JOURNAL OF BACrERIOLOGY, Sept. 1970, p. 679-685Copyright 0 1970 American Society for Microbiology

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

Galactose Transport in Saccharomyces cerevisiaeIII. Characteristics of Galactose Uptake in Transferaseless Cells: Evidence

Against Transport-Associated PhosphorylationSHOU-CHANG KUO' AND VINCENT P. CIRILLO

Department of Biochemistry, Division of Biological Sciences, State University of New York,Stony Brook, New York 11790

Received for publication 22 May 1970

The characteristics of the inducible galactose transport system in bakers' yeastwere studied in uridine diphosphate, galactose-1-phosphate uridylyl-transferaselesscells. Transferaseless cells transport galactose at the same initial rate as wild-typecells and accumulate a mixture of free galactose and galactose-l-phosphate. Theaddition of 14C-labeled galactose to cells preloaded with unlabeled galactose andgalactose-1-phosphate results in a higher rate of labeling of the free-sugar pool thanof the galactose-1-phosphate pool. These results support other evidence that galac-tose uptake in bakers' yeast is a carrier-mediated, facilitated diffusion and thatphosphorylation is an intracellular event after uptake of the free sugar.

Galactose metabolism in bakers' yeast dependson the induction of a transport carrier andthe galactose pathway enzymes: galactokinase[adenosine triphosphate, a - galactose-1 - phos -photransferase (EC 2.7.1.6)], transferase[uridine diphosphate, c-galactose-1-phosphateuridylyltransferase (EC 2.7.7.12)], and epi-merase [uridine diphosphoglucose (UDPG)4-epimerase (EC 5.1.3.2)] (10-12). Previousstudy of the characteristics of uptake of thenonmetabolized galactose analogues, D-fucoseand L-arabinose, showed that the process is afacilitated diffusion dependent only on the pres-ence of the transport gene product (i.e., thegalactose "carrier"; reference 8). Uptake of thenonmetabolized sugars was unaffected by thepresence or absence of galactokinase activity.These conclusions were extended to the uptakeof galactose itself in a study of the mechanism ofgalactose uptake by galactokinaseless cells (19).In galactokinaseless cells, galactose is recoveredas the free sugar and is never accumulated againsta concentration gradient even when the externalsugar concentration is varied from 10-1 to 1i-5 M.Furthermore, by measuring the rate of galactoseuptake under counterflow conditions, it wasdemonstrated that the rate of galactose uptakeby facilitated diffusion in galactokinaseless cellsis greater than the rate of galactose metabolismby wild-type cells over the same concentrationrange. Although the demonstration that the rateof galactose uptake by facilitated diffusion can

1 Present address: Microbiology Institute, Rutgers, The StateUniversity, New Brunswick, N.J. 08903.

account for the rate of galactose metabolismeliminates the necessity for the special transport-associated phosphorylation mechanism proposedfor galactose transport by Van Steveninck andhis colleagues (9, 23, 28), this study investigatesthe possible importance of the phosphorylationpathway in transferaseless mutants. The rate ofgalactose uptake by transferaseless cells is almostidentical with that of wild-type cells and resultsin the accumulation of a mixture of free galactoseand galactose-l-phosphate. The precursor rela-tions between galactose and galactose-l-phos-phate can be determined by measuring the relativerate of increase of the specific activity of carbon14 in the free-galactose and galactose-l-phosphatepool, when "IC-galactose is added to transferase-less cells preloaded with nonradioactive sugar.When this is done, galactose is found to belabeled before the galactose-l-phosphate, sug-gesting that even in cells with the enzymaticcapacity for galactose phosphorylation, galactoseis taken up into the cell by a nonphosphorylativepathway. The results of these experiments and adiscussion of the current status of the phos-phorylation theory for sugar uptake by yeastcells are presented below.

MATERIALS AND METHODSYeast strains. The inducible wild-type (346-3B)

and the UDPG, galactose-1-phosphate uridylyltrans-ferase-negative (103-IB) strains of Saccharomycescerevisiae used in this study were kindly provided byHoward C. Douglas of the Department of Micro-biology, University of Washington, Seattle, Wash.Media and growth of organism. The yeast cells were

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grown in induction medium (11, 12) containing 2%peptone (Difco), 1% Yeast Extract (Difco), 0.2%D-glucose, and 0.2% D-galactose as inducer. Thesugars and the sugar-free portions of the liquidmedium were autoclaved separately in double strengthat 120 C for 15 min and mixed aseptically before use.

Cells from a 24-hr Sabouraud Dextrose Agar(Difco) slant were used to inoculate 250 ml of liquidmedium contained in a 500-ml flask. After 24 hr ofincubation on a reciprocal water bath shaker at 30 C,the cells were harvested by centrifugation at 3,000 X gat 4 C. The cells were washed twice by resuspension in,and centrifugation from, 200 ml of glass-distilledwater. Washed cells were resuspended in 200 ml ofglass-distilled water and shaken at 30 C for 2 to 3 hrto deplete endogenous reserves.

Sugar uptake activity. Except where otherwiseindicated, 20% yeast suspensions in distilled water(wet weight/volume) were mixed with an equal volumeof 1 or 100 mm sugar, containing 0.1 ,uCi of D-galac-tose-1-'4C/ml, and incubated in a water bath main-tained 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 andwashed with two 5-ml portions of ice-cold distilledwater. For determination of total uptake, the filterand cells were transferred directly to scintillation vialscontaining 10 ml of Bray's solution (2). The radio-activity was measured in a Packard Tri-Carb liquidscintillation spectrometer. The apparent concentrationgradient of sugar is expressed as the ratio of concen-tration of sugar in the cell, C i, to that of the medium,C0. Cell water is assumed to be 50% of the packedcell volume (6).

Chromatographic separation of D-galactose ac-cumulation products. Washed cells were transferred to1 ml of absolute ethanol in a boiling-water bath.After evaporation of ethanol, 1 ml of distilled waterwas added and extraction was continued at roomtemperature for 2 hr. The cells were removed byfiltration and the extract was concentrated underreduced pressure. The concentrated extracts wereapplied to Whatman no. 1 filter paper and developedby ascending chromatography in the following solvent:7.5 parts 95% ethanol-3.0 parts 1 M ammoniumacetate (adjusted to pH 7.5 with ammonium hy-droxide) (20). The dried chromatograms were cut into1-cm strips and counted in 10 ml of Bray's solution.

Determination of-galactose-1-phosphate. A 0.5-mlamount of cell-free filtrate without concentration (seeabove) was passed through microcolumns of Dowex-l-X-10 (Cl- form). The columns were washed five timeswith 0.5 ml of distilled water, and the galactose-l-phosphate which is retained on the column was elutedwith five 0.5-ml portions of 3 N HCl (15). The effluentswere collected directly into scintillation vials. Thehydrochloric acid was removed by placing the scintil-lation vials in an oven at 80 C overnight before addi-tion of Bray's solution. The product was identified asgalactose-1-phosphate by its chromatographic be-havior and acid lability (20).

Sources of reagents. D-Galactose-1-'4C (specific

D CIRILLO J. BACTERIOL.

activity, 10 mCi/mmole) was purchased from Calbio-chem, Los Angeles, Calif. All other chemicals werereagent-grade.

D-Galactose was purified by incubation as a 5%h1solution with noninduced, wild-type cells for 2 hr at30 C and then by concentration to a syrup, clarifica-tion by charcoal, and recrystallization from alcohol(29).

RESULTSThe results of the previous paper of this series

(19) showed that the rate of galactose uptake bytransferaseless cells was identical to that ofmetabolizing cells. When -the nature of the intra-cellular accumulation products was examined,it was found that the intracellular radioactivityrepresented a mixture of free sugar and galactose-1-phosphate; however, the relative amount ofthe free sugar and its phosphorylated derivativevaries with the external.galactose concentration.At an external concentration of 1 mm, the ac-cumulated galactose is recovered almost exclu-sively as galactose-1-phosphate; less than 5 aclof the total radioactivity is recovered as the freesugar even after 1 hr of incubation at 25 C (Fig.1). However, when the external sugar concen-tration was increased to 100 mm, significantamounts of both free and phosphorylated sugarare found; initially, the free sugar occurs at ahigher concentration than the phosphorylatedsugar (Fig. 2A).To determine the precursor relationship be-

tween these two forms of the sugar, cells wereallowed to accumulate unlabeled free galactoseand galactose-1-phosphate by incubation withunlabeled sugar for 1 hr at 30 C followed by theaddition of "4C-labeled galactose. Most of thelabel is found as galactose-1-phosphate whenthe external galactose concentration for preload-ing and exchange was 1 mm (Fig. 1); however,

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TIME (minutes)90 ,20

FIG. 1. "C-D-galactose uptake and exchange bytransferaseless cells. Cells of strain 103-lB were

incubated at 10 C with I mM D-galactose for 120 min.D-Galactose-1-"4C was added at a final activity of 0.1ACi/ml at either zero time (uptake) or 60 miti (ex-change). Galactose (0, A) and galactose-l-phos-phate (-,A) are expressed as counts per min per S

juliters of cell water. The final cell concentration was 50,lAiters of cell water/mi.

*Galactosee-I- £ Galactose - I-P

Free goIlc,ose Free goloctos_ ----0----0-------- A a------- -a

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GALACTOSE TRANSPORT IN TRANSFERASELESS CELLS

A UPTAKE

'00

Gotlactoes -I-P Fro,: ---- 0

10'e := FDO. t--- Fro gdoctows

J0

10 20 30

TIME (mnuts)

FIG. 2. 14C-D-galactose uptaketransferaseless cells. All condition.in the legend to Fig. 1, except that t/concentration was 100 mM.

B EXCAtNGE nism associated with the steady-state and ex-change processes. The presence of phosphatase

- activity which hydrolyzes galactose-1-phosphatein yeast cells was observed in early experiments

G;alctoe-l-P~when the intracellular products of galactoseuptake were extracted by the Van Steveninckprocedure (24) in which the washed cells weresuccessively extracted for 1 hr at room tempera-

40 ture in 100% alcohol followed by another hourof extraction at room temperature after dilution

and exchange by of the alcohol with an equal volume of water.s were as des.cribed Galactose-1-phosphate is hydrolyzed during theye external galactose second hour of incubation in the 50% alcohol

by phosphatases which survive treatment for 1hr with absolute alcohol at room temperature.If the cells are treated with boiling alcohol or

the label appeared first in the free sugar whenpreloading and exchange were carried out with100 mm sugar. The virtual absence of free sugar

during initial uptake or during exchange in thecase of 1 mm galactose suggests that the smallamounts of galactose taken up by these cells arecompletely converted to galactose phosphateduring experimental manipulation; however, theresults with the 100 mm galactose are fairlyunambiguous. The specific activity of free galac-tose pool rises faster than that of galactose-l-phosphate. At 30 sec, the specific activity of thegalactose pool is over 7X higher than that ofgalactose-1-phosphate (Fig. 3). If there is onlyone kinetic pool of galactose-1-phosphate, theseresults mean that phosphorylation is not asso-ciated with the transport process, but is an eventwhich follows transport of the free sugar into thecell interior; if there is more than one pool forgalactose-1-phosphate, these results are equivocal.The exchange phenomenon illustrates another

important fact. At both 1 and 100 mm, the initialrate of uptake of labeled galactose by loadedcells is higher than the rate in unloaded cells.This shows that the plateau of the original uptakeis a steady state and not the result of the exhaus-tion of a necessary reactant in the cell. Further-more, the high exchange rate shows that thehigh intracellular concentration of phospho-rylated sugar does not inhibit the uptake processas would be expected if the intracellular phosphatewere a feedback inhibitor of the sugar uptakeprocess, as proposed by Sols (22) and Azam andKotyk (1).When the labeling is reversed in an exchange

experiment by preloading with labeled sugarand chasing with unlabeled sugar, all of theintracellular label is exchangeable whether it waspresent in the cell as free galactose or galactose-1-phosphate; however, only unphosphorylatedgalactose is found in the external medium. Thissuggests a very active dephosphorylation mecha-

extracted with 5% trichloroacetic acid, no hy-drolysis occurs.

DISCUSSIONThe hypothesis that monosaccharide transport

in bakers' yeast is associated with phosphoryla-tion was proposed about 15 years ago to explainwhat was then believed to be the following"facts." (i) The yeast cell is impermeable tosugars which could not be phosphorylated; (ii)the order of preference of the yeast cell for sugarsis the same as the substrate specificity of hexo-kinase; and (iii) uranyl ions (UO22+) which are

~ ~ ~ -1..............

1. Galactose -4--~~~~~~ 1

E tS

0.5- 5

~~~Galoctose/ GGIat oI..

0 5 10 15 20

TIME (minutes)

FIG. 3. Specific activity of free galactose and ga-

lactose-i-phosphate during exchange. The relativespecific activity of galactose (03) and galactose-l-phosphate (A) during the exchange experiment shownin Fig. 2 was calculated as the fraction of the specificactivity (counts per min per pmole) of the galactosein the medium. The total galactose concentration in thecells was found not to change during the exchangeperiod. The specific activity ratio (0) was calculatedas the specific activity of galactose divided by that ofgalactose-l-pkosphate for each experimental point.

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KUO AND CIRILLO

reversible inhibitors of sugar metabolism complexwith but do not cross the yeast cell membrane(5, 21). It was subsequently discovered that theyeast cell is, in fact, permeable to sugars whichare not phosphorylated and that nonmetabolizedsugars are transported by a nonconcentrativeprocess which is sensitive to uranyl ions butinsensitive to metabolic inhibitors like iodoacetate(IAA; 4, 6, 28). It was also found that the uptakeof the nonmetabolized sugars is competitivelyinhibited by the metabolized sugars and thatthe metabolized sugars can induce the uphillefflux of the nonmetabolized sugars from cellspreviously equilibrated with the nonmetabolizedsugars (i.e., counterflow). These newer findingsled to a rejection of the original phosphorylationhypothesis in favor of a carrier process whichmediates the downhill transport of free sugarsacross the cell membrane (i.e., facilitated diffu-sion; 6, 7, 22). The competitive inhibition betweenthe metabolized and nonmetabolized sugars andthe phenomenon of counterfiow showed that thetwo classes of sugars share the same carrier, butthe generally low affinity of the nonmetabolizedsugars (compared with that of the metabolizedsugars) suggested that the nonmetabolized sugarsare only marginal substrates of the carrier medi-ated process (6, 7, 22). The original studies in-volved the constitutive, glucose transport system;subsequently similar evidence was presentedfor the inducible, galactose transport system forwhich a separate, inducible carrier was proposed(8, 17).About 5 years ago the phosphorylation theory

for sugar uptake by yeast cells was revived byVan Steveninck and his colleagues on the basisof the effect of IAA and Ni2+ ions on the uptakeof glucose and galactose (26, 28). Although IAAdoes not inhibit the uptake of nonmetabolizedsugars and allows glucose and galactose (ininduced cells) to be taken up by facilitated diffu-sion, the initial rate of glucose or galactoseuptake in IAA-inhibited cells was reported to betoo low to account for the rate of sugar metab-olism by uninhibited cells. In addition, Ni2+ ionswere also reported to have a differential effecton the rate of uptake of metabolized and non-metabolized sugars. Thus, Ni2+ ions do not inhibitthe rate of uptake of nonmetabolized sugars,including galactose in noninduced cells, butstrongly inhibit the "initial rate" of uptake ofmetabolized sugars. Although the effect of Ni2+ions seems similar to that of IAA, it was reportedthe Ni2+ was not transported into the yeast celland acted exclusively at the cell surface. Further-more, the effect of both IAA and Ni2+ ions wasreported to be a conversion of the transport ofthe metabolized sugars from a high-affinity (low

Km and high Vmax) to a low-affinity process (highKm and low Vmax). These results suggested thattwo modes of transport can occur via the samecarrier: a low-affinity, carrier-mediated, facili-tated diffusion and a high-affinity metabolicallylinked, active transport as depicted in Fig. 4. Inthe active transport mechanism, the binding ofthe sugar, So, to the carrier at the outer face ofthe membrane, Co, is catalyzed by a sugar-specificenzyme, E (or "permease"), and a phosphatedonor, P, which is presumed to be membrane-associated polyphosphates generated by metabolicenergy. The sugar-carrier-phosphate complex,CSP, is the transported species which dissociatesinto free carrier, Co, and sugar phosphate whichenters the metabolic reactions of the cell (i.e.,glycolysis). Nonmetabolized sugars are trans-ported exclusively by the low-affinity, facilitateddiffusion pathway. Ni2+ is supposed to inhibitthe permease-catalyzed formation of the CSPcomplex; IAA results in the depletion of -P, thephosphate donor. In the presence of these in-hibitors, the metabolized sugars would be trans-

FIG. 4. Models of galactose transport in bakers'yeast. The carrier-mediated, facilitated diffusion pCth-way is indicated by solid arrows, and the permease-mediated, active transport pathway by double-linedarrows. S andSP are thefree andphosphorylated sugar,respectively; C an7d CS, thefree and the loaded carriers,respectively; E and ES, the permease and permease-sug,ar complex, respectively; -P is the phosphatedonor; and CSP is the permease catclyzed, carrier-sugar-phosphate complex which is involved in themembrane translocation step. The subscripts o and irefer to medium and cytoplasmic sides of the cellmembrane represented by the enclosing rectangle. Tlhekinases are hexokinase and galactokinase for glucoseand galactose phosphorylation, respectively (adaptedfrom reference 30).

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GALACTOSE TRANSPORT IN TRANSFERASELESS CELLS

ported by the Ni2+ and IAA-insensitive, low-affinity facilitated diffusion pathway. In thismodel, the high rate of uptake of the metabolizedsugars is accounted for by a phosphorylation"pump;" in the facilitated diffusion model (repre-sented by the heavy arrows in Fig. 4), the highrate of uptake of metabolized sugars is maintainedby a kinase trap (hexokinase or galactokinase forglucose or galactose, respectively). Thus, thepermease-phosphorylation model states thattransport is the rate-limiting step in sugar metab-olism, whereas the facilitated diffusion modelstates that the rate of intracellular phosphoryla-tion is the rate limiting step.

Before considering some inadequacies of themodel as applied to galactose, it must be pointedout that the validity of the data on Ni2+ andIAA-inhibited cells have recently been ques-tioned. Fuhrmann and Rothstein (13, 14) haverecently shown that Ni2+ ions do not act exclu-sively at the cell surface, as presumed by VanSteveninck and Booij (26), but are transportedinto the cell and block sugar metabolism byinhibition of alcohol dehydrogenase. As aninhibitor of glycolysis, therefore, Ni2+ ions affectsugar uptake by a mechanism comparable tothat of IAA (which inhibits triosephosphatedehydrogenase). With respect to the measure-ment of the "initial rate" of galactose uptake inthe presence of a metabolic block, the previouspaper of this series has shown that the low appar-ent rates of galactose uptake in galactokinaselesscells are an artifact resulting from the high rate ofequilibration between the cells and the medium(10). When the rate of uptake of "IC-labeledgalactose was measured in cells preloaded with alarge pool of nonradioactive galactose to serveas a trap for the labeled sugar, the initial rateof uptake was at least one order of magnitudegreater than that observed for unloaded cells. Itis thus questionable that Ni2+ ions and IAAreally convert the mode of transport of metabo-lized sugars from a high-affinity, active transportto a low-affinity, facilitated diffusion. The resultsfrom the studies on galactose uptake by galacto-kinaseless cells in the previous paper of thisseries suggest that both galactose and its non-metabolized analogues are transported by apassive, carrier-mediated, facilitated diffusionand that the rate of galactose uptake by metabo-lizing cells is galactokinase-limited, not transport-limited. [The consequences of this fact on thesugar pool size in metabolizing cells has beentreated elsewhere (9).] This conclusion is stronglysupported by the results of the present paper onthe rate of labeling of intracellular free galactoseand galactose-l-phosphate. It should be empha-sized, however, that the interpretation of the

results of the labeling experiments depends onthe important assumption that there is only asingle kinetic pool of galactose-1-phosphate. Ifthere were more than one pool, it would benecessary to measure the specific activity of theseparate pools. However, in view of the resultsof the counterflow experiments in kinaseless cells(19), the pool-labeling experiments support theconclusion that the intracellular galactose-1-phosphate is derived from intracellular freegalactose.

In addition to the questionable validity of theinterpretation of the effects of Ni2+ ions and IAAon galactose transport according to the VanSteveninck model, the model has other defi-ciencies. The "permease," although it is definedto be independent of the sugar kinases (hexo-kinase and galactokinase), acts as a phospho-transferase, carrying out the parallel function ofsugar phosphorylation by using polyphosphatedirectly or indirectly (e.g., phosphatidyl glycerol-phosphate derived from polyphosphate) as thephosphate donor (9, 26, 28). The model predictsthat galactokinaseless cells should either be ableto metabolize galactose by the transport-asso-ciated phosphorylation pathway or accumulategalactose by an active process. Neither of theseexpectations has been realized. Galactokinaselesscells transport galactose by a passive, carrier-mediated, facilitated diffusion mechanism. Fur-thermore, in this study it was found that, when"IC-labeled galactose was added to cells preloadedwith an unlabeled pool of galactose and galactose-1-phosphate, the specific activity of the free-sugarpool rose faster than that of the galactose-1-phosphate pool, suggesting that the sugar doesnot enter the cell as the phosphorylated derivative(19). The galactose system would appear to bequite different from the glucose system in whicha similar "precursor test" indicated that bothD-glucose and 2-deoxy-D-glucose are taken upby the cell as the phosphorylated derivative (24,25).The model also proposes that metabolizable

sugars inhibit the uptake of nonmetabolizedsugars by diverting the carrier from the facilitateddiffusion pathway to the active transport pathway.If this were true, it is difficult to understand thecounterflow phenomenon in which the additionof a metabolized sugar to cells preloaded withnonmetabolized sugars causes the nonmetabolizedsugars to leave the cell at the same rate as itsdownhill efflux into a sugar-free medium. Thediversion of the carrier to the active transportpathway should reduce the amount of carrieravailable for influx and efflux via the facilitateddiffusion pathway, unless the carrier efflux process(i.e., Ci -- CO) were proposed to be the same

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KUO AND CIRILLO

irrespective of whether the influx process was viathe passive or the active pathway. However, ifthe return mechanism is the same for both path-ways, it would be difficult to explain the proposeddifference in Vmax for the two pathways. Anotherdifficulty for the model is the fact that initialrate of efflux induced in the counterflow phe-nomenon is the same whether the experiment isperformed with untreated or IAA-inhibited cells.This latter difficulty is a reflection of the fact thatthe Ki for the inhibition of nonmetabolizedsugars by glucose or galactose is the same whetherthe inhibition is measured in IAA-treated oruninhibited cells. According to the model, themetabolizable sugars should be less effectiveinhibitors in IAA-treated cells, since in such cellsglucose and galactose are transported by facili-tated diffusion for which they have a markedlyreduced affinity than for the active transportpathway.

Finally, the observed increased rate of galactoseuptake in preloaded, transferaseless cells con-taining high concentrations of galactose-1-phos-phate suggests that the galactose transport systemcannot be considered to be under the feedbackcontrol of the intracellular sugar phosphateconcentration as proposed by Sols (22) for theglucose system. However, the net rate of sugaruptake and the size of the intracellular free sugarpool are related to the rate of intracellular phos-phorylation in a facilitated diffusion system bythe respective kinetic constants for the transportand phosphorylation reactions, as discussed in aseparate paper (Cirillo, J. Protozool., in press).From the present results, it must be concluded

that whatever validity the Van Steveninck modelmay still have for the constitutive, glucose trans-port system, it does not seem to apply to theinducible galactose system. Furthermore, thepool labeling experiments suggest that the galac-tose system is similar to the glucose system ofAspergillus, in which the free sugar and not thephosphorylated sugar enters the cell (3) andremoves the yeast galactose transport from thetype of phosphotransferase system employedby some bacteria (16, 18).

ACKNOWLEDGMENTS

The authors express their gratitude to Howard C. Douglas ofthe University of Washington School of Medicine in Seattle,Washington, for interest in this study and for generosity in makingavailable invaluable yeast mutants. We also express our gratitudeto Farooq Azam and Michael S. Christensen for assistance withseveral of these experiments.

This investigation was supported by Public Health Servicegrants GM-12743 from the National Institute of General MedicalSciences and FR-0767, from the Division of Research Facilitiesand Resources, and a grant-in-aid from the Graduate School ofthe State University of New York, Stony Brook, N.Y.

LITERATURE CITED

1. Azam, F., and A. Kotyk. 1967. Affinity and capacity of theglucose carrier in different physiological states of a synchro-nous culture of baker's yeast. Folia Microbiol. (Prague)12:115-120.

2. Bray, G. A. 1960. A simple efficient scintillator for countingaqueous samples in a liquid scintillation counter. Anal.Biochem. 1:279-285.

3. Brown, C. W., and A. H. Romano 1969. Evidence againstnecessary phosphorylation during hexose transport inAspergillus nidulans. J. Bacteriol. 100:1198-1203.

4. Burger, M., L. Hejmova, and A. Kleinzeller. 1959. Transportof some mono- and di-saccharides into yeast cells. Biochem.J. 71:233-242.

5. Cirillo, V. P. 1961. Sugar transport in microorganisms. Annu.Rev. Microbiol. 15:197-218.

6. Cirillo, V. P. 1962. Mechanism of glucose transport across theyeast cell membrane. J. Bacteriol. 84:485-491.

7. Cirillo, V. P. 1968. Relationship between sugar structure andcompetition for the sugar transport system in baker's yeast.J. Bacteriol. 95:603-611.

8. Cirillo, V. P. 1968. Galactose transport in Saccharomycescerevisiae. I. Non-metabolized sugars as substrates andinducers of the galactose transport system. J. Bacteriol.95:1727-1731.

9. Dierkauf, F. A., and H. L. Booij. 1968. Changes in the phos-phatide patterns of yeast cells in relation to active carbo-hydrate transport. Biochim. Biophys. Acta 150:214-225.

10. Douglas, H. C., and F. Condie. 1954. The genetic control ofgalactose utilization in Saccharomyces. J. Bacteriol. 68:662-670.

11. Douglas, H. C., and D. C. Hawthorne. 1964. Enzymatic ex-pression and genetic linkage of genes controlling galactoseutilization in Saccharomyces. Genetics 49:837-844.

12. Douglas, H. C., and D. C. Hawthorne. 1966. Regulation ofgenes controlling the synthesis of galactose pathway en-zymes in yeast. Genetics 54:911-916.

13. Fuhrmann, G.-F., and A. Rothstein. 1968. The transport ofZn+, Co+ and Ni+ into yeast cells. Biochim. Biophys.Acta 163:325-330.

14. Fuhrmann, G.-F., and A. Rothstein. 1968. The mechanism ofthe partial inhibition of fermentation in yeast by nickel ions.Biochim. Biophys. Acta 163:331-338.

15. Horowitz, E. B. 1962. A sensitive assay for galactokinase inEscherichia coli. Anal. Biochem. 3:498-513.

16. Kaback, H. R. 1968. The role of the phosphoenolpyruvate-phosphotransferase system in the transport of sugars byisolated membrane preparations of Escherichia coli. J. Biol.Chem. 243:3711-3724.

17. Kotyk, A., and C. Haskovec. 1968. Properties of the sugarcarrier in baker's yeast. III. Induction of the galactose car-rier. Folia Microbiol. (Prague) 13:12-19.

18. Kundig, W., F. D. Kundig, B. Anderson, and S. Roseman.1966. Restoration of active transport of glycosides in Es-cherichia coli by a component of a phosphotransferasesystem. J. Biol. Chem. 241:3243-3246.

19. Kuo, S. C., M. S. Christensen, and V. P. Cirillo. 1970. Galac-tose transport in Saccharomyces cerevisiae. 11. The charac-teristics of galactose uptake and exchange in galactokinase-cells. J. Bacteriol. 103:671-678.

20. Palladini, A. C., and L. F. Leloir. 1952. Studies on uridine-diphosphate-glucose. Biochem. J. 51:426-430.

21. Rothstein, A. 1954. Enzyme systems of the cell surface in-volved in the uptake of sugars by yeast. Symp. Soc. Exp.Biol. 8:165-201.

22. Sols, A. 1967. Regulation of carbohydrate transport andmetabolism in yeast, p. 47-66. In A. K. Mills and Sir H.Krebs (ed.), Aspects of yeast metabolism. BlackwellScientific Publishers, Oxford.

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