SUGAR TRANSPORT BY SACCHAROMYCES CEREVISIAE

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Studies of sugar transport into the yeast cell have shown that the ... transport process, and the transported sugar is ... produced by the addition of 1 ml of clarified,.
SUGAR TRANSPORT BY SACCHAROMYCES CEREVISIAE PROTOPLASTS VINCENT P. CIRILLOl Department of Microbiology, Seton Hall College of Medicine and Dentistry, Jersey City, New Jersey

Received for publication July 18, 1962

inoculated with 10 ml of the overnight culture, and incubated at 30 C for 3.5 hr; this provided a culture of actively budding cells necessary for optimal protoplast formation. The yeast from the 3.5-hr culture was harvested at 3,000 X g, washed twice in distilled water, and resuspended in 10 ml of buffered mannitol (15% mannitol in 0.06 M Na2HPO, plus 0.02 M citric acid at pH 5.5). Protoplasts were produced by the addition of 1 ml of clarified, cysteine-treated snail enzyme, and incubated overnight. After overnight incubation, the protoplasts were washed three times in 0.6 M NaCl. Studies of sugar transport into the yeast cell Since 0.6 M NaCl is equivalent in osmotic preshave shown that the transport process involves sure to 1.1 M sugar, it will be referred to as 1.1 an interaction of the transported sugar with the osmolar NaCl. The snail enzyme (Suc digestif d'Helix pomatia) cell surface (Rothstein, 1954; Robertson and Halvorson, 1957; Burger, Hejmova, and Klein- was purchased from L'Industrie Biologique zeller, 1959; Avigad, 1960; Kotyk, 1961; De La Francaise, Sein, France. The digestive juice was Fuente and Sols, 1962; Scharff, 1961; Scharff and clarified by centrifugation at 3,000 X g for 15 Kremer, 1962; and Cirillo, 1961a,b, 1962). The min, and the thiomersalate preservative was present paper shows that yeast protoplasts retain neutralized by the addition of 0.1 volume of 1% the capacity to transport sugars, and that the cysteine. Sugar transport by protoplasts. Sugar transport transported sugar is osmotically free. The cell were carried out by mixing 0.1 ml experiments in involved the not wall is, therefore, directly transport process, and the transported sugar is of yeast protoplasts with 5 ml of 10% L-sorbose in 1.1 osmolar NaCl. The tubes were incubated not significantly bound to intracellular sites. in a water-bath shaker at 30 C. The incubation MATERIALS AND METHODS was stopped by centrifugation at 3,000 X g for Preparation of yeast protoplasts. A pure culture 1 to 2 mia, and the cells were washed with three of Saccharomyces cerevisiae, isolated from a cake 5-ml portions of 1.1 osmolar NaCl at 4 C. Before of Anheuser-Busch baker's yeast, was main- the last wash, a sample of protoplasts was retained on Sabouraud's dextrose agar (Difco). moved for hemocytometer count. The packed Yeast protoplasts were prepared by the pro- cells from the last centrifugation were lysed in cedure described by Marini, Arnow, and Lampen 5 ml of distilled water. A duplicate set of tubes, (1961). The growth from an 18-hr Sabouraud's using whole cells from the same culture from slant was used to inoculate 100 ml of liquid me- which protoplasts were prepared, were also exdium (peptone, 5 g/liter; yeast extract, 3 g/liter; posed to sugar in the same manner. After addiand glucose, 10 g/liter), and grown on a shaker tion of 5 ml of distilled water to the washed cells, overnight at 30 C. Fresh medium (100 ml) was both the lysed protoplasts and the whole cells I U.S. Public Health Service Career Developwere placed in a boiling-water bath for 20 min. The hot-water extracts were clarified by cenment Awardee. 1251

ABSTRACT CIRILLO, VINCENT P. (Seton Hall College of Medicine and Dentistry, Jersey City, N.J.). Sugar transport by Saccharomyces ceremsiae protoplasts. J. Bacteriol. 84:1251-1253. 1962.-By the use of Saccharomyces cerevisiae protoplasts, the L-sorbose transport mechanism has been associated with the protoplast membrane rather than the cell wall. The osmotic fragility of protoplasts in L-sorbose solutions shows that the transported sugar remains osmotically active.

J. BACTERIOL.

CIRILLO

1252 TABLE 1. Sorbose transport by protoplasts Cell type

Whole cells

Protoplasts

Uranyl ion*

Rate of

Inhibition

transportt Ihbto

0 10 100

48 47

0 10 100

33

17 4 0

3 65 88 100

* Expressed as ,umoles per 1010 cells. as mg per 1010 cells per hr.

t Expressed

OSMOLAR/TY OF EXTERNAL NoC/ 0.8 1.0 0.9

0)

0.3 0.1 0.2 OSMOTIC PRESSURE DIFFERENCE FIG. 1. Per cent protoplast lysis in NaCi solutions of different osmotic pressure. The right ordinate and upper abscissa relate cell count as a function of osmolarity of the external medium between 0.7 and 1.1 osmolar NaCl. The left ordinate and lower abscissa relate per cent lysis to the difference in osmotic pressure between cells and medium (assuming that the intracellular osmotic pressure is equal to 1.1 osmolar NaCl).

trifugation, diluted, and assayed for sorbose by the alcoholic anthrone method (Wise et al., 1955).

The rate of sugar transport is expressed as mg of sugar per 1010 cells per hr; 1010 cells are equivalent to 1 ml of packed whole cells. RESULTS AND DISCUSSION

Sugar uptake by protoplasts. The data in Table 1 show that protoplasts retain the capacity to transport L-sorbose at nearly the same rate as whole cells. Since sugar transport by whole cells is quite sensitive to uranyl ion, the sensitivity of sugar transport by protoplasts was compared with that of whole cells. The data in Table 1

show that protoplasts are tenfold more sensitive to uranyl ion than are whole cells. Osmotic activity of the transported sugar. Protoplast cell counts after L-sorbose transport experiments were found to be below the initial counts. This indicated that the transported sugar was osmotically active, and had caused cell lysis due to an increase in intracellular osmotic pressure. The percentage of protoplast lysis could, therefore, give an estimate of the intracellular concentration of osmotically free sugar. To permit such an estimate, the percentage of lysis was determined at decreasing external concentrations of NaCl (Fig. 1). The data of Fig. 1 show that a 0.4 osmolar reduction of the external medium causes 50% lysis. Since the minimal NaCl concentration of the external medium which did not cause cell lysis was 1.1 osmolar, the intracellular osmolarity can be assumed to be equivalent to 1.1 osmolar NaCl. The percentage of lysis of yeast protoplasts in a sorbose solution will, therefore, give the osmotic pressure difference between the intracellular and extracellular environment. When yeast protoplasts were incubated in 1.1 M L-sorbose for 1 hr, lysis was found to be 53%. From Fig. 1, this is equivalent to an osmotic pressure difference of 0.4 M between the protoplasts and the medium. Since L-sorbose is a nonmetabolizable sugar, and the volume of the external medium is so large compared to the cell volume, the external sorbose concentration remains unchanged during the experiment (Cirillo, 1961a, 1962). The internal osmolarity of the protoplasts was, therefore, increased by 0.4 M, and represents the amount of free intracellular sugar. This means that the sugar has reached 36% equilibration with the medium in 1 hr, which is the amount of sorbose uptake predicted by previous experiments in which the intracellular sugar was analyzed chemically (Cirillo, 1962). The transported sugar is, therefore, osmotically free, and not bound to extra- or intracellular sites. Similar results were reported with bacterial protoplasts for sugar (Sistrom, 1958) and organic acid (Wachsman and Storek, 1960) transport. ACKNOWLEDGMENTS

The technical assistance of Marilyn Funkhouser in these experiments is acknowledged with pleasure. It is also a pleasure to acknowledge the assistance of J. 0. Lampen of the Institute

VOL. 84, 1962

L-SORBOSE TRANSPORT IN YEAST PROTOPLAST

of Microbiology, Rutgers University, in the preparation and use of yeast protoplasts. This investigation was supported by a research grant (8443) from the U.S. Public Health Service. LITERATURE CITED AVIGAD, A. 1960. Accumulation of trehalose and sucrose in relation to the metabolism of aglucosides in yeast of defined genotype. Biochim. et Biophys. Acta 40:124-134. BURGER, M., L. HEJMOVA, AND A. KLEINZELLER. 1959. Transport of some monosaccharides into yeast cells. Biochem. J. 71:233-242. CIRILLO, V. P. 1961a. The transport of nonfermentable sugars across the yeast cell membrane, p. 343-351. In A. Kleinzeller and A. Kotyk [ed.], Membrane transport and metabolism. Academic Press, Inc., New York. CIRILLO, V. P. 1961b. The mechanism of sugar transport into the yeast cell. Trans. N.Y. Acad. Sci., Ser. II 23:725-734. CIRILLO, V. P. 1962. Mechanism of glucose transport across the yeast cell membrane. J. Bacteriol. 84:485-491. DE LA FUENTE, G., AND A. SOLS. 1962. Transport of sugars in yeast. II. Mechanisms of utilization of disaccharides and related glycosides. Biochim. et Biophys. Acta 56:49-62. KOTYK, A. 1961. The effect of oxygen on transport phenomena in a respiration-deficient mutant of baker's yeast, p. 352-360. In A. Klein-

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zeller and A. Kotyk [ed.],Membrane transport and metabolism. Academic Press, Inc., New York. MARINI, F., P. ARNOW, AND J. 0. LAMPEN. 1961. The effect of monovalent cations on the inhibition of yeast metabolism by nystatin. J. Gen. Microbiol. 24:51-62. ROBERTSON, J. J., AND H. 0. HALVORSON. 1957. The components of maltozymase in yeast and their behavior during deadaptation. J. Bacteriol. 73:186-198. ROTHSTEIN, A. 1954. Enzyme systems of the cell surface involved in the uptake of sugars by yeast. Symp. Soc. Exptl. Biol. 8:165-201. SCHARFF, T. G. 1961. Evidence for hexose transport in acetone-dried yeast. Arch. Biochem. Biophys. 95:329-335. SCHARFF, T. G., AND E. H. KREMER, III. 1962. A tentative mechanism for the anaerobic transport of glucose, fructose and mannose in yeast. Arch. Biochem. Biophys. 97:192-198. SISTROM, W. B. 1958. On the physical state of the intracellularly accumulated substrates of ,Bgalactoside-permease in Escherichia coli. Biochim. et Biophys. Acta 29:579-587. WACHSMAN, J. T., AND R. STORCK. 1960. Propionate induced lysis of protoplasts of Bacillus megaterium. J. Bacteriol. 80:600-606. WISE, C. S., R. J. DIMLER, H. A. DAVIS, AND C. E. RIST. 1955. Determination of easily hydrolyzable fructose units in dextran preparations. Anal. Chem. 27:33-36.