Fluorescent measurement of the intracellular pH during sporulation of Saccharomyces cerevisiae

FEMS Microbiology Letters 153 (1997) 17^23

Fluorescent measurement of the intracellular pH during sporulation of Saccharomyces cerevisiae Juan Carlos Aon a

a ;b

, Sonia Cortassa b * ;

è gicas (INSIBIO, CONICET-UNT), Departamento de Bioqu| èmica de la Nutricio è n, Instituto Superior de Investigaciones Biolo

èmica Biolo è gica `Dr. Bernabe è Bloj', Facultad de Bioqu| èmica, Qu| èmica y Farmacia, Universidad Nacional de Tucuma è n, Instituto de Qu| è n, Argentina San Miguel de Tucuma

b

è gico de Chascomu è s (INTECH), Casilla Correo 164, 7130 Chascomu è s, Buenos Aires, Argentina Instituto Tecnolo

Received 6 January 1997; revised 6 April 1997; accepted 6 April 1997

Abstract

This work reports the intracellular pH (pHi ) dynamics of Saccharomyces cerevisiae cells in sporulation medium. Cells loaded with the pH-sensitive dye carboxy-seminaphthorhodafluor-1 (C.SNARF-1) exhibited an alkalization of the pHi following the extracellular pH during sporulation in the absence of buffer and almost no change in pHi or vpH when sporulation was carried out in buffered medium. The results indicate that the pH gradient does not appear to be directly involved in the regulation of acetate uptake during sporulation. However, the alkalization of pHi by eliciting a decrease in metabolic fluxes could account for a lower demand for acetate. Keywords : Saccharomyces cerevisiae

; Fluorescent dye; Intracellular pH; Sporulation

1. Introduction

During sporulation the yeast Saccharomyces ceredisplays a typical pattern of metabolic £uxes [1,2]. S. cerevisiae cells of various strains exhibited a maximum in acetate consumption rate 4^8 h after transfer to sporulation medium and an abrupt decrease thereafter. Additionally, we observed a correlation of the acetate consumption rate during exposure to sporulation medium with the frequency of asci per cell [1]. We attempted to ¢nd out how the acetate consumption rate is regulated. The question arose whether the observed decrease of substrate visiae

* Corresponding author. Tel.: +54 (241) 24049; Fax: +54 (241) 24048; E-mail: [email protected]

consumption after 8 h in sporulation medium was associated with the concomitant medium alkalization. Thus, we performed our sporulation experiments in bu¡ered medium. The results indicate that the acetate consumption rate in bu¡ered sporulation medium followed the same time course as in nonbu¡ered medium although the absolute values were larger [1]. Then, our results in bu¡ered medium did not con¢rm the conjecture that the decrease in acetate uptake would occur due to the alkalization of sporulation medium. The hypothesis has been put forward that some H‡ acetate symport may accomplish, at least in part, the transport of acetate [3], although it is generally accepted that this substrate would enter the cell in its protonated form by passive di¡usion, the driving force being the gradient across

0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 1 8 4 - 5

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J.C. Aon, S. Cortassa / FEMS Microbiology Letters 153 (1997) 17^23

the plasma membrane. If there is an acetate symport, the transport would be dependent on the proton motive force and, more precisely, on its vpH component. In this work, we investigated the dynamics of the intracellular pH (pHi ) in the time lapse of the increase and subsequent decrease in acetate consumption rate. Besides, as a large alkalization of the extracellular medium was registered, we wondered how yeast cells manage to keep the pHi within the physiological range. The measurement of pHi was based upon a pHdependent £uorescent signal of cells loaded with £uorescein derivatives [4]. The advantage of this method as a tool for characterizing the pHi is that it is rapid, inexpensive and simple. From all the £uorescent compounds tested up to now, it appears that carboxy-seminaphthorhoda£uor-1 (C.SNARF-1) is the most suitable dye to measure pHi in S. cerevisiae cells [5]. However, other £uorescent compounds may also be suitable. Recently, the relationship between cellular viability and pHi has been investigated in S. cerevisiae cells loaded with 5- and 6-carboxy£uorescein [6] and the proton £uxes were studied with pyranine loaded yeast cells [7]. Fluorescent pH determinations have the disadvantage that the £uorescent intensity of the signal depends on many parameters and variables, apart from pHi . Among those parameters are the concentration of the dye, which in turn depends on the C.SNARF-1.acetoxymethyl ester (C.SNARF-1.AM) uptake capacity and de-esteri¢cation rate of the probe by the cells, variations in the lamp strength, bleaching e¡ects and many other factors hard to control. To overcome these drawbacks the ratio of £uorescence emission intensity between two wavelengths is usually measured [5]. This ratio makes it possible to calibrate the cell suspension in order to estimate the pHi from the £uorescence ratio. 2. Materials and methods

2.1. Growth and sporulation conditions S. cerevisiae strains were grown in yeast nitrogen base medium plus 1% (w v31 ) potassium acetate supplemented with uracil (20 Wg ml31 ) and 0.1% yeast extract. The pH of the medium was adjusted to 4.5.

The cultures were performed in £asks under a gas: liquid phase ratio of 10:1, in an orbital shaker thermostated at 30³C. Yeast cultures of di¡erent S. cerevisiae strains grown to mid-exponential phase, as described above, were harvested by centrifugation, washed twice with sterile water, and resuspended in sporulation medium at a cell density of 2U107 cells ml31 . The sporulation medium contained 1% (w v31 ) potassium acetate supplemented with 0.1% yeast extract. Uracil was added at a concentration of 5 mg ml31 . The pH of the medium was adjusted at the beginning of the incubation to 7.0 with KOH. The cells were incubated in Erlenmeyer £asks containing a 10:1 ratio of gas:liquid phase, in an orbital shaker thermostated at 30³C. 2.2. Incorporation and leakage of the £uorescent compound C.SNARF.1 into yeast cells

Loading of S. cerevisiae cells with C.SNARF1.AM was carried out by addition of the compound to the growth medium followed by incubation during 12 h. The usual loading protocols used by various authors [5] with cells resuspended in bu¡ered medium did not result in a signi¢cant increase in the £uorescence. However, cellular esterases were able to hydrolyze the acetomethoxy ester as could be judged by the increase in £uorescence registered upon incubation of the esteri¢ed compound with a CH1211 S. cerevisiae cell extract. After the incubation period the cells were harvested and resuspended in sporulation medium as described in Section 2.1. The loaded cells resuspended in either bu¡ered or non-bu¡ered sporulation medium lose less than 25% of the initial £uorescence associated with cells after 24 h of incubation. Even 3^4 days later cells appeared as £uorescent under the microscope. 2.3. Fluorescence measurements

Intact or Triton X-100 permeabilized cells were diluted to a concentration of 1U107 cells ml31 either in bu¡ered sporulation medium or in 0.2 M HEPES/ KOH and transferred to the spectro£uorometer cuvette. The emission of £uorescence in the range 550^ 700 nm was recorded upon excitation at 535 nm in an Aminco-Bowman SPF spectro£uorometer (Silver Spring, USA) working with 10 nm wavelength width.

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Fig. 1. pH sensitivity of C.SNARF-1 staining of S. cerevisiae cells. A: Fluorescence emission spectra at pH 5.3 and 8.2. C.SNARF-1.AM loaded cells were permeabilized with 0.2% Triton X-100 and analyzed for their £uorescence emission in the wavelength range 550^700 nm after excitation at 535 nm. B: Cells loaded with the £uorescent pH indicator and exposed to sporulation conditions during 10 h were subjected to microscopic examination after dilution in sporulation medium bu¡ered at pH 7.0. Cells were photographed by £uorescence emission after excitation with blue light in a Cannon photomicroscope epi£uorescence at 100U magni¢cation with a Kodak 400 ASA ¢lm. 2.4. Microscopy of £uorescent loaded cells

3. Results and discussion

Cells loaded with the £uorescent pH indicator were diluted in sporulation medium bu¡ered at pH 7.0 and visualized after excitation with blue light in a Cannon epi£uorescence photomicroscope. Photomicrographs were taken at 40 or 100U magni¢cation with a Kodak 400 ASA ¢lm.

When S. cerevisiae strain CH1211 was incubated with 20 WM C.SNARF-1.AM in MOPS or HEPES bu¡er for up to 5 h, negligible £uorescence was observed associated with the cells. However, permeabilized cells were able to hydrolyze the acetoxymethyl ester bond of the £uorescent compound which resulted in an increase in £uorescence (results not

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Fig. 2. Ratio of £uorescence emission intensity of 580 over 610 nm. A solution of C.SNARF-1 obtained after incubation of C.SNARF1.AM with permeabilized cells was subjected to changes in the medium pH and its £uorescence intensity registered as described in the text (¢lled squares). Cells loaded with the £uorescent compound were permeabilized, resuspended in 0.2 M HEPES/KOH and subjected to successive pH changes in the extracellular medium (open squares). Continuous and dashed lines belong to the best ¢t of experimental points based on Eq. 1 (see text).

shown). This result suggested that in the presence of bu¡er the C.SNARF-1.AM uptake rate of this particular yeast strain was extremely low. Thus, the cells were loaded with the esteri¢ed form of C.SNARF-1 during 12 h growth in minimal medium with acetate as carbon source under the conditions described in Section 2. This procedure yielded £uorescently labelled yeast cells as can be observed in Fig. 1B. After incubation, the cells were harvested and resuspended in potassium acetate sporulation medium at a cell density of 2U107 cells ml31 , as previously described [1]. That the dye was distributed inside the cell and not adsorbed to the cell wall was con¢rmed by the fact that digestion of the cell wall of the loaded cells with lyticase (Sigma Chemical Co.) released 15^20% of the £uorescence which may be due to some cell lysis occurring during cell wall digestion. To corroborate whether the internalized £uorescent dye was sensitive to pH changes, cells were permeabilized by treatment with 0.2% Triton X-100, permeabilized cells were diluted to a concentration of 1U107 cells ml31 either in bu¡ered sporulation medium or in 0.2 M HEPES/KOH and transferred to a spectro£uorometer cuvette. The emission of £uorescence in the range 550^700 nm was recorded upon excitation at 535 nm. Fig. 1A shows the spectrum of the £uorescent compound when the external

pH was adjusted to 5.3 or 8.2. The spectrum of C.SNARF-1 exhibited a peak of £uorescence emission at 580 nm with a shoulder at 610 nm (Fig. 1A). The emission at 580 nm decreased at alkaline pH and the shoulder at 610 nm increased markedly (Fig. 1A). To evaluate pH sensitivity of the £uorescent ratio of C.SNARF-1 incorporated into yeast cells, a permeabilized cell suspension resuspended in bu¡ered sporulation medium was subjected to successive pH changes (Fig. 2). At each pH value the £uorescence intensity was recorded at 580 and 610 nm. The continuous and dashed lines in Fig. 2 correspond to the ¢tting of the experimental points with an equation based on the acid-base equilibrium of £uorescent species, ruled by the following equation taken from [8]: Ro R1 1 Ratio Ro 3 1 10 pK 3pH ˆ

‰

…

‡

‡

…

†

0

†

… †

Š

where Ro

ˆ

FIP C:SNARF ÿ 1 at 580 nm FIP C:SNARF ÿ 1 at 610 nm

(FIP=£uorescence intensity of protonated),

FEMSLE 7612 20-10-97

2

… †

J.C. Aon, S. Cortassa / FEMS Microbiology Letters 153 (1997) 17^23

21

Fig. 3. Intracellular and extracellular pH changes during exposure of yeast cells to sporulation conditions. Cells loaded with C.SNARF-1 were collected at mid-exponential phase of growth on acetate and transferred to sporulation medium in the absence (A) or presence (B) of 0.2 M HEPES/KOH pH 7.0. Samples were taken at regular intervals to monitor the £uorescence intensity ratio between 580 and 610 nm. From the latter £uorescence ratio, the pHi was determined by applying Eqs. 1^4. Intracellular (¢lled squares), extracellular pH (empty squares) and pH gradient across the membrane (¢lled diamonds) are mean values þ S.E.M. from duplicate determinations of two independent experiments.

R1

ˆ

cell suspension exhibited the same shape as that

FII C:SNARF ÿ 1 at 580 nm FII C:SNARF ÿ 1 at 610 nm

…3†

(Fig. 2). In order to calculate the ratio, we assumed that the absolute values of the £uorescence intensity

(FII=£uorescence intensity of ionized), and

  FII C:SNARF ÿ 1 at 610 nm pK ˆ pK log FIP C:SNARF ÿ 1 at 610 nm 0

3

shown by a solution of de-esteri¢ed C.SNARF-1

at both 580 nm and 610 nm increased by the same …4†

factor since they were higher in permeabilized cells than in intact cells. The increase in the £uorescence upon addition of Triton X-100 agreed with previous

with pK=7.33.

reports of an increase in £uorescence intensity of As the pH was increased, the £uorescence ratio at

C.SNARF-1 upon addition of saponin [5]. Both

580 nm in relation to 610 nm of the permeabilized

curves exhibited a decreasing ratio between pH 6.5

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and 8.5 which could be adjusted by a typical pH law in which the pK of the C.SNARF-1 would be the same within cells and in solution but the maximal and minimal ratios achieved at extreme pH (lower than 6 and higher than 9) were di¡erent (see Eqs. 1^4 above). The dynamics of the pHi during sporulation conditions was investigated by loading cells with C.SNARF-1.AM during growth on minimal medium. Cells were harvested at mid-exponential phase and transferred to potassium acetate sporulation medium where the progress of the £uorescence intensity ratio was followed from 1 to 10 h. Fig. 3 shows the pHi time course of S. cerevisiae cells challenged with sporulation medium from time zero in the absence (panel A) or presence (panel B) of 0.2 M HEPES/ KOH, pH 7.0. Previously, we showed that in buffered potassium acetate medium the ¢nal sporulation frequency was the same as in the absence of bu¡er but the appearance of asci was delayed in the presence of bu¡er [1]. The pHi of yeast cells followed the same alkalization tendency exhibited by the extracellular medium both in the presence and in the absence of bu¡er. In the presence of bu¡er, pHi increased less than the extracellular pH (pHo ) resulting in a small decrease in vpH. In the absence of bu¡er, the pHi increased from pH 7.4 to 8.2. The pH gradient across the membrane inverted after pHi V7.5 (Fig. 3A). In fact, at 3, 6 and 9.5 h of exposure to sporulation conditions, the gradient was negative, i.e. more acidic inside, indicating intracellular accumulation of H‡ . On the other hand, in the presence of buffered sporulation medium the vpH component of the proton motive force was always positive and relatively constant (Fig. 3B). Concerning the question motivating this work, the results obtained do not support the idea that the decrease in the acetate consumption rate might be due to a decrease in the vpH fueling its transport. Both in the presence and in the absence of bu¡er, the time courses of acetate consumption rate were similar but with di¡erences in the absolute values [1]. In the same period, in which ¢rst an increase followed by a decrease of acetate uptake was recorded, no apparent changes were observed in the pH gradient across the plasma membrane (Fig. 3). Moreover, the vpH in the presence of bu¡er was relatively constant

over the same time span where acetate uptake varied widely (see Fig. 3 and [1]). However, alkalization of cytoplasm could bring about decreased metabolic rates. In fact, an increase in pHi could be one of the mechanism explaining the decrease observed in anabolic £uxes, namely through gluconeogenesis [1,2]. Thus, pHi could indirectly regulate acetate uptake through a decrease in the demand of acetate for anabolism. This point deserves further research. The consumption of acetate by yeast is followed by a release of CO2 produced in catabolism [9]. Such a release of CO2 is important to avoid the huge alkalinization that would otherwise occur due to the consumption of a weak acid, such as acetate, in the ¢rst hour after transfer to non-bu¡ered sporulation medium. According to the rate of O2 consumption determined in sporulating cultures, either loaded with the £uorescent compound or unloaded, it could be estimated that all the CO2 produced by the operation of the tricarboxylic acid cycle should be dissolved in the medium to bu¡er pHo at the observed value (results not shown). This CO2 would avoid further alkalization produced by the disappearance of the acetate from the sporulation medium. On the other hand, in bu¡ered medium, we calculated that approximately 50% of the CO2 produced should stay in the medium to account for the measured pHo values. The inversion of the pH gradient observed in sporulation medium in the absence of bu¡er would certainly a¡ect the transport of those molecules requiring a proton motive force for their uptake, e.g. amino acids. The results of this work provide a likely explanation for the decrease in incorporation of radiolabelled precursors of proteins and nucleic acids during the progress of sporulation [10,11]. This decreased incorporation could, however, be overcome by addition of bu¡er [11] which keeps the pH gradient relatively constant at least during 10 h of exposure to the sporulation medium (Fig. 3B). Previous reports showed heterogeneous distribution of the £uorescent probe [4,5]. It has been observed with £uorescent dyes, such as £uoresceins and diacetyl£uorescein, that the pH in the immediate neighbourhood of the plasma membrane was relatively similar to the pHo in contrast with the alkaline center of the cell [4]. However, in the presence of

FEMSLE 7612 20-10-97

J.C. Aon, S. Cortassa / FEMS Microbiology Letters 153 (1997) 17^23 bu¡ered medium this heterogeneous pH distribution disappears within 15 to 20 min. Besides, and

microspectro£uorimetric

31

techniques

cans

[12].

Under

our

experimental

indicated

Candida albi-

conditions,

we

were unable to distinguish whether di¡erent populations would exhibit di¡erent internal pH. However, microscopic examination of the £uorescent labelled cells

revealed

a

rather

homogeneous

color

in

cell

cytoplasm (Fig. 1B). In agreement with previous results [5], we observed exclusion of the dye from the vacuole

and

a

relative

homogeneous

staining

Taken together, these data indicate that it is quite

vpH

[1] Aon,

J.C.

and

sporulation of

Cortassa,

S.

(1996)

Metabolic

Saccharomyces cerevisiae

rates

during

on acetate. Antonie

van Leeuwenhoek 69, 257^265. [2] Aon, J.C., Rapisarda, V.A. and Cortassa, S. (1996) Metabolic rates

regulate

cerevisiae.

the

success

of

sporulation

Saccharomyces

in

Exp. Cell Res. 222, 157^162.

[3] Serrano, R. (1991) Transport across yeast vacuolar and plasma membranes. In : Genome Dynamics, Protein Synthesis and Energetics. The Molecular and Cellular Biology of the Yeast

Saccharomyces

(Broach, J.R., Pringle, J.R. and Jones, E.W.,

Eds.), Vol. 1, pp. 523^585. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [4] Slavik, J. and Kotyk, A. (1984) Intracellular pH distribution and transmembrane pH pro¢le of yeast cells. Biochim. Bio-

throughout the cytoplasm.

unlikely that the

References

P-NMR

the existence of cell populations with di¡erent internal pH in germinating blastospores of

23

across the plasma membrane

phys. Acta 766, 679^684. [5] Haworth, R.S., Lemire, B.D., Crandall, D., Cragoe, E.J. and Fliegel, L. (1991) Characterization of proton £uxes across the

Saccharomyces cerevisiae.

plays a role in the decrease of the acetate consump-

cytoplasmic membrane of the yeast

tion rate observed during sporulation. Also, either

Biochim. Biophys. Acta 1098, 79^89.

passive or facilitated di¡usion appears unrelated to the pH changes in the extra- or intracellular medium

[6] Imai, T. and Ohno, T. (1995) The relationship between viability and intracellular pH in the yeast

siae.

Saccharomyces cerevi-

Appl. Environ. Microbiol. 61, 3604^3608.

suggesting that the decrease in acetate consumption

[7] Pen ì a, A., Ramirez, J., Rosas, G. and Calahorra, M. (1995)

rate may obey some intracellular regulation ; among

Proton pumping and the internal pH of yeast cells, measured

them, the high pHi could play a role in triggering a

with

general decrease of metabolic £uxes, such as the gluconeogenic £ux which may in turn decrease the demand of acetate.

pyramine

introduced

by

electroporation.

J.

Bacteriol.

177, 1017^1022. [8] Optiz, N., Merten, E. and Acker, H. (1994) Evidence for redistribution-associated intracellular p

K

shifts of the pH-sensi-

tive £uoroprobe carboxy-SNARF-1. P£u ë gers Arch. 427, 332^ 342. [9] Duggan, P.F. (1964) The uptake of potassium by bakers yeast

Acknowledgments

from potassium acetate. Biochim. Biophys. Acta 88, 223^224. [10] Curiale, M.S., Petryna, M.M. and Mills, D. (1976) Ribonucleic acid synthesized in meiotic cells of

The

authors

are

grateful

to

Dr.

M.A.

Aon

for

S. cerevisiae :

e¡ect of

culture medium pH. J. Bacteriol. 126, 661^667.

suggestions and critical reading of the manuscript.

[11] McCusker, J.H. and Haber, J.E. (1977) E¤cient sporulation

This work was supported by a grant of the Funda-

of yeast in media bu¡ered near pH 6. J. Bacteriol. 132, 180^

è n Antorchas to S. Cortassa. S.C. is a career memcio ber

of

the

`Consejo

Nacional

de

Investigaciones

185. [12] Rabaste, F., Sancelme, M., Delort, A.M., Blais, J. and Bolart, J. (1995) Intracellular pH of

Candida albicans

blastospores as

è¢cas y Te è cnicas' (CONICET, Argentina) and Cient|

measured by laser microspectro£uorimetry and

J.C.A. is a fellow of CONICET.

ochim. Biophys. Acta 1268, 41^49.

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31

P-NMR. Bi-