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Correlation between resistance to ethidium bromide and changes in monovalent cation uptake in yeast
ARCHIVESOF BIOCHEWTRY AND BIOPHYSICS Vol. 217, No. 1, August, pp. 30-36, 1982
Correlation
between Resistance to Ethidium Bromide and Changes Monovalent Cation Uptake in Yeast’
in
A. BRUNNER, N. CARRASCO, AND A. PEfiA’ Deportamento de Mb&i&gia Centro de Investigaciones en Fisiologia Celulur, and Departamento Bioquimica, Facuhd de Medic&a, Universidad Notional Automma de Mexico, 0.4510 Mexiw,
de
D. F., Mexico
Received September 28, 1981, and in revised form February 25, 1982
A mutant of Kluyveronayces la&k resistant to ethidium bromide was studied and found to have an impairment to transport the dye. As described for other mutants of this kind, the fluorescence changes of the dye that are observed when the cells transport it, were not observed in the mutant strain. Simultaneous to this difficulty to take up the mutagen, the cells showed a diminished ability to take up monovalent cations, as compared to the wild-type strains. The defect of the mutant strain does not seem to reside in the capacity to pump out protons, which also indicates that it has no alterations of the general energy conversion systems. This view is also supported by the fact that the growth yields are similar in both the mutant and the wild-type strains. Both ethidium and K+ fail to stimulate respiration of the mutant yeast when present in the medium, as compared to the wild-type strains. The mutant strain shows a normal cation content, which indicates that the impairment to take up monovalent cations, although much decreased, may still be enough to maintain a normal content of cations within the cells. According to the investigation carried out, the mutant cells seem to be normal, expect for the fact that they are unable to transport both ethidium and K+ from the medium. The data support the hypothesis that ethidium bromide and K+ may be transported by the same system in yeast. The mutagenic properties of ethidium bromide (EB)3 and other intercalating drugs (1) have been studied widely, particularly because of the selectivity of these agents in impairing mitochondrial functions (l-3). The basis for the action of EB seems to be its ability to intercalate with the DNA molecules (4-6), but additional factors may be involved which have to do with particular characteristics of the in-
teraction of EB with mitochondria (7-9). Mutants of Kluyvermyces lads that are resistant to higher concentrations of EB than wild-type strains have been described (10). These mutants, in many cases, show cross resistance to decamethylene biguanidine and to octylguanidine (10-12). These molecules have both shown effects on K+ uptake by yeast (13). Also, a brief report has been published (12) on a difficulty of the mutants to take up the mutagen. Since a series of interactions of the mutagen with the cell have to take place in order to obtain the final effect on mitochondrial DNA (9), several explanations can be given for the resistance to the drug. Besides this, the uptake of EB in yeast cells has been found to show interesting characteristics; it seems that this trans-
* This work was partially supported by Grants 1646, PCCBNAL-790256 and 1669 of the Consejo Nacional de Ciencia y Tecnologia of Mexico. ‘Author to whom correspondence should be sent: Centro de investigaciones en Fisiologa Celular, Apartado Postal 70-600,045lO Mexico, D. F., Mexico. 3Abbreviations used: EB, ethidium bromide; CTAB, cetyltrimethylammonium bromide; TEA, triethanolamine; Mes, 2-(N-morpholino) ethanesulfonic acid. 0003-9861/82/090030-07$02.00/O Copyright All rights
Q 1992 by Academic Press, Inc. of reproduction in any form reserved.
30
ETHIDIUM
RESISTANCE AND CATION UPTAKE
port has some relationship to the system for monovalent cation uptake (14). With all these antecedents, it was of interest to compare some of the interactions of EB with a K. lactis EB-resistant mutant with its parental strains, including the measurement of the dye uptake. Data were found which indicated a relationship between resistance to the drug and an alteration in the ability of the cells to take up monovalent cations. However, the study of several other mutants and revertants indicated that other factors are involved in resistance, but there is a close relation between the capacities of the cells to take up ethidium and Kf.
31
dye, previously equilibrated to the water bath temperature. Aliquots were then taken to separate the cells in a microfuge. The concentration of the remaining dye in the supernatant was measured by the absorbance of the supernatant (475 nm) with that of a standard curve of the dye. The uptake of “Rb+ was measured by incubating the cells with the isotope, filtering, and washing with cold 5 mM RbCl to eliminate trapped and bound 88Rb+. The radioactivity taken up by the cells was measured by means of an end window-gas flow detector or by scintillation counting. To get the values of K,,, and V, the x2 method was used. RESULTS
As previously mentioned a significant difference can be consistently observed between the parental strains and the reMATERIALS AND METHODS sistant mutant, with respect to the ability to grow at higher EB concentrations in the Three strains of K. lactis were employed throughmedium. The mutant used in most of this out most of this work. The two sensitive ones were work, KA6-GA, carries either a single nuW6OOB,designated in the paper as Si, and WM37, clear or several closely linked nuclear mudesignated as Sa. These haploid strains were kindly tations that confer resistance to ethidium supplied by Dr. James R. Mattoon. The strain resisbromide. The mutant was derived from a tant to EB was KA6-8A, and was obtained by a cross cross between WGOOBR:(Y,Adei, AdeP,Leu, between W6OOBR1,a spontaneous mutant of W6OOB, EBR, and WM37: a, His, (10, ll), which is resistant to EB, and WM37. The general characteristics of this type of strain have been described before a mutant resistant to EB reported previ(10, 11). Strain KAG-8A was purified and a resistant ously (lo), and can grow in glycerol meclone selected before initiating the work. Other mudium containing 10 PM EB, whereas sentants from our collection were also used in one set sitive strains can only tolerate 5 PM EB.
of experiments (see Table III). Cells were grown in the medium described by De Kloet et al (15) for approximately 24 h in a gyrotory shaker at 250 rpm. A constant amount of cells from this culture was used, either immediately or after up to 1 week, to inoculate 1 liter of medium, which was incubated in the shaker at 30°C for 24 h. After growth, the cells were collected, washed twice with water by centrifugation, and resuspended in water to give a ratio of 0.5 g of wet weight/ml of the suspension. The cation content of the cells was extracted with 30% perchloric acid. The cation concentrations were then measured in the extracts by flame photometry. K+ movements were monitored by means of a cationic electrode (Beckman 39047) connected to an electrometer, amplifier, and recording system. This same instrument was used to measure H+ concentrations, using a pH combination electrode. Fluorescence changes of EB were measured in a Mark I Farrand spectrofluorometer with a recorder attached. Respiration was followed by means of a Clark oxygen electrode and an adequate power source and recording device. Ethidium bromide uptake was measured by adding the cells to the incubation mixture containing the
EB Uptake by the Wild-Type and the Mutant Strains Since the mutant resistant to EB used in this work (KA6-8A) is different from those employed previously (12), we first determined a defect in EB transport. While EB was taken up normally by the wildtype strains (S, and S,), it is not taken up normally by the mutant. It should be mentioned also that the experimental conditions are somewhat different than those employed before (12), especially because the cells were not starved. After 15 min, the Si, Sz, and R strains transported 792, 577, and 25 nmol. g-’ of EB, respectively. Comparative
Fluorescence Changes of EB
Under the same conditions used to measure the uptake of EB, after the addition of the dye, both wild-type strains
32
BRUNNER, CARRASCO, AND PENA
showed an increase of fluorescence (results not shown). The resistant mutant, on the other hand, did not show any increase of fluorescence aside from the initial one that can be observed in the absence of the cells. This is in full agreement with the data reported for other mutants (12) studied previously. Uptake of Monovalent Cations and Eflects of EB
TABLE I CONTENTS OF SEVERAL CATIONS IN THE THREE STRAINS OF K. lactic USED IN THE WORK pm01 (g of yeast)-’
Strain
K+
Na+
Ca”
Sl SZ R
78-70 65-80 10-14
9.8-10.6 11.0-13.8 7.5-9.3
32-40 22.5-27.5 26.8-22.0
Note. The cations were extracted from a suspension
It has been previously reported that EB containing 250 mg of yeast, with 30% perchloric acid. seems to show a rather specific interaction The contents of the cations were measured by flame with the system for monovalent cation photometry. Results of two experiments are given. transport in yeast (14). This fact suggested the possibility that the EB-resistant mutant, which has an impaired ability to wild-type and the resistant strains when transport the mutagen, might also have the detergent CTAB, that disrupts the a defect in monovalent cation transport. cells, was added (Fig. 1, last three tracThe measurement of the influx of K+ (Fig. ings). Measurements were also made of 1) revealed that the resistant mutant of the content of several cations, including K. luctis was defective when compared K+ in the three strains used. Table I shows with the wild-type strains. The influx of that there was no difference in the content K+ was studied with 1 InM K+ added to the of the three cations determined. Figure 2 incubation medium (First three tracings also shows the ability of both the sensitive of Fig. l), or with endogenous K+ (tracings and the resistant strains to take up %Rb+. 4 to 6 of Fig. 3) with similar results. It is This experiment may give more informaimportant that similar amounts of mono- tion about the in$ux of the monovalent valent cations are released from both the cation. The initial rates of uptake are much higher for the wild-type that for the EB resistant strains. EB and other cationic dyes (16), when added to yeast can produce not only the inhibition of K+ uptake, but also the efflux of the internal cation from the cells, in a p process that requires energy (16-18). Since this effect may be due to an interaction of the dyes with the monovalent transport system of yeast, the effect of EB was tested. Figure 3 shows that especially at the lower EB:yeast ratios, the dye can produce a large efflux of K+ in the wild-type strains, but not in the resistant one. As the amount of cells was decreased, and the amount of dye per cell increased, EB could FIG. 1. K+ movements by the S and R strains of also produce the efflux of the monovalent K. &z&s in a medium containing 1 mkt KC1 (first three cation in the resistant strain. tracings) or with the endogenous content of monoExperiments were carried out to detervalent cations (tracings 4 to 6). Incubation conditions: 20 mM MES-TEA buffer, pH 6.0; 100 mrd glucose; 1.0 mine the kinetic constants of the three strains for the uptake of Rb+. As expected mM or no added KCI; yeast cells, 100 mg; final volume, (Fig. 4), there was a large change, both in 5.0 ml; temperature 25°C. In the last three tracings, substrate was omitted, and only 10 mg of yeast were the Km and the V values of the R strain. added after the inclusion of 400 PM CTAB. A large number of experiments had to be
ETHIDIUM
RESISTANCE
AND CATION
33
UPTAKE
The Proton Pumping Capacity of the Strains
I
+ L
/R 4
8
12
min
FIG. 2. Transport rate of %Rb+ by the S and R strains of K. lactis Incubation: 100 m?d glucose; 20 mM ME&TEA buffer, pH 6.0; 4.0 mM %RbCl yeast, 100 mg; final volume, 2.0 ml; temperature 25°C. After equilibrating the temperature of the medium the incubation was started by the addition of the yeast cells. Then aliquots were taken, filtered, and washed several times with 5 mM RbCl. The filters were dried and counted in scintillation vials.
accumulated to obtain satisfactory correlation coefficients, due to the variability of the results in different experiments.
It has been postulated that yeast cells can transport monovalent cations because of the existence of a proton pump, driven by ATP (19-30), and that a low pH can function only if K+ is added to the medium, but at high values of pH it can function in the absence of cations. It seems that at high pH values, proton pumping can become “uncoupled” from monovalent cation transport, and be coupled, perhaps to anion extrusion by the cells (19). This circumstance offers the possibility of assaying the proton pumping activity of the cells, free from the ion uptake capacity. Although the ability to pump protons is the driving force for monovalent cation uptake, it can work independently. It was decided to study the proton pumping capacity of the cells under conditions where it is uncoupled from monovalent cation Km S,
Vm
mMycq~l~~mg.~m,n~-’ -00.73 -00.39-00.96-21
015 I/[R~+],
FIG. 3. Effects of EB (200 PM) on the endogenous K+ movements of the three strains of K. kzctis in a K+ free medium. Incubation: 8 mM maleate-TEA buffer, pH 6.0; 40 mM glucose; final volume, 5.0 ml; temperature 25°C. Yeast cells were added after equilibrating the temperature of the medium. EB and CTAB were added where indicated at 200 PM concentration.
r
n ’
1.0 mM-’
FIG. 4. Kinetic analysis of the transport of =Rb+ by the S and R strains of K. lactis. Incubation conditions were as for Fig. 2, but yeast cells were added to the medium without %Rb+. After 2 min. the isotope was added, an aliquot was taken after 2 more min, filtered, and treated as described under Materials and Methods.
34
BRUNNER,
CARRASCO,
AND PENA
with Saccharomyces cerewieiae (14), in both wild-type strains used in this work, respiration was stimulated by the addition of EB or K+. Studies on Rev&ants We studied several revertants of our R strain, as well as several other K. Zactis strains, in which it was expected to know if the lesions can exist independently of one another. The data of Table III show that first, resistance is not necessarily equivalent to inability of the cells to transport EB into the cells. There are five strains that are sensitive to EB, with a low transport of the dye. Second, except for one strain, there is a constant absence of the capacity of the cells to transport both EB and monovalent cations, as determined by the uptake of %Rb+. In this latter respect strain KD5 is the exception, because it can transport EB and not Rb+. However, this uptake of EB in strain KD5 is different to that observed in yeast cells (14); it does not require a substrate and it is not affected by 10 mM KCl.
FIG. 5. Measurement of H+ extrusion by the S and R strains of K. kzctis at pH 7.5. Incubation conditions: 6.5 mM MES-TEA buffer, pH 7.5; 135 mM ethanol; approximately 1.5 mM HsOs; yeast, 250 mg; final volume, 6.2 ml; temperature 25°C. Yeast cells of the different strains were added where indicated and the pH was recorded. A calibration curve was constructed by the addition of HCl to the incubation medium.
uptake, i.e., at pH 7.5 with ethanol as substrate, to avoid the extensive formation of CO2 that is produced when glucose is fermented. The results of Fig. 5 show clearly that there are only small differences in the ability of the three strains studied to pump protons.
DISCUSSION
The data presented seem to indicate a close relationship between the transport of monovalent cations and EB by yeast cells, as was suggested before (14). The data in this paper which support this are: (a) the low capacity of a mutant resistant to EB to take up the dye, coincident with a low capacity to take up monovalent cations; (b) the absence of fluorescence
Eflects of EB on Respiratim Another similarity between EB and K+ is revealed in the stimulation that both agents can produce on respiration. This stimulation was also studied in all strains. The results of Table II show that neither EB nor K+ can stimulate respiration in the R mutant. On the other hand, as found TABLE
II
EFFECTSOFEB AND K+ ONRESPIRATIONBYWILD-TYPEANDEB RESISTANTSTRAINSOFK. la&s
Strain
S2 SZ R
Control respiration with no K+ added (natg of Oz. 50 mg-‘)
10 mM KC1
200 PM EB
300 PM EB
740-720 546-795 770-690
1.17-1.17 1.28-1.33 1.03-0.98
1.08-1.05 1.08-1.23 0.98-0.95
1.03-1.05 1.07-1.13 0.95495
Ratio to the control
without
any addition
Note. Incubation: 20 mM MES-TEA buffer, pH 6.0; 108 mM ethanol; yeast cells, 50 mg; final volume, 5.0 ml; temperature 25°C. Respiration rates were measured at 1 min after the addition of yeast. Values of two experiments are presented.
ETHIDIUM
35
RESISTANCE AND CATION UPTAKE TABLE III
CORRELATIONBETWEENTHEABILITYOFSEVERALSTRAINSOFK. l&is AND MONOVALENTCATIONS
TOTRANSPORTEB
Strain
Sensitivity to EB
EB uptake (nmol (1200 mg 15 min)-‘)
%Rb+ uptake (peq (100 mg 5 min)-‘)
KA6-8A (B) R-l a,His,EBS,acrR KA6-8A (B) R-2 a,His,EBS,AcriR KA6-8A(B) R-3 a,His,EBS,AcriR KA6-8A(F)i a,His,EBR,AcriS KA6-8A(F)s a,His,EBR,AcriS KA6-8A a,His,EBS,AcriR KD5 cu,His,EBS,AcriR KA5-4C a,Adez,EBS,AcriR WM 37 a,His,EBS,AcriR W600 B ol,Ade,Adez,Leu,EBS,AcriR KA68A (R) a,His,EBR,AcriS
S S S R R S S S S S R
35 33 46 23 26 32 170 29 176 115 27
0.22 0.21 0.25 0.18 0.19 0.21 0.22 0.22 1.90 2.43 0.19
Note. Incubation conditions: EB transport: 100 mM glucose, 20 mbf MES-TEA buffer, pH 6.0; 266 j,tM EB; yeast cells, 1200 mg; final volume, 6 ml. Aliquots were taken at 15 min, centrifuged in the microfuge for 20 s, and EB remaining in the supernatant was measured by absorbance in the spectrophotometer. @Rb+uptake was measured in the same medium, but in 2.0 ml of final volume, and adding 200 mg of yeast cells. After 5 min, an aliquot was taken and filtered and washed as described under Materials and Methods. The filters were dried and counted.
changes observed for EB in the resistant mutant, as compared with the two wildtype strains; (c) the incapability of EB to produce the efflux of monovalent cations in the resistant mutant within moderate concentration ranges; and (d) the absence of stimulation of respiration produced both by EB and K+ in the mutant, as compared with the normal effect observed in the wild-type strains. It is difficult to postulate a mechanism for the inhibition of monovalent cation uptake produced by EB, especially because of the complex structure of this dye. However, the data of this paper add further support to the idea that the mutagen can be taken up by the cells through the same system as monovalent cations. No other lesion could be demonstrated besides the inability to transport EB and K+ by the R mutant. The kinetic data for the uptake of Rb+, comparing the wild-type and EB resistant strains suggest a profound alteration of the transport system. Starting with the curves of Fig. 4, in which 4 mM Rb+ was used, a concentration well above the Km for the transport of the cation in the wild-
type strains, a large decrease was observed in the rate of transport of the cation. The changes of the kinetic constants for the EB resistant mutant are large, both for Km and V. The content of cations of the mutant, almost normal in comparison with the wild-type strains was rather unexpected (Table I and Fig. 1). However, it must be mentioned that the ability of the cells to transport monovalent cations is very much diminished, but not absent. It is possible that the wild-type strains have an excess capacity to transport monovalent cations, and that in the EB-resistant mutant, the low rate of uptake of monovalent cations may be enough to maintain the normal content of them inside the cells. Besides, the normal content of other cations in the cells is in further agreement with the generally normal conditions of the mutant strain. The data obtained with a series of different strains, as well as revertants of the R strain, indicate that the resistance does not have to be due to the alteration of the cells in their ability to transport the dye. There must be some other lesion that was
36
BRUNNER,
CARRASCO,
not detected and may be worthy of further study. One of the strains (KD5) shown in Table III seems to behave against the rule that EB and monovalent cation uptake are altered simultaneously. However, in the analysis of the characteristics of the transport of EB, it was found that it does not depend on the presence of substrate and it is not affected by Kf, as is the case with yeast cells (14). This means probably that this mutant has some other lesion that increases the uptake of EB by a mechanism different to that described previously. With the exception of the KD5 strain, in all of the other, there was a coincidence between the alteration in the transport of EB and monovalent cations, which supports our view of a close connection between both transport systems. REFERENCES 1. MARKOVICH, H. (1951) Ann. Inst. Pastew 81,452468. 2. BULBER, C. J. E. A. (1964) Antonie van Leeuwnhoek 30,1-g. 3. SLONIMSKI, P., PERRODIN, G., AND CROFT, J. (1968) Biochem Biophys. Res. Commun 30,
232-239. 4. WARING, M. M. (1965) J. MoL Biol 13, 269-282. 5. TSAI, C. C., JAIN, S. C., AND SOBELL, H. M. (1977) J. Mol BioL 114,301-315. 6. JAIN, S. C., TSAI, S. C., AND SOBELL, H. M. (1977) J. MoL BioL 114, 317-331. 7. BASTOS, R. N., AND MAHLER, H. R. (1974) BioL Ch 249, 6617-6627. 8. PEGA, A., CHAVEZ, E., C~RABEZ, A., AND TUENA DE G~MEZ, M. (1977) Arch. Biochem Biophys. 180,522-529. 9. PEGA, A., CLEMENTE, S. M., BORBOLLA, M., CARRASCO,N., AND URIBE, S. (1980) Arch. Biochem Biophys. 201,420-428.
AND PE&A
10. BRIJNNER, A., MAS, J., C~LIS, E., AND MAITOON, J. R. (1973) Biochem Biophys. Res. Commu~ 53,638-644. 11. C&IS, E., MAS, J., AD BRUNNER, A. (1975) Gem%
Res. 25, 59-69. 12. C~LIS, E., MAS, J., AND BRUNNER, A. (1975) FEBS Leti!. 57, 241-244. 13. PEGA, A. (1973) FEBS Lett. 34,117-119. 14. PEGA, A., AND RAM~REZ, G. (1975) J. Membr. BioL 22,369-384. 15. DE KLOET, S. R., VAN WERMESKERKEN, R. K. A., AND KONINGSBERGER, V. V. (1961) Biochim
Biophys. Acta 47,138-143. 16. PENA, A., MORA, M. A., AND CARRASCO, N. (1979) J. Membr. Bid 47, 261-284. 17. PEGA, A. (1978) J. Membr. BioL 42,199-213. 18. BORBOLLA, M., AND PE~A, A. (1980) J. Membr.
BioL 54,149-156. 19. PEGA, A., CINCO, G., G~MEZ-PUYOU, A., AND TIJENA, M. (1972) Arch. Biochem Biophys 153, 413-425. 20. PEGA, A., CINCO, G., G~MEZ-P~ou, A., AND TIJENA, M. (1969) B&him Biophgs. Acta 180, l-8. 21. PE~A, A. (1975) Arch. B&hem, Biophys. 167,397409. 22. PENA, A. (1976) in Mitochondria, Bioenergetics, Biogenesis and Membrane Structure (Packer, L., and Gbmez-Puyou, A., eds.), pp. 21-30, Academic Press, New York. 23. DELHEZ, J., DUFOUR, J. P., THINES, D., AND GOFFEAU, A. (1977) Eur. J. Biochem. 79,319-328. 24. MALPARTIDA, F., AND SERRANO, R. (1980) FEBS Z&t 11,69-72. 25. DUFOUR, J. P., BOUTRY, M., AND GOFFEAU, A. (1980) .I BioL Chem 255, 5735-5741. 26. DUFOUR, J. P., AND GOFFEAU, A. (1980) Eur. J. Biochem 105,145-X4. 27. DUFOUR, J. P., AND GOFFEAU, A. (1980) J. Bid Chem 255, 10,591-10,598. 28. AMORY, A., FOURY, F., AND GOFFEAU, A. (1980)
J. BioL Chem 255,9353-9357. 29. SERRANO, R. (1980) Eur. J. Biochem. 105,419~442. 30. MALPARTIDA, F., AND SERRANO, R. (1981) J. BioL Chem, 256,4175-4177.
Correlation
between Resistance to Ethidium Bromide and Changes Monovalent Cation Uptake in Yeast’
in
A. BRUNNER, N. CARRASCO, AND A. PEfiA’ Deportamento de Mb&i&gia Centro de Investigaciones en Fisiologia Celulur, and Departamento Bioquimica, Facuhd de Medic&a, Universidad Notional Automma de Mexico, 0.4510 Mexiw,
de
D. F., Mexico
Received September 28, 1981, and in revised form February 25, 1982
A mutant of Kluyveronayces la&k resistant to ethidium bromide was studied and found to have an impairment to transport the dye. As described for other mutants of this kind, the fluorescence changes of the dye that are observed when the cells transport it, were not observed in the mutant strain. Simultaneous to this difficulty to take up the mutagen, the cells showed a diminished ability to take up monovalent cations, as compared to the wild-type strains. The defect of the mutant strain does not seem to reside in the capacity to pump out protons, which also indicates that it has no alterations of the general energy conversion systems. This view is also supported by the fact that the growth yields are similar in both the mutant and the wild-type strains. Both ethidium and K+ fail to stimulate respiration of the mutant yeast when present in the medium, as compared to the wild-type strains. The mutant strain shows a normal cation content, which indicates that the impairment to take up monovalent cations, although much decreased, may still be enough to maintain a normal content of cations within the cells. According to the investigation carried out, the mutant cells seem to be normal, expect for the fact that they are unable to transport both ethidium and K+ from the medium. The data support the hypothesis that ethidium bromide and K+ may be transported by the same system in yeast. The mutagenic properties of ethidium bromide (EB)3 and other intercalating drugs (1) have been studied widely, particularly because of the selectivity of these agents in impairing mitochondrial functions (l-3). The basis for the action of EB seems to be its ability to intercalate with the DNA molecules (4-6), but additional factors may be involved which have to do with particular characteristics of the in-
teraction of EB with mitochondria (7-9). Mutants of Kluyvermyces lads that are resistant to higher concentrations of EB than wild-type strains have been described (10). These mutants, in many cases, show cross resistance to decamethylene biguanidine and to octylguanidine (10-12). These molecules have both shown effects on K+ uptake by yeast (13). Also, a brief report has been published (12) on a difficulty of the mutants to take up the mutagen. Since a series of interactions of the mutagen with the cell have to take place in order to obtain the final effect on mitochondrial DNA (9), several explanations can be given for the resistance to the drug. Besides this, the uptake of EB in yeast cells has been found to show interesting characteristics; it seems that this trans-
* This work was partially supported by Grants 1646, PCCBNAL-790256 and 1669 of the Consejo Nacional de Ciencia y Tecnologia of Mexico. ‘Author to whom correspondence should be sent: Centro de investigaciones en Fisiologa Celular, Apartado Postal 70-600,045lO Mexico, D. F., Mexico. 3Abbreviations used: EB, ethidium bromide; CTAB, cetyltrimethylammonium bromide; TEA, triethanolamine; Mes, 2-(N-morpholino) ethanesulfonic acid. 0003-9861/82/090030-07$02.00/O Copyright All rights
Q 1992 by Academic Press, Inc. of reproduction in any form reserved.
30
ETHIDIUM
RESISTANCE AND CATION UPTAKE
port has some relationship to the system for monovalent cation uptake (14). With all these antecedents, it was of interest to compare some of the interactions of EB with a K. lactis EB-resistant mutant with its parental strains, including the measurement of the dye uptake. Data were found which indicated a relationship between resistance to the drug and an alteration in the ability of the cells to take up monovalent cations. However, the study of several other mutants and revertants indicated that other factors are involved in resistance, but there is a close relation between the capacities of the cells to take up ethidium and Kf.
31
dye, previously equilibrated to the water bath temperature. Aliquots were then taken to separate the cells in a microfuge. The concentration of the remaining dye in the supernatant was measured by the absorbance of the supernatant (475 nm) with that of a standard curve of the dye. The uptake of “Rb+ was measured by incubating the cells with the isotope, filtering, and washing with cold 5 mM RbCl to eliminate trapped and bound 88Rb+. The radioactivity taken up by the cells was measured by means of an end window-gas flow detector or by scintillation counting. To get the values of K,,, and V, the x2 method was used. RESULTS
As previously mentioned a significant difference can be consistently observed between the parental strains and the reMATERIALS AND METHODS sistant mutant, with respect to the ability to grow at higher EB concentrations in the Three strains of K. lactis were employed throughmedium. The mutant used in most of this out most of this work. The two sensitive ones were work, KA6-GA, carries either a single nuW6OOB,designated in the paper as Si, and WM37, clear or several closely linked nuclear mudesignated as Sa. These haploid strains were kindly tations that confer resistance to ethidium supplied by Dr. James R. Mattoon. The strain resisbromide. The mutant was derived from a tant to EB was KA6-8A, and was obtained by a cross cross between WGOOBR:(Y,Adei, AdeP,Leu, between W6OOBR1,a spontaneous mutant of W6OOB, EBR, and WM37: a, His, (10, ll), which is resistant to EB, and WM37. The general characteristics of this type of strain have been described before a mutant resistant to EB reported previ(10, 11). Strain KAG-8A was purified and a resistant ously (lo), and can grow in glycerol meclone selected before initiating the work. Other mudium containing 10 PM EB, whereas sentants from our collection were also used in one set sitive strains can only tolerate 5 PM EB.
of experiments (see Table III). Cells were grown in the medium described by De Kloet et al (15) for approximately 24 h in a gyrotory shaker at 250 rpm. A constant amount of cells from this culture was used, either immediately or after up to 1 week, to inoculate 1 liter of medium, which was incubated in the shaker at 30°C for 24 h. After growth, the cells were collected, washed twice with water by centrifugation, and resuspended in water to give a ratio of 0.5 g of wet weight/ml of the suspension. The cation content of the cells was extracted with 30% perchloric acid. The cation concentrations were then measured in the extracts by flame photometry. K+ movements were monitored by means of a cationic electrode (Beckman 39047) connected to an electrometer, amplifier, and recording system. This same instrument was used to measure H+ concentrations, using a pH combination electrode. Fluorescence changes of EB were measured in a Mark I Farrand spectrofluorometer with a recorder attached. Respiration was followed by means of a Clark oxygen electrode and an adequate power source and recording device. Ethidium bromide uptake was measured by adding the cells to the incubation mixture containing the
EB Uptake by the Wild-Type and the Mutant Strains Since the mutant resistant to EB used in this work (KA6-8A) is different from those employed previously (12), we first determined a defect in EB transport. While EB was taken up normally by the wildtype strains (S, and S,), it is not taken up normally by the mutant. It should be mentioned also that the experimental conditions are somewhat different than those employed before (12), especially because the cells were not starved. After 15 min, the Si, Sz, and R strains transported 792, 577, and 25 nmol. g-’ of EB, respectively. Comparative
Fluorescence Changes of EB
Under the same conditions used to measure the uptake of EB, after the addition of the dye, both wild-type strains
32
BRUNNER, CARRASCO, AND PENA
showed an increase of fluorescence (results not shown). The resistant mutant, on the other hand, did not show any increase of fluorescence aside from the initial one that can be observed in the absence of the cells. This is in full agreement with the data reported for other mutants (12) studied previously. Uptake of Monovalent Cations and Eflects of EB
TABLE I CONTENTS OF SEVERAL CATIONS IN THE THREE STRAINS OF K. lactic USED IN THE WORK pm01 (g of yeast)-’
Strain
K+
Na+
Ca”
Sl SZ R
78-70 65-80 10-14
9.8-10.6 11.0-13.8 7.5-9.3
32-40 22.5-27.5 26.8-22.0
Note. The cations were extracted from a suspension
It has been previously reported that EB containing 250 mg of yeast, with 30% perchloric acid. seems to show a rather specific interaction The contents of the cations were measured by flame with the system for monovalent cation photometry. Results of two experiments are given. transport in yeast (14). This fact suggested the possibility that the EB-resistant mutant, which has an impaired ability to wild-type and the resistant strains when transport the mutagen, might also have the detergent CTAB, that disrupts the a defect in monovalent cation transport. cells, was added (Fig. 1, last three tracThe measurement of the influx of K+ (Fig. ings). Measurements were also made of 1) revealed that the resistant mutant of the content of several cations, including K. luctis was defective when compared K+ in the three strains used. Table I shows with the wild-type strains. The influx of that there was no difference in the content K+ was studied with 1 InM K+ added to the of the three cations determined. Figure 2 incubation medium (First three tracings also shows the ability of both the sensitive of Fig. l), or with endogenous K+ (tracings and the resistant strains to take up %Rb+. 4 to 6 of Fig. 3) with similar results. It is This experiment may give more informaimportant that similar amounts of mono- tion about the in$ux of the monovalent valent cations are released from both the cation. The initial rates of uptake are much higher for the wild-type that for the EB resistant strains. EB and other cationic dyes (16), when added to yeast can produce not only the inhibition of K+ uptake, but also the efflux of the internal cation from the cells, in a p process that requires energy (16-18). Since this effect may be due to an interaction of the dyes with the monovalent transport system of yeast, the effect of EB was tested. Figure 3 shows that especially at the lower EB:yeast ratios, the dye can produce a large efflux of K+ in the wild-type strains, but not in the resistant one. As the amount of cells was decreased, and the amount of dye per cell increased, EB could FIG. 1. K+ movements by the S and R strains of also produce the efflux of the monovalent K. &z&s in a medium containing 1 mkt KC1 (first three cation in the resistant strain. tracings) or with the endogenous content of monoExperiments were carried out to detervalent cations (tracings 4 to 6). Incubation conditions: 20 mM MES-TEA buffer, pH 6.0; 100 mrd glucose; 1.0 mine the kinetic constants of the three strains for the uptake of Rb+. As expected mM or no added KCI; yeast cells, 100 mg; final volume, (Fig. 4), there was a large change, both in 5.0 ml; temperature 25°C. In the last three tracings, substrate was omitted, and only 10 mg of yeast were the Km and the V values of the R strain. added after the inclusion of 400 PM CTAB. A large number of experiments had to be
ETHIDIUM
RESISTANCE
AND CATION
33
UPTAKE
The Proton Pumping Capacity of the Strains
I
+ L
/R 4
8
12
min
FIG. 2. Transport rate of %Rb+ by the S and R strains of K. lactis Incubation: 100 m?d glucose; 20 mM ME&TEA buffer, pH 6.0; 4.0 mM %RbCl yeast, 100 mg; final volume, 2.0 ml; temperature 25°C. After equilibrating the temperature of the medium the incubation was started by the addition of the yeast cells. Then aliquots were taken, filtered, and washed several times with 5 mM RbCl. The filters were dried and counted in scintillation vials.
accumulated to obtain satisfactory correlation coefficients, due to the variability of the results in different experiments.
It has been postulated that yeast cells can transport monovalent cations because of the existence of a proton pump, driven by ATP (19-30), and that a low pH can function only if K+ is added to the medium, but at high values of pH it can function in the absence of cations. It seems that at high pH values, proton pumping can become “uncoupled” from monovalent cation transport, and be coupled, perhaps to anion extrusion by the cells (19). This circumstance offers the possibility of assaying the proton pumping activity of the cells, free from the ion uptake capacity. Although the ability to pump protons is the driving force for monovalent cation uptake, it can work independently. It was decided to study the proton pumping capacity of the cells under conditions where it is uncoupled from monovalent cation Km S,
Vm
mMycq~l~~mg.~m,n~-’ -00.73 -00.39-00.96-21
015 I/[R~+],
FIG. 3. Effects of EB (200 PM) on the endogenous K+ movements of the three strains of K. kzctis in a K+ free medium. Incubation: 8 mM maleate-TEA buffer, pH 6.0; 40 mM glucose; final volume, 5.0 ml; temperature 25°C. Yeast cells were added after equilibrating the temperature of the medium. EB and CTAB were added where indicated at 200 PM concentration.
r
n ’
1.0 mM-’
FIG. 4. Kinetic analysis of the transport of =Rb+ by the S and R strains of K. lactis. Incubation conditions were as for Fig. 2, but yeast cells were added to the medium without %Rb+. After 2 min. the isotope was added, an aliquot was taken after 2 more min, filtered, and treated as described under Materials and Methods.
34
BRUNNER,
CARRASCO,
AND PENA
with Saccharomyces cerewieiae (14), in both wild-type strains used in this work, respiration was stimulated by the addition of EB or K+. Studies on Rev&ants We studied several revertants of our R strain, as well as several other K. Zactis strains, in which it was expected to know if the lesions can exist independently of one another. The data of Table III show that first, resistance is not necessarily equivalent to inability of the cells to transport EB into the cells. There are five strains that are sensitive to EB, with a low transport of the dye. Second, except for one strain, there is a constant absence of the capacity of the cells to transport both EB and monovalent cations, as determined by the uptake of %Rb+. In this latter respect strain KD5 is the exception, because it can transport EB and not Rb+. However, this uptake of EB in strain KD5 is different to that observed in yeast cells (14); it does not require a substrate and it is not affected by 10 mM KCl.
FIG. 5. Measurement of H+ extrusion by the S and R strains of K. kzctis at pH 7.5. Incubation conditions: 6.5 mM MES-TEA buffer, pH 7.5; 135 mM ethanol; approximately 1.5 mM HsOs; yeast, 250 mg; final volume, 6.2 ml; temperature 25°C. Yeast cells of the different strains were added where indicated and the pH was recorded. A calibration curve was constructed by the addition of HCl to the incubation medium.
uptake, i.e., at pH 7.5 with ethanol as substrate, to avoid the extensive formation of CO2 that is produced when glucose is fermented. The results of Fig. 5 show clearly that there are only small differences in the ability of the three strains studied to pump protons.
DISCUSSION
The data presented seem to indicate a close relationship between the transport of monovalent cations and EB by yeast cells, as was suggested before (14). The data in this paper which support this are: (a) the low capacity of a mutant resistant to EB to take up the dye, coincident with a low capacity to take up monovalent cations; (b) the absence of fluorescence
Eflects of EB on Respiratim Another similarity between EB and K+ is revealed in the stimulation that both agents can produce on respiration. This stimulation was also studied in all strains. The results of Table II show that neither EB nor K+ can stimulate respiration in the R mutant. On the other hand, as found TABLE
II
EFFECTSOFEB AND K+ ONRESPIRATIONBYWILD-TYPEANDEB RESISTANTSTRAINSOFK. la&s
Strain
S2 SZ R
Control respiration with no K+ added (natg of Oz. 50 mg-‘)
10 mM KC1
200 PM EB
300 PM EB
740-720 546-795 770-690
1.17-1.17 1.28-1.33 1.03-0.98
1.08-1.05 1.08-1.23 0.98-0.95
1.03-1.05 1.07-1.13 0.95495
Ratio to the control
without
any addition
Note. Incubation: 20 mM MES-TEA buffer, pH 6.0; 108 mM ethanol; yeast cells, 50 mg; final volume, 5.0 ml; temperature 25°C. Respiration rates were measured at 1 min after the addition of yeast. Values of two experiments are presented.
ETHIDIUM
35
RESISTANCE AND CATION UPTAKE TABLE III
CORRELATIONBETWEENTHEABILITYOFSEVERALSTRAINSOFK. l&is AND MONOVALENTCATIONS
TOTRANSPORTEB
Strain
Sensitivity to EB
EB uptake (nmol (1200 mg 15 min)-‘)
%Rb+ uptake (peq (100 mg 5 min)-‘)
KA6-8A (B) R-l a,His,EBS,acrR KA6-8A (B) R-2 a,His,EBS,AcriR KA6-8A(B) R-3 a,His,EBS,AcriR KA6-8A(F)i a,His,EBR,AcriS KA6-8A(F)s a,His,EBR,AcriS KA6-8A a,His,EBS,AcriR KD5 cu,His,EBS,AcriR KA5-4C a,Adez,EBS,AcriR WM 37 a,His,EBS,AcriR W600 B ol,Ade,Adez,Leu,EBS,AcriR KA68A (R) a,His,EBR,AcriS
S S S R R S S S S S R
35 33 46 23 26 32 170 29 176 115 27
0.22 0.21 0.25 0.18 0.19 0.21 0.22 0.22 1.90 2.43 0.19
Note. Incubation conditions: EB transport: 100 mM glucose, 20 mbf MES-TEA buffer, pH 6.0; 266 j,tM EB; yeast cells, 1200 mg; final volume, 6 ml. Aliquots were taken at 15 min, centrifuged in the microfuge for 20 s, and EB remaining in the supernatant was measured by absorbance in the spectrophotometer. @Rb+uptake was measured in the same medium, but in 2.0 ml of final volume, and adding 200 mg of yeast cells. After 5 min, an aliquot was taken and filtered and washed as described under Materials and Methods. The filters were dried and counted.
changes observed for EB in the resistant mutant, as compared with the two wildtype strains; (c) the incapability of EB to produce the efflux of monovalent cations in the resistant mutant within moderate concentration ranges; and (d) the absence of stimulation of respiration produced both by EB and K+ in the mutant, as compared with the normal effect observed in the wild-type strains. It is difficult to postulate a mechanism for the inhibition of monovalent cation uptake produced by EB, especially because of the complex structure of this dye. However, the data of this paper add further support to the idea that the mutagen can be taken up by the cells through the same system as monovalent cations. No other lesion could be demonstrated besides the inability to transport EB and K+ by the R mutant. The kinetic data for the uptake of Rb+, comparing the wild-type and EB resistant strains suggest a profound alteration of the transport system. Starting with the curves of Fig. 4, in which 4 mM Rb+ was used, a concentration well above the Km for the transport of the cation in the wild-
type strains, a large decrease was observed in the rate of transport of the cation. The changes of the kinetic constants for the EB resistant mutant are large, both for Km and V. The content of cations of the mutant, almost normal in comparison with the wild-type strains was rather unexpected (Table I and Fig. 1). However, it must be mentioned that the ability of the cells to transport monovalent cations is very much diminished, but not absent. It is possible that the wild-type strains have an excess capacity to transport monovalent cations, and that in the EB-resistant mutant, the low rate of uptake of monovalent cations may be enough to maintain the normal content of them inside the cells. Besides, the normal content of other cations in the cells is in further agreement with the generally normal conditions of the mutant strain. The data obtained with a series of different strains, as well as revertants of the R strain, indicate that the resistance does not have to be due to the alteration of the cells in their ability to transport the dye. There must be some other lesion that was
36
BRUNNER,
CARRASCO,
not detected and may be worthy of further study. One of the strains (KD5) shown in Table III seems to behave against the rule that EB and monovalent cation uptake are altered simultaneously. However, in the analysis of the characteristics of the transport of EB, it was found that it does not depend on the presence of substrate and it is not affected by Kf, as is the case with yeast cells (14). This means probably that this mutant has some other lesion that increases the uptake of EB by a mechanism different to that described previously. With the exception of the KD5 strain, in all of the other, there was a coincidence between the alteration in the transport of EB and monovalent cations, which supports our view of a close connection between both transport systems. REFERENCES 1. MARKOVICH, H. (1951) Ann. Inst. Pastew 81,452468. 2. BULBER, C. J. E. A. (1964) Antonie van Leeuwnhoek 30,1-g. 3. SLONIMSKI, P., PERRODIN, G., AND CROFT, J. (1968) Biochem Biophys. Res. Commun 30,
232-239. 4. WARING, M. M. (1965) J. MoL Biol 13, 269-282. 5. TSAI, C. C., JAIN, S. C., AND SOBELL, H. M. (1977) J. Mol BioL 114,301-315. 6. JAIN, S. C., TSAI, S. C., AND SOBELL, H. M. (1977) J. MoL BioL 114, 317-331. 7. BASTOS, R. N., AND MAHLER, H. R. (1974) BioL Ch 249, 6617-6627. 8. PEGA, A., CHAVEZ, E., C~RABEZ, A., AND TUENA DE G~MEZ, M. (1977) Arch. Biochem Biophys. 180,522-529. 9. PEGA, A., CLEMENTE, S. M., BORBOLLA, M., CARRASCO,N., AND URIBE, S. (1980) Arch. Biochem Biophys. 201,420-428.
AND PE&A
10. BRIJNNER, A., MAS, J., C~LIS, E., AND MAITOON, J. R. (1973) Biochem Biophys. Res. Commu~ 53,638-644. 11. C&IS, E., MAS, J., AD BRUNNER, A. (1975) Gem%
Res. 25, 59-69. 12. C~LIS, E., MAS, J., AND BRUNNER, A. (1975) FEBS Leti!. 57, 241-244. 13. PEGA, A. (1973) FEBS Lett. 34,117-119. 14. PEGA, A., AND RAM~REZ, G. (1975) J. Membr. BioL 22,369-384. 15. DE KLOET, S. R., VAN WERMESKERKEN, R. K. A., AND KONINGSBERGER, V. V. (1961) Biochim
Biophys. Acta 47,138-143. 16. PENA, A., MORA, M. A., AND CARRASCO, N. (1979) J. Membr. Bid 47, 261-284. 17. PEGA, A. (1978) J. Membr. BioL 42,199-213. 18. BORBOLLA, M., AND PE~A, A. (1980) J. Membr.
BioL 54,149-156. 19. PEGA, A., CINCO, G., G~MEZ-PUYOU, A., AND TIJENA, M. (1972) Arch. Biochem Biophys 153, 413-425. 20. PEGA, A., CINCO, G., G~MEZ-P~ou, A., AND TIJENA, M. (1969) B&him Biophgs. Acta 180, l-8. 21. PE~A, A. (1975) Arch. B&hem, Biophys. 167,397409. 22. PENA, A. (1976) in Mitochondria, Bioenergetics, Biogenesis and Membrane Structure (Packer, L., and Gbmez-Puyou, A., eds.), pp. 21-30, Academic Press, New York. 23. DELHEZ, J., DUFOUR, J. P., THINES, D., AND GOFFEAU, A. (1977) Eur. J. Biochem. 79,319-328. 24. MALPARTIDA, F., AND SERRANO, R. (1980) FEBS Z&t 11,69-72. 25. DUFOUR, J. P., BOUTRY, M., AND GOFFEAU, A. (1980) .I BioL Chem 255, 5735-5741. 26. DUFOUR, J. P., AND GOFFEAU, A. (1980) Eur. J. Biochem 105,145-X4. 27. DUFOUR, J. P., AND GOFFEAU, A. (1980) J. Bid Chem 255, 10,591-10,598. 28. AMORY, A., FOURY, F., AND GOFFEAU, A. (1980)
J. BioL Chem 255,9353-9357. 29. SERRANO, R. (1980) Eur. J. Biochem. 105,419~442. 30. MALPARTIDA, F., AND SERRANO, R. (1981) J. BioL Chem, 256,4175-4177.