Folia Microbiol. ~3, 286--291 (1978)
Stimulation of Amino Acid Transport in Saccharomyces cerevisiae by Metabolic Inhibitors J. ttORXK, A. KOTYK and L. l~irtovX Laboratory for Cell Membrane Transport, Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4
Received October 28, 1977
ABSTRACT. Inhibitors of energy metabolism (3-ehlorophenylhydrazonomalononitrile, antimycin A, iodoacetamide, dieyclohexylcarbodiimide) but not of transport (uranyl ions) stimulate a t low concentrations the uptake of L-leucine, L-glutamic acid, L-arginine and, to a lesser degree, of 2-aminoisobutyric acid in Savcha~'omyees cerevisiae. The effect is apparent only after augmenting the energy reserves of cells b y preincubation with D-glucose or, more strikingly, with ethanol. I t is absent in a m u t a n t (opt) lacking the translocation system for A D P - - A T P in mitochondria. The presence of two different energy reserves for amino acid transport is indicated (one in energy-poor, the other in energy.rich cells). The stimulating effect appears to be caused by a retarded degradation of the transport proteins as occurs at a lowered level of mitochondria-produced A_TP.
The practically unidirectional transport of amino acids in baker's yeast ( K o t y k and Rihov~, 1972a) mediated b y at least ten systems of different specificity (e.g., Grenson et al., 1970; K o t y k et al., 197 la) proceeds against considerable concentration gradients b y energy-dependent mechanisms. Some authors (e.g., Seaston-et al., 1973, 1976) assume t h a t the actual energy coupling with transport goes at the expense of a proton gradient (although this is apparently not as simple as with the sodiumnonelectrolyte ,coupling in animal cells). It appeared t h a t more light could be shed on the problem b y investigating the metabolic sources of energy in the context of amino acid transport, a partial aspect of this approach being the application of metabolic inhibitors. In connection with this line of research it was observed t h a t amino acid uptake is anomalously stimulated b y low inhibitor concentrations -the details are the subject of this communication. MATERIALS AND METHODS
A distillery strain of Saccharomyces cerevisiae ~ K o t y k et al., 1971a) and the opt m u t a n t of the same species (a kind gift of Dr. Sublk, Bratislava) were used. The cells were maintained on wort agar slopes and propagated in glucose--yeast extract synthetic media, stationary-phase cells being used after vigorous stirring on a magnetic stirrer for 2 h to deplete the endogenous reserves of the yeast. Transport of amino acids was followed at 30 ~ aerobically in a Dubnoff incubator. After a 1-h preincubation in 1 ~ D-glucose the yeast cells were washed with distilled water, resuspended in water to a density of 10--15 mg dry weight per ml and labelled amino acid with inhibitor and cycloheximide (0.4 m ~ final concentration) were added. Six suspension samples were taken at 30-s intervals, filtered through membrane filters (0.45 ~m pore diameter; Synthesia, Czechoslovakia) and washed twice
A M I N O A C I D T R A N S P O R T I N 8. C E R E V I S I A E
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with 1 ml ice-cold water. Filters with washed cells were placed in scintillation vials containing a toluene scintillation liquid. Counting was done in an Isocap 27: liquid scintillation counter. Chemicals. Antimycin A and dicyclohexylcarbodiimide (DCCD) were from Serva (FRG), 3-chlorophenylhydrazonomalononitrile (commercially carbonyl cyanide mchlorophenylhydrazone; CCCP) from Sigma (USA) and cycloheximide (Actidione) from the Upjohn Co. (USA). All other chemicals were of reagent-grade purity and were supplied by Lachema (Czechoslovakia). Uniformly 14C-labelled amino acids were obtained from the Institute for Research, Production and Uses of Radioisotopes (Czechoslovakia), 2-aminoisobutyric acid from the Radiochemical Centre (Great Britain). RESULTS
To obtain more quantitative information on inhibitor effects (older data can be found in Kotyk et al., 1971b) different inhibitor concentrations were applied and the initial rate of uptake was studied. Table I shows that all the metabolic inhibitors used, i.e. CCCP (uncoupler of oxidative phosphorylation), antimycin (respiratory chain inhibitor), DCCD (inhibitor of H+-translocating adenosinetriphosphatase), iodoacetamide (inhibitor of glycolysis) and uranyl ions (inhibitor of membrane transport of amino acids) inhibit the uptake of L-leucine (and other amino acids; see below) at relatively high concentrations. On the other hand, with the exception of uranyl ions, they stimulate the uptake at rather low concentrations. The stimulation as such is not additive, combinations of inhibitors resulting always in a relative depression (Table II). This suggests that the inhibitors affect a common effeetor of amino acid transpol~. The nonspecific action of the inhibitors was further supported by their effect on the TABL~ I. Effect of inhibitors on t h e initial rate of u p t a k e of 10 m:~ L-leucine Inhibitor
Concentration
~M
100
None
CCCP
DCCD
Antimycin
Percent uptake
0.1 1 10 20 200 2000 0.3 3 30
127.9 111.8 29.9 130.1 107.4 49.3 127.7 132.4 30.8
Iodoacetamide
5 50 500
122.4 111.0 77.7
I~ranyl n i t r a t e
1 100 10000
94.3 24.5 23.9
288
J . HOR/~K E T AL.
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T ~ L E II. Effects of inhibitor combinations in s t i m u l a t i n g concentrations on the initial rate of u p t a k e of 10 m ~ L-leucine Inhibitor
Percent uptake
~-ono 0.1 pLM CCCP q- 0.3 ~ a n t i m y c i n 0.1 [z~ CCCP q- 20 ~ZMDCCD 20 ~M DCCD + 0.3 ~M a n t i m y c i n
100 112 93 104
half-saturation constant K T of transport. The values obtained over the concentration range from 0.25 to 5 mM b y plotting according to Eisenthal and Cornish-Bowden (1975) are summarized in Table III. Both in the stimulating and in the inhibitory concentrations of inhibitors, the KT is practically unaffected. The exceptions formed b y uranyl ions and, to a lesser degree, by DCCD are a p p a r e n t l y due to their action directly at the membrane. TABLE I I L Effect of inhibitors on the KT of u p t a k e of I,-leucine Inhibitor
KT
Concentration ~
m~
0 0.1 10 0.3 30 100 20 2000
0.29 0.23 0.27 0.29 0.31 1.38 0.19 0.81
None CCCP CCCP AJatimyein Antimyein Uranyl nitrate DCCD DCCD
I t followed from earlier experiments (Hors and K o t y k , 1977) that in an Arrhenius plot of the m a x i m u m rate of uptake there is a break, possibly corresponding to the transition point of membrane lipids, at 18--20 ~ The position of this point, arrived at b y measurements at 2 ~ intervals between 13 and 31 ~ is not markedly altered b y any of the inhibitors tested; likewise, the apparent activation energies of L-leucine t r a n s p o r t remain at 31-- 40 k J mo1-1 above and at 78--87 k J nlo1-1 below the transition t e m p e r a t u r e (Table IV). TABLE IV. Effect of inhibitors on the activition energy Ea a n d t h e transition t e m p e r a t u r e Tt of u p t a k e of 10 m ~ L-leucine
Ea Inhibitor
Tt ~
k J tool-I above Tt
~one 0.I ~
CCOP
0.3 ~tM a n t i m y c i n 20 ~zM DCCD
19 18 18 18
34 3.1 33 40
!
below Tt 82 82 78 87
1978
AMINO ACID TRANSPORT
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TABLE V. Effect of i n h i b i t o r s on t h e initial r a t e s of u p t a k e o f 10 mM a m i n o a c i d s u t i l i z i n g s y s t e m s o t h e r t h a n t h e g e n e r a l a m i n o acid p e r m e a s e (in ,ereent of control w i t h o u t inhibitor) Inhibitor
Concentration
P e r c e n t u p t a k e of
~z~ 0.1 1 10
CCCP
20 200 2000
DCCD
Antimycin
0.3 3 30
argminc
g l u t a m i e acid
AIB a
146 116 114
116 110 103
141 140 92
126 117 91
108
112 114 20
129 124 46
105
a A I B 2 - a m i n o i s o b u t y r i c acid.
To answer the possible objection that the stimulating effect is specific for t h e general amino acid permease and m a y have to do with a derepression of its synthesis (this transport system probably is the only one transporting leucine in baker's yeast) representatives of other transport systems were tested, viz. L-glutamic acid, L-arginine, and 2-aminoisobutyric acid. The effects oftheinhibitors are much the same as with leucine (Table V). tABLE VI. Effect o f i n h i b i t o r s on t h e initial r a t e of u p t a k e of.10 mM L-leucine a f t e r different p r e i n c u b a t i o n s (in p e r c e n t of c o n t r o l w i t h o u t inhibitor) P r e i n c u b a t e d in Inhibitor
0.1 y.M CCCP 0.3 ~ a n t i m y e i n 20 tz~ D C C D
~vater
1 % D-glucose
1% ethanol
94 90 103
128 128 130
156 166 171
Table VI shows the stimulation of the initial rate of L-leucine transport after a 1-h preincubation with D-glucose (as used in the rest of the experiments), with ethanol (to emphasize the role of mitochondrially produced energy reserves) and in water (where only some "basal" t y p e of energy reserve is present). The stimulating effects are seen to be the more pronounced the greater the role of mitochondrial energy production. It should be noted, however, that in absolute terms preincuTABLE V I I . I n i t i a l r a t e of u p t a k e of 10 m ~ n-leucine in t h e opl m u t a n t in t h e p r e s e n c e o f i n h i b i t o r s (in p e r c e n t o f c o n t r o l w i t h o u t inhibitor) Inhibitor
0.3 ~ a n t i m y c i n 0.1 tim CCCP 20 ~M D C C D
Percent uptake
85 100 80
29@ J. HORA-K E T A L .
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TABLE V I I I . Initial r a t e of u p t a k e of 10 mM T.-leucine in t h e presence of dicyclohexylcarbodiimide (DCCD) Concentration of DCCD
W i t h o u t eycloheximide
W i t h 0.4 m ~ eyeloheximide
0 20 ~ 2000 ~M
100 102 68
100 139 73
bation with glucose results in much higher uptake rates than preincubation with ethanol (17 times and 8 times, respectively, compared with the control preincubated in water). The role of mitoehondrial energy transformation (concretely in the form stored in ATP) is further stressed b y the results obtained with the opl mutant. This m u t a n t lacks the A D P - - A T P trans]oeating system so that A T P produced in mitochondria is unavailable for processes taking place in the eytosol or elsewhere in the cell. Table V I I indicates that the stimulating effects of inhibitors are absent in this mutant. DISCUSSION
The amount of available data notwithstanding (cf. Seaston et al., 1973, 1976) there is no clear evidence on either the ultimate source of energy for amino acid transport in yeast or on its actual coupling with energy at the molecular level. Earlier studies ( K o t y k and l~ihovA, 1972b) showed a significant correlation between the amount of acid-insoluble high-energy phosphate (most of it is highmolecular weight polyphosphate located in plasma membranes) and the capacity of amino acid transport. At a later date (Hors and Kotyk, 1977) it appeared that an amino acid m a y be driven uphill b y two different sources of energy, one of them apparently b e i n g a mitoehondrial product. There are indications that the energy source produced or made available after preincubation with glucose (in a great part) or ethanol (exclusively) m a y be mitochondrial A T P as in the opl m u t a n t the corresponding stimulation of subsequent uptake is absent. Transport without preincubation with an energy source is much less sensitive to the presence of functional mitochondrial ATP-producing systems. This is parallelled, among other things, b y the high temperature effect on glucose-stimulated transport and a low one on the nonstimulated transport (HorAk and K o t y k , 1977). The new finding made here that metabolic inhibitors at low concentrations stimulate leucine uptake is again restricted to the preincubated cells, and, even more strikingly, the stimulation occurs after preincubation with ethanol (i.e., after a more or less exclusive production of A T P at the mitoehondrial level). Again significantly, it does not appear in the oiD1 mutant. Likewise, in the case of 2-aminoisobutyric acid it is very little pronounced, in keeping with the finding t h a t the uptake of A I B is not much stimulated b y preincubation with glucose (by mere 50 %; Hors and K o t y k , 1977). The stimulation itself is a perplexing problem at present. As it is caused b y a variety of inhibitors acting at different levels one is forced to speculate that the only function which t h e y all can ultimately affect is the production of (mitochondrial) ATP. Two explanations m a y be offered here. I f an energy source (say, glucose or its derivative) is metabolized energy can be usefully stored in two ways, one in
1978
AMINO ACID T R A N S P O R T I N S. C E . R E V I S I A E
2?|
ATP mainly produced in the mitochondria, the other in polyphosphate which is formed (or elongated) in a reaction branching off from glycolysis at the site of 1,3-diphosphoglyceric acid (cf. Kulaev, 1975). If the flow of material to the mitochondria is blocked by low inhibitor concentrations more would be channelled into the polyphosphate branch. I f this happens to be the source of energy for amino acid transport an increase would be easily explained. However, this explanation has the drawback that stimulation is observed even with CCCP (an uncoupler which increases the flow of substrate through the mitochondrial respiratory chain) and with iodoacetamide which acts primarily at the level of glyceraldehyde-phosphate dehydrogenase (i.e., before the branching off toward polyphosphate). The other explanation (and the one favoured here) is that the degradation of transport systems (apparently by intracellular proteases) is stimulated by the presence of mitochondrial ATP. Inhibitors of its production may thus retard the degradation and, in the presence of cycloheximide when no renewal of the degraded proteins is possible this is reflected in a relatively higher capacity of these proteins (involved in amino acid transport in this particular case) than in the absence of inhibitors. This view is substantially supported by the experiment shown in Table VIII where no stimulation of leucine transport is observed in the absence of cycloheximide. The hypothesis that mitochondrial ATP may regulate the degradation of proteins is strengthened by an early observation (Poncovs and Kotyk, 1968) that low concentrations of glucose retard while those of 2-deoxy-D-arabino-hexose stimulate amino acid uptake. Likewise, de-induction of an inducible maltose (Alonso and Kotyk, 1978) and trehalose (Kotyk and Michaljani6ovs in press) transport proteins in yeast in the presence of cycloheximide is greatly enhanced by the presence of glucose. It thus appears that the stimulating effect of low inhibitor doses on amino acid transport is a consequence of a slower breakdown of the responsible transport protein(s). REFERENCES ALONSO A., KOTYK A.: A p p a r e n t half-lives of sugar t r a n s p o r t proteins in baker's yeast. Folia 2klicrobiol. 23, 118 (1978). EISEN~AL R., CORNISH-BOWDEN A.: The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 139, 717 (1974). GBENSO~r M., H o u C., CRXBEEL M. : Multiplicity of the amino acid permeases in Saccharomyces cerevisiar IV. Evidence for a general amino acid pormease. J. BacterioL 103, 770 (1970). H O R ~ J., KOTX~ A.: Temperature effects in amino acid t r a n s p o r t b y Saccharomyccs cerevisiae. Exp. Mycology 1, 63 (1977). KOTYK A., PONEC M., ~i~OVA[ L.: U p t a k e of amino acids b y actidione-treated yeast cells. I. Specificity of carriers. Felix* MicrobioL 16, 432 (1971a). KOTYK A., I~fHOVA[ L., PO~r M.: U p t a k e of amino acids b y aetidione-treated yeast cells. II. Effect of incubation conditions a n d metabolic inhibitors. Folia Microbiot. 16, 445 (1971b). KOTYK A., l~igo~r L. : Transport of ce-aminoisobutyrlc acid in Saccharomyces cerevisiae. Feedback control. Biochi~n. Biophys. Acta 288, 380 (1972a). KOTYK A., ~IHOVA~ L.: Energy requirements for amino acid u p t a k e in Saccharomyces cerevisiae. Folia Microbiol. 17, 353 (1972b). KOTY~ A., MIC~-LJANI~O~rX D.: Transport of trehalose in baker's yeast. J. Gen. Microbiol. in press. KULAEV I. S. : Biochemistry of inorganic polyphosphate. Rev. Physiol. Biochem. Pharmacol. 73, 131 (1975). PONCOVA~ M., KOTYK A. : I n t e r a c t i o n of amino acids a n d sugars in t r a n s p o r t in Saccharomyces cerevisiae. Folia MicrobioL 13, 529 (1968). SEASTON A., I~rKSO~r C., EDDY A. A.: The absorption of protons with specific amino acids a n d carbohydrates b y yeast. Biochem. J. 134, 1031 (1973). SEASTON A., C~RR G., EDDY A. A.: The concentration of glycine b y preparations of the yeast Saccharomyces carlsbergensis depleted of adenosine triphosphate. Effect of p r o t o n gradients a n d uncoupling agents. Biochem. J. 154, 669 (1976).
Stimulation of Amino Acid Transport in Saccharomyces cerevisiae by Metabolic Inhibitors J. ttORXK, A. KOTYK and L. l~irtovX Laboratory for Cell Membrane Transport, Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4
Received October 28, 1977
ABSTRACT. Inhibitors of energy metabolism (3-ehlorophenylhydrazonomalononitrile, antimycin A, iodoacetamide, dieyclohexylcarbodiimide) but not of transport (uranyl ions) stimulate a t low concentrations the uptake of L-leucine, L-glutamic acid, L-arginine and, to a lesser degree, of 2-aminoisobutyric acid in Savcha~'omyees cerevisiae. The effect is apparent only after augmenting the energy reserves of cells b y preincubation with D-glucose or, more strikingly, with ethanol. I t is absent in a m u t a n t (opt) lacking the translocation system for A D P - - A T P in mitochondria. The presence of two different energy reserves for amino acid transport is indicated (one in energy-poor, the other in energy.rich cells). The stimulating effect appears to be caused by a retarded degradation of the transport proteins as occurs at a lowered level of mitochondria-produced A_TP.
The practically unidirectional transport of amino acids in baker's yeast ( K o t y k and Rihov~, 1972a) mediated b y at least ten systems of different specificity (e.g., Grenson et al., 1970; K o t y k et al., 197 la) proceeds against considerable concentration gradients b y energy-dependent mechanisms. Some authors (e.g., Seaston-et al., 1973, 1976) assume t h a t the actual energy coupling with transport goes at the expense of a proton gradient (although this is apparently not as simple as with the sodiumnonelectrolyte ,coupling in animal cells). It appeared t h a t more light could be shed on the problem b y investigating the metabolic sources of energy in the context of amino acid transport, a partial aspect of this approach being the application of metabolic inhibitors. In connection with this line of research it was observed t h a t amino acid uptake is anomalously stimulated b y low inhibitor concentrations -the details are the subject of this communication. MATERIALS AND METHODS
A distillery strain of Saccharomyces cerevisiae ~ K o t y k et al., 1971a) and the opt m u t a n t of the same species (a kind gift of Dr. Sublk, Bratislava) were used. The cells were maintained on wort agar slopes and propagated in glucose--yeast extract synthetic media, stationary-phase cells being used after vigorous stirring on a magnetic stirrer for 2 h to deplete the endogenous reserves of the yeast. Transport of amino acids was followed at 30 ~ aerobically in a Dubnoff incubator. After a 1-h preincubation in 1 ~ D-glucose the yeast cells were washed with distilled water, resuspended in water to a density of 10--15 mg dry weight per ml and labelled amino acid with inhibitor and cycloheximide (0.4 m ~ final concentration) were added. Six suspension samples were taken at 30-s intervals, filtered through membrane filters (0.45 ~m pore diameter; Synthesia, Czechoslovakia) and washed twice
A M I N O A C I D T R A N S P O R T I N 8. C E R E V I S I A E
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with 1 ml ice-cold water. Filters with washed cells were placed in scintillation vials containing a toluene scintillation liquid. Counting was done in an Isocap 27: liquid scintillation counter. Chemicals. Antimycin A and dicyclohexylcarbodiimide (DCCD) were from Serva (FRG), 3-chlorophenylhydrazonomalononitrile (commercially carbonyl cyanide mchlorophenylhydrazone; CCCP) from Sigma (USA) and cycloheximide (Actidione) from the Upjohn Co. (USA). All other chemicals were of reagent-grade purity and were supplied by Lachema (Czechoslovakia). Uniformly 14C-labelled amino acids were obtained from the Institute for Research, Production and Uses of Radioisotopes (Czechoslovakia), 2-aminoisobutyric acid from the Radiochemical Centre (Great Britain). RESULTS
To obtain more quantitative information on inhibitor effects (older data can be found in Kotyk et al., 1971b) different inhibitor concentrations were applied and the initial rate of uptake was studied. Table I shows that all the metabolic inhibitors used, i.e. CCCP (uncoupler of oxidative phosphorylation), antimycin (respiratory chain inhibitor), DCCD (inhibitor of H+-translocating adenosinetriphosphatase), iodoacetamide (inhibitor of glycolysis) and uranyl ions (inhibitor of membrane transport of amino acids) inhibit the uptake of L-leucine (and other amino acids; see below) at relatively high concentrations. On the other hand, with the exception of uranyl ions, they stimulate the uptake at rather low concentrations. The stimulation as such is not additive, combinations of inhibitors resulting always in a relative depression (Table II). This suggests that the inhibitors affect a common effeetor of amino acid transpol~. The nonspecific action of the inhibitors was further supported by their effect on the TABL~ I. Effect of inhibitors on t h e initial rate of u p t a k e of 10 m:~ L-leucine Inhibitor
Concentration
~M
100
None
CCCP
DCCD
Antimycin
Percent uptake
0.1 1 10 20 200 2000 0.3 3 30
127.9 111.8 29.9 130.1 107.4 49.3 127.7 132.4 30.8
Iodoacetamide
5 50 500
122.4 111.0 77.7
I~ranyl n i t r a t e
1 100 10000
94.3 24.5 23.9
288
J . HOR/~K E T AL.
Vol, 23
T ~ L E II. Effects of inhibitor combinations in s t i m u l a t i n g concentrations on the initial rate of u p t a k e of 10 m ~ L-leucine Inhibitor
Percent uptake
~-ono 0.1 pLM CCCP q- 0.3 ~ a n t i m y c i n 0.1 [z~ CCCP q- 20 ~ZMDCCD 20 ~M DCCD + 0.3 ~M a n t i m y c i n
100 112 93 104
half-saturation constant K T of transport. The values obtained over the concentration range from 0.25 to 5 mM b y plotting according to Eisenthal and Cornish-Bowden (1975) are summarized in Table III. Both in the stimulating and in the inhibitory concentrations of inhibitors, the KT is practically unaffected. The exceptions formed b y uranyl ions and, to a lesser degree, by DCCD are a p p a r e n t l y due to their action directly at the membrane. TABLE I I L Effect of inhibitors on the KT of u p t a k e of I,-leucine Inhibitor
KT
Concentration ~
m~
0 0.1 10 0.3 30 100 20 2000
0.29 0.23 0.27 0.29 0.31 1.38 0.19 0.81
None CCCP CCCP AJatimyein Antimyein Uranyl nitrate DCCD DCCD
I t followed from earlier experiments (Hors and K o t y k , 1977) that in an Arrhenius plot of the m a x i m u m rate of uptake there is a break, possibly corresponding to the transition point of membrane lipids, at 18--20 ~ The position of this point, arrived at b y measurements at 2 ~ intervals between 13 and 31 ~ is not markedly altered b y any of the inhibitors tested; likewise, the apparent activation energies of L-leucine t r a n s p o r t remain at 31-- 40 k J mo1-1 above and at 78--87 k J nlo1-1 below the transition t e m p e r a t u r e (Table IV). TABLE IV. Effect of inhibitors on the activition energy Ea a n d t h e transition t e m p e r a t u r e Tt of u p t a k e of 10 m ~ L-leucine
Ea Inhibitor
Tt ~
k J tool-I above Tt
~one 0.I ~
CCOP
0.3 ~tM a n t i m y c i n 20 ~zM DCCD
19 18 18 18
34 3.1 33 40
!
below Tt 82 82 78 87
1978
AMINO ACID TRANSPORT
I N S. C E R E V I S I A E
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TABLE V. Effect of i n h i b i t o r s on t h e initial r a t e s of u p t a k e o f 10 mM a m i n o a c i d s u t i l i z i n g s y s t e m s o t h e r t h a n t h e g e n e r a l a m i n o acid p e r m e a s e (in ,ereent of control w i t h o u t inhibitor) Inhibitor
Concentration
P e r c e n t u p t a k e of
~z~ 0.1 1 10
CCCP
20 200 2000
DCCD
Antimycin
0.3 3 30
argminc
g l u t a m i e acid
AIB a
146 116 114
116 110 103
141 140 92
126 117 91
108
112 114 20
129 124 46
105
a A I B 2 - a m i n o i s o b u t y r i c acid.
To answer the possible objection that the stimulating effect is specific for t h e general amino acid permease and m a y have to do with a derepression of its synthesis (this transport system probably is the only one transporting leucine in baker's yeast) representatives of other transport systems were tested, viz. L-glutamic acid, L-arginine, and 2-aminoisobutyric acid. The effects oftheinhibitors are much the same as with leucine (Table V). tABLE VI. Effect o f i n h i b i t o r s on t h e initial r a t e of u p t a k e of.10 mM L-leucine a f t e r different p r e i n c u b a t i o n s (in p e r c e n t of c o n t r o l w i t h o u t inhibitor) P r e i n c u b a t e d in Inhibitor
0.1 y.M CCCP 0.3 ~ a n t i m y e i n 20 tz~ D C C D
~vater
1 % D-glucose
1% ethanol
94 90 103
128 128 130
156 166 171
Table VI shows the stimulation of the initial rate of L-leucine transport after a 1-h preincubation with D-glucose (as used in the rest of the experiments), with ethanol (to emphasize the role of mitochondrially produced energy reserves) and in water (where only some "basal" t y p e of energy reserve is present). The stimulating effects are seen to be the more pronounced the greater the role of mitochondrial energy production. It should be noted, however, that in absolute terms preincuTABLE V I I . I n i t i a l r a t e of u p t a k e of 10 m ~ n-leucine in t h e opl m u t a n t in t h e p r e s e n c e o f i n h i b i t o r s (in p e r c e n t o f c o n t r o l w i t h o u t inhibitor) Inhibitor
0.3 ~ a n t i m y c i n 0.1 tim CCCP 20 ~M D C C D
Percent uptake
85 100 80
29@ J. HORA-K E T A L .
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TABLE V I I I . Initial r a t e of u p t a k e of 10 mM T.-leucine in t h e presence of dicyclohexylcarbodiimide (DCCD) Concentration of DCCD
W i t h o u t eycloheximide
W i t h 0.4 m ~ eyeloheximide
0 20 ~ 2000 ~M
100 102 68
100 139 73
bation with glucose results in much higher uptake rates than preincubation with ethanol (17 times and 8 times, respectively, compared with the control preincubated in water). The role of mitoehondrial energy transformation (concretely in the form stored in ATP) is further stressed b y the results obtained with the opl mutant. This m u t a n t lacks the A D P - - A T P trans]oeating system so that A T P produced in mitochondria is unavailable for processes taking place in the eytosol or elsewhere in the cell. Table V I I indicates that the stimulating effects of inhibitors are absent in this mutant. DISCUSSION
The amount of available data notwithstanding (cf. Seaston et al., 1973, 1976) there is no clear evidence on either the ultimate source of energy for amino acid transport in yeast or on its actual coupling with energy at the molecular level. Earlier studies ( K o t y k and l~ihovA, 1972b) showed a significant correlation between the amount of acid-insoluble high-energy phosphate (most of it is highmolecular weight polyphosphate located in plasma membranes) and the capacity of amino acid transport. At a later date (Hors and Kotyk, 1977) it appeared that an amino acid m a y be driven uphill b y two different sources of energy, one of them apparently b e i n g a mitoehondrial product. There are indications that the energy source produced or made available after preincubation with glucose (in a great part) or ethanol (exclusively) m a y be mitochondrial A T P as in the opl m u t a n t the corresponding stimulation of subsequent uptake is absent. Transport without preincubation with an energy source is much less sensitive to the presence of functional mitochondrial ATP-producing systems. This is parallelled, among other things, b y the high temperature effect on glucose-stimulated transport and a low one on the nonstimulated transport (HorAk and K o t y k , 1977). The new finding made here that metabolic inhibitors at low concentrations stimulate leucine uptake is again restricted to the preincubated cells, and, even more strikingly, the stimulation occurs after preincubation with ethanol (i.e., after a more or less exclusive production of A T P at the mitoehondrial level). Again significantly, it does not appear in the oiD1 mutant. Likewise, in the case of 2-aminoisobutyric acid it is very little pronounced, in keeping with the finding t h a t the uptake of A I B is not much stimulated b y preincubation with glucose (by mere 50 %; Hors and K o t y k , 1977). The stimulation itself is a perplexing problem at present. As it is caused b y a variety of inhibitors acting at different levels one is forced to speculate that the only function which t h e y all can ultimately affect is the production of (mitochondrial) ATP. Two explanations m a y be offered here. I f an energy source (say, glucose or its derivative) is metabolized energy can be usefully stored in two ways, one in
1978
AMINO ACID T R A N S P O R T I N S. C E . R E V I S I A E
2?|
ATP mainly produced in the mitochondria, the other in polyphosphate which is formed (or elongated) in a reaction branching off from glycolysis at the site of 1,3-diphosphoglyceric acid (cf. Kulaev, 1975). If the flow of material to the mitochondria is blocked by low inhibitor concentrations more would be channelled into the polyphosphate branch. I f this happens to be the source of energy for amino acid transport an increase would be easily explained. However, this explanation has the drawback that stimulation is observed even with CCCP (an uncoupler which increases the flow of substrate through the mitochondrial respiratory chain) and with iodoacetamide which acts primarily at the level of glyceraldehyde-phosphate dehydrogenase (i.e., before the branching off toward polyphosphate). The other explanation (and the one favoured here) is that the degradation of transport systems (apparently by intracellular proteases) is stimulated by the presence of mitochondrial ATP. Inhibitors of its production may thus retard the degradation and, in the presence of cycloheximide when no renewal of the degraded proteins is possible this is reflected in a relatively higher capacity of these proteins (involved in amino acid transport in this particular case) than in the absence of inhibitors. This view is substantially supported by the experiment shown in Table VIII where no stimulation of leucine transport is observed in the absence of cycloheximide. The hypothesis that mitochondrial ATP may regulate the degradation of proteins is strengthened by an early observation (Poncovs and Kotyk, 1968) that low concentrations of glucose retard while those of 2-deoxy-D-arabino-hexose stimulate amino acid uptake. Likewise, de-induction of an inducible maltose (Alonso and Kotyk, 1978) and trehalose (Kotyk and Michaljani6ovs in press) transport proteins in yeast in the presence of cycloheximide is greatly enhanced by the presence of glucose. It thus appears that the stimulating effect of low inhibitor doses on amino acid transport is a consequence of a slower breakdown of the responsible transport protein(s). REFERENCES ALONSO A., KOTYK A.: A p p a r e n t half-lives of sugar t r a n s p o r t proteins in baker's yeast. Folia 2klicrobiol. 23, 118 (1978). EISEN~AL R., CORNISH-BOWDEN A.: The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 139, 717 (1974). GBENSO~r M., H o u C., CRXBEEL M. : Multiplicity of the amino acid permeases in Saccharomyces cerevisiar IV. Evidence for a general amino acid pormease. J. BacterioL 103, 770 (1970). H O R ~ J., KOTX~ A.: Temperature effects in amino acid t r a n s p o r t b y Saccharomyccs cerevisiae. Exp. Mycology 1, 63 (1977). KOTYK A., PONEC M., ~i~OVA[ L.: U p t a k e of amino acids b y actidione-treated yeast cells. I. Specificity of carriers. Felix* MicrobioL 16, 432 (1971a). KOTYK A., I~fHOVA[ L., PO~r M.: U p t a k e of amino acids b y aetidione-treated yeast cells. II. Effect of incubation conditions a n d metabolic inhibitors. Folia Microbiot. 16, 445 (1971b). KOTYK A., l~igo~r L. : Transport of ce-aminoisobutyrlc acid in Saccharomyces cerevisiae. Feedback control. Biochi~n. Biophys. Acta 288, 380 (1972a). KOTYK A., ~IHOVA~ L.: Energy requirements for amino acid u p t a k e in Saccharomyces cerevisiae. Folia Microbiol. 17, 353 (1972b). KOTY~ A., MIC~-LJANI~O~rX D.: Transport of trehalose in baker's yeast. J. Gen. Microbiol. in press. KULAEV I. S. : Biochemistry of inorganic polyphosphate. Rev. Physiol. Biochem. Pharmacol. 73, 131 (1975). PONCOVA~ M., KOTYK A. : I n t e r a c t i o n of amino acids a n d sugars in t r a n s p o r t in Saccharomyces cerevisiae. Folia MicrobioL 13, 529 (1968). SEASTON A., I~rKSO~r C., EDDY A. A.: The absorption of protons with specific amino acids a n d carbohydrates b y yeast. Biochem. J. 134, 1031 (1973). SEASTON A., C~RR G., EDDY A. A.: The concentration of glycine b y preparations of the yeast Saccharomyces carlsbergensis depleted of adenosine triphosphate. Effect of p r o t o n gradients a n d uncoupling agents. Biochem. J. 154, 669 (1976).
