose by mutants of the galactose pathway. L-Arabinose uptake was measured in mutants deficient in the transport carrier (ga 2), galactokinase (ga 1), or transferase (ga 7). It was found that the rate of L-arabinose uptake in transport-negative cells is no greater than that in noninduced cells (Fig. IA), whereas uptake in galactokinaseless or transferaseless cells is a nonconcentrative process essentially similar to that of wild-type cells. Chromatography of the cell extracts from these experiments revealed only free, unmodified L-arabinose, irrespective of the genotype of the cells used. This is consistent with the previous report of this series (10) that L-arabinose is transported by facilitated diffusion. When D-galactose uptake was measured in these mutants, a similar pattern was obtained. Although the rate of galactose uptake by carriernegative cells is not significantly greater than that of noninduced cells, the rates of uptake by kinaseless (Fig. 2) and transferaseless (Fig. 1B) cells are both initially greater than that of the noninduced controls. However, there is a great difference between the pattern of galactose uptake

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GALACTOSE TRANSPORT IN GALACTOKINASELESS CELLS

673

by the kinaseless and tra nsferaseless cells. The rate of galactose uptake Iby induced kinaseless cells (Fig. 2), although iniitially higher than that of the noninduced kinasele-ss cells, falls nearly to 0 zero within the first minutte of uptake, although 0.25the intracellular galactose concentration is still only 0.3 to 0.4 that of the cexternal medium; after 10 min of uptake, the Ci/Clo ratio is still less than 0.5. The rate of galactose uptake by transferaseless cells, on the other hatnd, is essentially equal 0 10 5 to that of wild-type cells fc)r at least 10 min (Fig. TIME (minutes) 1B). Furthermore, whereaiS the galactose taken FIG. 2. "4C-galactose uptake by induced (0) and up by the kinaseless cells is, exclusively free galac- noninduced (0) galactokinaseless cells. All conditions tose, the galactose taken ujp by the transferaseless similar to those described in the legend (Fig. 1). cells is converted to galac:tose-1-phosphate at a rate comparable to that c)f wild-type cells (20). In no case was there an,y indication of either 25 C. If facilitated diffusion is the mechanism active transport (i.e., accuimulation of free sugar of galactose transport by metabolizing cells, it against a concentration difference) or conversion is necessary to show that the rate of galactose to the phosphorylated dlerivative by galacto- uptake by this process is at least equal to the rate kinaseless cells, even when the external galactose of galactose metabolism by induced, wild-type concentration was varied from 10- to 10-1 M. cells. It was the apparent failure of this test by These data show that in Igalactokinaseless cells, iodoacetate-inhibited cells which led Van Stevegalactose, like its nonmet abolized analogues, is ninck to conclude that facilitated diffusion could transported by a facilitaLted diffusion process not account for the rate of galactose uptake mediated by the product c)f the Ga 2 gene. The during metabolism (27, 28). However, it proved apparent concentration of s,ugar against a gradient difficult to measure the initial rate of "C-labeled in transferaseless cells (i..e., Ci/C0 > 1.0) is galactose uptake by galactokinaseless cells since misleading since the intrac ellular sugar is largely equilibration was reached too quickly. However, galactose-1-P (20). this difficulty could be overcome by using cells Exchange transfer in galactokinaseless cells at preloaded with a high concentration of unlabeled galactose. The large pool of unlabeled galactose effectively traps the labeled sugar, allowing the A L- ARABINOSE rate of uptake of labeled galactose to be 4.0-B D-ALACTO initial measured in rapidly equilibrating, kinaseless cells. The results of such an experiment are shown in 3.0- Fig. 3. Washed galactokinaseless cells were incubated with nonradioactive 100 mm galactose for 1 hr at 25 C followed by centrifugation and 2.0ao .5' resuspension in a medium of 1 mm "C-labeled galactose. Control cells were suspended in the -.0 1 mm "C-labeled galactose solution without 0.5 preloading. Galactose uptake by the preloaded 0 5 10 cells shows an overshoot phenomenon (Fig. 3A), 1 3 5 10 20 30 40 50 60 in which the initial rate of uptake is clearly one TIME (n FIG. 1. D-Galactose and L-arabinose uptake by order of magnitude greater than that in unloaded various strains of Saccharomyces. Uptake was meas- cells followed by rapid loss of label from the cells. ured at 30 C from an externa l concentration of I mM. At its maximum, the intracellular concentration D-Galactose-1-14C was presi ent at 0.1 ,uCi/ml; L- of label was about 1.2 times that of the external Arabinose-1-"4C was present at 1.0 ,uCi/ml. The ap- medium (Fig. 3A). The difference in the rate of parent concentration ratio, Cj,IC,, is the ratio of counts uptake by preloaded induced and noninduced in an equal volume of cell water (yeast wet weight cells (Fig. 3A versus 3B) shows that the pheX 0.5) and external medium. The final cell concentra- nomenon is specific for the galactose carrier tion was 125 Iliters of cell induced wild type, 346-3B; (O transferaseless, (0) induced galactokinaselesss, 346-3C; (0) induced transportless, 346-3D; (A) noninduced wild type 346-3B.

103-lB;

since the entry of galactose into noninduced cells occurs via the constitutive, glucose carrier (9). The rapid loss of label after the initial burst of uptake cannot be accounted for by the net

674


TIME (mbwu)

FIG. 3. Galactose uptake and exchange by induced (A) and noninduced (B) galactokinaseless (346-3C) cells at 25 C. "4C-D-galactose uptake was measured from an external galactose concentration of I mM (0.1 uCi/ml) by using cells not previously incubated with galactose (uptake, A) and cells previously incubated with 100 mM unlabeled galactose at room temperature (exchange transfer, 0). Both the induced and noninduced loaded cells contained approximately 50 pumoles of unlabeled galactose/ml of cell water at the time of transfer to the radioactive I mM galactose medium. The induced cells were preloaded for I hr; the noninduced cells, in which galactose uptake occurs via the constitutive, glucose carrier, were preloaded for 4 hr. The dotted line shows the level of intracellular label which represents diffusion equilibrium with the external medium.

efflux of galactose from a high intracellular concentration at the low extracellular concentration, although net galactose loss does take place. When the cells are preloaded in 100 mm galactose, the intracellular galactose concentration is approximately 50 mm. When these cells are suspended in the 1 mm "4C-galactose exchange medium, there is a net efflux of the intracellular pool (Fig. 4). At zero time, the Ci/Co ratio is 50. After an initially rapid efflux which reduces the Ci/Co ratio to 28 by 1 min, the subsequent efflux is much slower; at 5 min, the Ci/Co ratio is still about 24. This pattern of efflux is characteristic for yeast cells (18, 26). The maximal concentration ratio for the labeled sugar is only 1.2 at 10 sec of exchange when the concentration ratio of unlabeled sugar is 45; at 5 min, the concentration ratio for the labeled sugar is about 0.25, whereas that of the unlabeled sugar is 25. Thus, the intracellular specific activity never reaches that which would be expected if all of the intracellular sugar were exchangeable; if equilibration occurred, the concentration gradients for total sugar and for label would be the same. The significance of this apparent "compartmentation" is not clear, but it appears to be a concentration-dependent phenomenon (see below). Exchange transfer at 0 C. The rate of uptake

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of labeled galactose by preloaded cells at 25 C occurs too rapidly to measure an initial rate; therefore, the rate of exchange was measured at successively lower temperatures. It was discovered that the rate of exchange is less affected by reduced temperature by comparison with the effect of temperature on uptake by unloaded cells. Furthermore, as the temperature is reduced, the maximal concentration ratio of labeled galactose increases and the subsequent efflux is reduced, until at 0 C the maximal concentration ratio for labeled galactose is 3.5 and there is no observable efflux after 10 min (Fig. 5). Except for the absence of the overshoot, all of the characteristics of the exchange phenomenon observed at 25 C are also seen at 0 C. (i) The maximal concentration ratio of labeled sugar is approximately 3.5 (Fig. 5A); (ii) the rate of exchange is proportionately greater than the rate of uptake by unloaded cells (Fig. 5A); (iii) the rate of exchange by induced cells is even more than one order of magnitude greater than that by noninduced cells, although both were loaded to the same level (Fig. 5A versus 5B); (iv) the apparent intracellular specific activ-

40

0)

3 0

0

t

2

0

a

0 0

0

0

LN

0)

TIME (minutes)

FIG. 4. Relative concentration ratio of '4C-labeled and total sugar during exchange by induced (346-3C) cells at 25 C. The concentration ratio for '4C-labeled galactose was calculated from the data of Fig. 3A Jor the exchange experiment. The concentration ratio for total galactose was determined in a parallel experiment in which the cells were preloaded in a '4C-labeled 100 mM galactoae solution and suspended in a nonradioactive I mm galactose solution; the concentration ratio was calculated from the radioactivity contained in equal volumes (SO slditers) of cell water and suspending medium. Note the difference in scale for 14C-galactose and total galactose.

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-INDUED CELLS

B NON-

A INDUCED CELLS 8.75- .

E

EXCHANGE

IN.-

z 0 0 0

5.0EXTERNAL MEDRJM CONCENTRJtATION

2

EX(,CHIANGE

UPTKE 5

675


oFa4r10

5

e

IS

20

TIME (miuts)

FIG. 5. Galactose uptake and exchange by 346-3C cells at 0 C. The cells were preloaded at room temperature; however, the uptake and exchan ge were measured at 0 C. All other conditions as described for Fig. 3.

ity reaches a maximum of about 11% of that of the medium (Fig. 6). The ap parently higher specific activity reached at low(er temperatures (i.e., 11% at OC versus 2.5% after 10 sec at 25 C) may be due to slower losrs of sugar from the exchangeable "compartment.' Exchange in the absence of ncet efflux. In the previous sections, exchange waws measured between labeled galactose at a low external concentration (1 mM) and unlabeled galIactose at a high intracellular concentration (40 tto 50 mM). Although this procedure provided optimal conditions to measure the initial raLte of galactose uptake, the intracellular galactos e concentration is decreasing continuously durinig the exchange process, and the exchange betwe en the cells and medium appeared incomplete (F'ig. 4 and 6). It 4*

was decided to study the time course and extent of exchange at various temperatures in the absence of net sugar loss from cells into the medium. The same concentration was used for preloading and exchange, namely 100 mm. In the experiment shown in Fig. 7, preloading was carried out by incubation in unlabeled 100 mm galactose for 10 min at 25 C; exchange was measured by using '4C-labeled 100 mM galactose at various temperatures. Uptake by unloaded cells is shown at the same temperatures. Several aspects of the results of this experiment are significant. (i) No overshoot is expected and none is observed in the absence of an imposed concentration asymmetry between cells and medium; (ii) the exchange process is much less temperature-sensitive than net uptake; (iii) all of the intracellular sugar pool seems to be exchangeable irrespective of the temperature of exchange. The incomplete exchange observed in the previous experiments (Fig. 4 and 6) is apparently a consequence of the sugar concentration asymmetry between the cells and medium, for which no explanation is immediately obvious. D-Fucose exchange. If the exchange phenomenon is mediated by the galactose carrier, it should be exhibited by the use of galactose analogues. This expectation was realized when a comparison was made of the rate of uptake of 3H-labeled D-fucose by D-fucose-loaded and unloaded cells. The results of such an experiment (Fig. 8) show that the rate of uptake by the loaded cells is much greater than that of the unloaded cells. It has also been shown that the rate of D-fucose uptake can be enhanced by preloading the cells with D-galactose and vice versa.

80

14C- golocl

-os

A UPTME

70

B EXCHANGE

S

60 Oa0~

3

U 0 a

50 o

I:

A0 0

2

-4

Toa/gi

8.

octose

d

40

0 2

3o

0

0

*-10

-20 U TM (m.)

FIG. 7. Effect of temperature on galactose uptake and exchange by kinaseless cells (346-3C). (A) Uptake was measured with unloaded cells from an external TIME (minutes) concentration of 14C-labeled 100 mm galactose containing 0.1 ACi/ml. (B) Exchange was measured by cells FiG. 6. Relative concentration ratio of "4C-labeled galactose and total galactose during exchange by in- after preloading with nonradioactive 100 mv galactose duced (346-3C) cells at 0 C. All operations as described for 10 min at 25 C. All other conditions, except for temperature, were as for Fig. 3. for Fig. 4.

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J. BACTERIOL.

account for the rate of galactose metabolism by induced, wild-type cells. DISCUSSION From measurements of the "initial rate" of galactose uptake by facilitated diffusion by using iodoacetate-inhibited, wild-type cells, Van Steveninck and his colleagues concluded that galactose uptake by facilitated diffusion could not account for the rate of galactose uptake by metabolizing cells (27-30). However, as seen in the experiments reported above, it is very difficult to measure the initial rate of galactose uptake by nonmetabolizing cells unless an intracellular "trap" is provided. Galactose uptake by unloaded galactokinaseless cells shows a rapid equilibration at a low, apparent intracellular concentration, with the net rate of uptake falling to zero within 1 min. If the initial rate of galactose uptake were defined TIME (minutes) as the amount of galactose taken up in 1 min, for FIG. 8. Uptake and exchange Of D-fucose by in- example, the initial rate of uptake would be duced 346-3C cells at 25 C. 3H-labeled D-fucose uptake underestimated by at least one order of magniand exchange were measured from I mM D-fucose containing 0.2 ,uCi/ml. The cells for the exchange experiment were preloaded with unlabeled 100 mm D-fucose for I hr at 25 C. All other operations were as for galactose uptake and exchange, described in the legend of Fig. 3.

Effect of UO?2+ and iodoacetate on exchange. As a final correlation between the exchange phenomenon and carrier-mediated uptake, the effect of uranyl ion which is an inhibitor of carriermediated uptake was tested on the exchange phenomenon. The data presented in Fig. 9 show that the exchange phenomenon shows the same uranyl ion sensitivity exhibited by other sugar transport phenomena in yeast (5, 11, 12). Finally, it is significant that 10-' M iodoacetate, which has no affect on carrier-mediated uptake, also has no affect on the exchange process. Relative rate of galactose transport by facilitated diffusion and its rate of metabolism. Since we established that the rate of galactose uptake by preloaded galactokinaseless cells is a measure of the rate of galactose uptake by carrier-mediated, facilitated diffusion, it remains only to compare this rate with that of galactose metabolism at similar external concentrations. Exchange by kinaseless cells and metabolism by wild-type cells were measured at 10 C, at which temperature both processes occur at conveniently measurable rates over the concentration range from 105 to 10-1 M. The results are presented in Table 2. It is clear that the rate of galactose uptake by carriermediated, facilitated diffusion at each concentration is from 4 to 10 times greater than the rate of galactose metabolism. Transport by this process is, therefore, more than adequate to

E 0

F0

o

5 TIME (minutes)

10

FIG. 9. Effect of uranyl ion on galactose uptake ant exchange by 346-3C cells at 0 C. Galactose uptake and exchange were measured at 0 C in 0.1 m acetate buffer (pH 4.0). At the arrows, uranyl nitrate was added to a final concentration of I mm; sodium nitrate was added to the control suspensions. To remove the radioactivity introduced by the uranyl ions, the cells were washed with ice-cold 0.01 m phosphate buffer (pH 7.0) instead of distilled water used in other experiments. All other conditions for uptake and exchaange as described for Fig. 3.

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tude. The longer the initial interval used to calculate the initial rate, the greater the error. Van Steveninck and Rothstein used a 10-min interval (27). The experiments with preloaded, galactokinaseless cells, however, show that the rate of galactose transport by facilitated diffusion is more than high enough to account for the rate of galactose uptake by metabolizing cells. The large intracellular pool of unlabeled sugar serves as a trap equivalent to that provided by intracellular phosphorylation by galactokinase positive cells. This is shown by the fact that the rate of galactose uptake by transferaseless cells which are normal for the carrier and galactokinase is identical to that of wild-type cells. The fact that facilitated diffusion can account for the rate of galactose uptake by metabolizing cells removes the necessity for proposing a phosphorylative pathway for galactose uptake by metabolizing cells, but it does not necessarily exclude the phosphorylation mechanism. However, direct experimental evidence against the phosphorylative pathway by using transferaseless cells is presented in the next paper of this series. When "IC-labeled galactose was added to transferaseless cells containing an unlabeled pool of free galactose and galactose-1-phosphate, the specific activity rose faster in the free galactose than in the galactose phosphate pool (20). There seems to be no experimental support, therefore, for the proposal that galactose uptake by bakers' yeast is a phosphorylation-associated process. The exchange phenomenon in yeast cells exhibits two significant features which should be emphasized. (i) The activation energy for exchange is significantly lower than that for net transport, and (ii) a large portion of the intracellular sugar is nonexchangeable when the external sugar concentration is much lower than that of the intracellular pool. The observation that exchange phenomena show a lower activation energy than net transport was extensively studied by Burger and Lacko (3, 21-23) for the facilitated diffusion of sugars in human erythrocytes. They showed that, although net transport is completely inhibited at 0 C, exchange occurs at a very high rate. They also showed that exchange between pairs of sugars (such as between D-galactose and D-fucose in the present case) is only exhibited by sugars sharing a common carrier. They proposed that the low activation energy for exchange may result from the fact that a carrier with a sugar molecule already attached is held in a more favorable orientation for interaction with a second displacing sugar than an unloaded carrier, since an unloaded carrier would be expected to be associated with molecules other than sugars or with nonmobile components of the cell mem-

677

TABLE 2. Comparison of galactose exchange by galactokinaseless (346-3C) cells and the rate of metabolism by wild-type (346-3B) cells" Molarity

105 lo-, 10-3 10-2 10-1

Kinaseless cells (exchange rate)b

37 360

3,500 20,000

45,000

Wild-type

Ratio of rate to

exchange

cells (uptake rate)b

metabolic uptake

3.8 47 470 4,000 11,500

9.7 7.6 7.4 5.0 3.9

a "Exchange" in kinaseless cells: washed cell suspensions were incubated with nonradioactive 100 mm galactose for 10 min at 25 C. The cells were then centrifuged, washed, and suspended in the indicated concentrations of D-galactose-1-'4C at 10 C. "Uptake" in wild-type cells was measured at 10 C from the various external concentrations of D-galactose-1-'4C at 10 C. At 10 sec, a portion was collected on a membrane filter (Millipore Corp.), washed, and counted. b Expressed as micromoles of '4C-galactose per gram (dry weight) of yeast after 10 sec at 10 C.

brane. The high energy of activation for binding to unloaded carriers would be due to the necessity to break these alternative associations (23). More recently, the phenomenon of exchange transfer has been used to study the facilitated diffusion of the lactose transport system in Escherichia coli in which energy coupling for active transport has been inhibited (17, 31). Very similar results were obtained. Several years ago, Britten and McClure, studying the active transport of amino acids in E. coli, described an energy-independent exchange transfer (2). They found that the relative rates of exchange and net uptake at 0 C were 0.18 and 0.0074, respectively; the corresponding rates at 25 C were 0.61 and 2.0. They proposed that the metabolic-energy requirement for amino acid accumulation represented an energy-dependent activation of specific amino acid-binding sites to which the intracellular amino acids are adsorbed; they further proposed that exchange between free and adsorbed amino acids is an energy-independent process. Thus, no active accumulation can occur at 0 C but exchange occurs rapidly. The large pool size involved in the sugar exchange presented in the present study and the fact that the sugars are osmotically active (7) preclude an adsorption model in which the energy of activation for binding to unoccupied intracellular sites is greater than that for exchange between a free and bound sugar. However, if one restricts the binding sites to those of the membrane carrier, we return to the model

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proposed by Burger and Lacko. We feel this is the best model to explain our present results. The difference in the fraction of intracellular sugar pool which exchanges with external sugar when the external sugar concentration is lower or equal to that of the intracellular pool is difficult to explain. Incomplete exchange, representing an intracellular "compartmentation," is not unexpected since there is considerable evidence for such compartmentation from studies on the efflux kinetics of various sugars from yeast (18, 19, 26), but the explanation of why this compartmentation is not evident when the external sugar concentration is equal to that of the intracellular pool will depend on future studies. ACKNOWLEDGMENTS The authors express their gratitude to Howard C. Douglas of the University of Washington School of Medicine in Seattle, Washington, for interest in this study and for generosity in making available invaluable yeast mutants. We also express our gratitude to Alberto Sols, Instituto Maranon, Centro de Investigaciones, Biologicas, C.S.I.C., Madrid, Spain, for valuable suggestions regarding the exchange experiments. This investigation was supported by Public Health Service grants GM-12743 from the National Institute of General Medical Sciences and FR-0767 from the Division of Research Facilities and Resources, and a grant-in-aid from the Graduate School of the State University of New York, Stony Brook, N.Y. LITERATURE CITED 1. Bray, G. A. 1960. A simple efficient scintillator for counting aqueous samples in a liquid scintillation counter. Anal. Biochem. 1:279-285. 2. Britten, R. J., and F. T. McClure. 1962. The amino acid pool in Escherichia coli. Bacteriol. Rev. 26:292-335. 3. Burger, M. 1961. Transport of sugars across cellular and biological membranes. p. 455-456. In A. Kleinzeller and A. Kotyk (ed.), Membrane transport and metabolism. Academic Press Inc., New York. 4. Burger, M., L. Hejmova, and A. Kleinzeller. 1959. Transport of some mono- and di-saccharides into yeast cells. Biochem. J. 71:233-242. 5. Cirillo, V. P. 1961. Mechanism of sugar transport into the yeast cell. Trans. N. Y. Acad. Sci. Ser. II 23:725-734. 6. Cirillo, V. P. 1962. Mechanism of glucose transport across the yeast cell membrane. J. Bacteriol. 84:485-491. 7. Cirillo, V. P. 1962. Sugar transport by yeast protoplasts. J. Bacteriol. 84:1251-1253. 8. Cirillo, V. P. 1967. A comparison of sugar uptake by glucose grown and galactose grown baker's yeast. Abh. Deut. Akad. Wiss. Berlin KI. Med. 1966 Nr. 6, p. 153-159, 369-370. 9. Cirillo, V. P. 1968. Relationship between sugar structure and competition for the sugar transport system in baker's yeast. J. Bacteriol. 95:603-611. 10. Cirillo, V. P. 1968. Galactose transport in Saccharomyces cerevisiae. I. Nonmetaboilzed sugars as substrates and inducers of the galactose transport system. J. Bacteriol. 95: 1727-1731. 11. Cirillo V. P. and P. 0. Wilkins. 1964. Use of uranyl ion in membrane transport studies. J. Bacteriol. 87:232-233. 12. Dennis C., A. Rothstein, and E. Meier. 1954. The relation-

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