Journal of General Microbiology (1982), 128, 2303-2312.
Printed in Great Britain
2303
The Requirement of Oxygen for the Active Transport of Sugars into Yeasts
By J . A. B A R N E T T * A N D A . P. S I M S
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U . K .
(Received 25 February 1982)
The inducible systems of uptake of P-D-galactopyranosides into Kluyveromyces fragilis, Kluyveromyces marxianus and Debaryumyces polymorphus were studied under aerobic and anaerobic
conditions, using both lactose and the non-metabolized analogue, methyl 1-thio-P-D-galactopyranoside (TMG). A common carrier served both substrates, and aerobic entry was by active
transport, involving proton symport. The rate of uptake and the final equilibrium concentration
were decreased on adding N,N’-dicyclohexylcarbodiimide,diethylstilbestrol or antimycin A,
and active transport was completely abolished by carbonyl cyanide m-chlorophenylhydrazone.
Uptake under anaerobic conditions differed markedly from that occurring aerobically : TMG
was not concentrated anaerobically, even by strongly fermenting yeasts. Instead, it was transported into the cells by facilitated diffusion, which could sustain a rate of entry of over half the
maximum rate observed aerobically. That this difference between aerobic and anaerobic transport into yeasts might apply to glycosides in general, was suggested by the finding that a strain of
Saccharomyces cerevisiae also took up maltose by active transport under aerobic conditions, but
by facilitated diffusion anaerobically. By contrast, the amino acid, 2-aminoisobutyric acid, was
concentrated by Kluyveromyces fragilis, even under anaerobic conditions. The significance of
these findings in relation to the Kluyver effect is discussed.
INTRODUCTION
All species of yeasts are reported to utilize D-glucose aerobically and nearly half of these can
ferment it anaerobically, probably to carbon dioxide and ethanol. By contrast, although many
species grow aerobically on disaccharides, only a few can ferment these anaerobically. For
example, although more than half the yeast species can use such substrates as maltose,
a,a-trehalose or cellobiose aerobically (three disaccharides composed solely of D-glucose
monomers), only 10% or fewer can ferment them (Barnett, 1976, 1981).
Such observations led Sims & Barnett (1978) to investigate the Kluyver effect, namely, that
certain yeasts can utilize particular disaccharides aerobically, but not anaerobically, although
these yeasts can use one or more of the component hexoses anaerobically. These authors
suggested an explanation for the Kluyver effect in terms of the requirement of oxygen for entry
of these sugars into many yeasts and, accordingly, have now investigated the effects of changes
between aerobic and anaerobic conditions on the transport of certain glycosides into four yeasts.
The present paper describes the results of this investigation.
METHODS
Yeasts,growth and preparation of suspensions. The following yeasts were used : Kluyveromyces fragilis NCYC 100
(lactose, aerobic growth
fermentation +); Kluyveromyces marxianus NCYC 111 (lactose, aerobic growth
fermentation - ) ; Debaryomyces polymorphus CBS 4349 (lactose, aerobic growth , fermentation - ) ; Succharomyces cerevisiae, a hex2 mutant (SMC-1 B/3) with unregulated maltose uptake (Entian, 1980). (The name Kluyuero-
+,
~~
+
+,
~~
Abbreviations: AIB, 2-aminoisobutyric acid; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD,
N,N’-dicyclohexylcarbodiimide; SHAM, salicylhydroxamic acid ; TMG, methyl 1-thio-P-D-galactopyranoside;
TPP+, tetraphenylphosphonium ion.
0022-1287/82/0001-0430 $02.00 01982 SGM
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J . A . B A R N E T T A N D A . P . SIMS
myces fragilis has been retained here to avoid confusion, although this species is now included in Kluyveromyces
marxianus.)
The yeasts were maintained on slopes of Difco Bacto YM agar. For studies of the lactose transport system, the
yeasts were grown on Difco Bacto Yeast Nitrogen Base without amino acids (N-base) (pH 4.9, to which D
glucose, succinate or lactose was added at a concentration of 25 mM. Strain SMC-1 B/3 was cultivated on a medium
of Difco Bacto yeast extract (1 %, w/v), Difco Bacto peptone (2%, w/v) and 650 mM-ethanol (Entian, 1980). For
measuring amino acid uptake, Kluyveromycesfragilis was grown on Difco Bacto Yeast Carbon Base with 8 mMglutamate. Incubations were at 25 "C, each in 200 ml medium contained in a conical flask (500 ml capacity),
agitated at 300 rev. min-' in a New Brunswick incubator.
Washed yeast suspensions were usually prepared from exponentially growing cells, as described by Sims &
Barnett (1978).
Anaerobic conditions. Anaerobic incubations were in a water-jacketed glass chamber of about 50 ml total
capacity, with a tight-fitting rubber stopper (Suba-Seal) covered with a layer of silicone oil. Through the SubaSeal, the chamber was gassed with argon (about 5 ml min-l) by means of a cannula, and a hypodermic needle
provided an exit port. Fine plastic tubing, attached to the cannula, bubbled the argon from the bottom of the vessel
through the suspending medium, which was stirred with a Teflon-covered magnet. An oxygen electrode, placed
low in the vessel, monitored the oxygen concentration. The output from the oxygen electrode (Beckman 39065
Polarographic Oxygen Sensor) was amplified so that oxygen concentrations of >0.15 nmol ml-1 could be
measured reliably. A side-arm, with Suba-Seal port, provided for sampling by syringe. Commercial argon,
nominally >99.999% pure, was passed through a Nilox de-oxygenator (Jencons, Heme1 Hempstead, Herts.)
before use.
Measurements of sugar and amino acid transport. Anaerobic uptake of radioactively-labelled compounds was
measured as follows. A plastic hypodermic syringe of 1.0 ml capacity with needle (size no. 18) was calibrated and,
after flushing with argon, used for sampling from the side-arm of the anaerobic chamber. The contents of the
syringe were discharged quickly on to a cellulose acetate filter (25 mm diameter, 1.2 pm pore size), the yeast
washed twice on the filter with 3.5 ml ice-cold 0.1 M-KH~PO,,and the filter with yeast placed in 14 ml of
scintillation fluid (7 g PPO, 500 ml Triton X-100, 1 1 toluene) plus 1 ml water. The scintillation vials were counted
for 10 min in a Philips liquid scintillation counter (PW4700) programmed for quench correction. The methods
used for measuring aerobic transport were described fully by Sims & Barnett (1978).
Measurement of proton movements. Measurements were made of proton uptake by a washed suspension of
Kluyveromyces fragilis (10 mg dry wt yeast ml-I) in 0.3 m-Tris/citrate buffer (pH 4.9, stirred magnetically at
25 "C. The changes in pH, accompanying the uptake of sugars, were detected with a combination electrode (glass/
reference; CMAW of Russell pH, Auchtermuchty, Fife, Scotland), connected to an ion activity meter (Philips
PW9414), which controlled a recorder (JJ Instruments CR600) via a sensitive amplifier (Vibron Electrometer EIL
33B). A full-scaledeflectionof the recorder (200 mm) corresponded to a ApH of 0.25. The apparatus was calibrated
and used as described by Eddy & Nowacki (1971): the relationship between ApH and [H+]was established by
adding known amounts of 2.5 mM-HC1to the buffer. After adding sugar to the yeast, proton uptake was linear for
at least 30 s; the values for proton uptake were calculated from these initial rates.
Measurements of /?-D-galactosidase(EC 3 . 2 .I . 23) activity. fl-D-Galactosidaseactivity was measured for cells
made permeable, as described by Sims & Barnett (1978) for other glycosidases. 4-Nitrophenyl 8-Dgalactopyranoside was used at 3.3 mM.
Measurements of ethanol in the suspending medium. Ethanol in the suspending medium was measured enzymically
(Bernt & Gutman, 1974).
Measurements of intracellular maltose. These were made with strain SMC-1 B/3 of Saccharomyces cerevisiae to
estimate the accumulation of free maltose in the cells. Samples of about 4.5 mg dry wt of yeast were removed at 10
min intervals after adding 0.5 mM-[U-14C]maltose.Each yeast sample was washed thoroughly with ice-cold 0.1 MKH2P04 to remove exogenous maltose and placed in 10 ml60% (w/v) ethanol at 0 "C, in order to extract the
endogenous sugars. The extract was taken to dryness with a rotary evaporator. Each extract was re-dissolved in 2-0
ml25 mM-sodium phosphate buffer (pH 5.8) and 100 units of glucose oxidase were added. Incubation was at 35 "C
for 2 h. Each incubation mixture was chromatographed on a column of Dowex AG 1-X8 (acetate) 2-5 cm x 0.5
cm, to remove the D-gluconateformed by the oxidation of D-glucose. The effluent, free of D-glucose, was incubated
for 2 h at 35 "C with 10 units of a-D-glucosidaseand then with 100 units of glucose oxidase. The D-gluconate, thus
derived from the intracellular maltose, was adsorbed on a column of Dowex AG l-X8; the column was washed
with 3 x 1.0 m15 mM-sodium acetate (pH 4.8), eluted with 2 mlO.2 M-KNO, and the radioactivity of the eluate
determined. These conditions ensured a quantitative conversion of maltose to D-glucose and D-glUCOSe to Dgluconate. The a-D-glucosidase used did not hydrolyse a,a-trehalose.
Determination of yeast cell-water. The method used for estimating cell-water was based on that of Conway &
Downey (1950). Yeast, harvested from a culture in the exponential phase of growth, was equilibrated with 0.1 MKH2P04,containing about 1 pCi [3H]inulinml-l (37 kBq ml-l). The suspension was then centrifuged in a tapered
glass tube, supported in a rubber bung, into a thick-walled vinyl tube of constant internal diameter (1.65 mm). The
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2305
Transport of sugars into yeasts
tube and its contents were frozen in liquid nitrogen, and measured lengths cut to estimate the radioactivity of the
contents and the dry weight of the yeast.
Chemicals. Where possible, the chemicals were of analytical grade and were mostly from BDH or Sigma,
including 4-nitrophenyl fi-Pgalactopyranoside, methyl 1-thio-fi-D-galactopyranoside(TMG), 2-aminoisobutyric
acid (AIB), polyoxyethylene lauryl ether (Brij 35), carbonyl cyanide m-chlorophenylhydrazone (CCCP),
diethylstilbestrol, N,N'-dicyclohexylcarbdiimide (DCCD), salicylhydroxamic acid (SHAM), antimycin A,
rotenone, glucose oxidase (EC 1.1.3.4) and a-D-glucosidase (EC 3 . 2 . 1 . 2 0 ) from yeast. The anion-exchange resin,
Dowex AG 1-X8 (200-400 mesh) came from Bio-Rad, maltose free of D-glucose from Merck, [U-14C]maltose,2amino[ l-14C]isobutyric acid ([ 14C]AIB)and [3H]inulin from Amersham, and [14C]methyl 1-thio-fi-D-galactopyranoside ([ 14C]TMG) from New England Nuclear.
RESULTS
Characterization of the aerobic lactose transport system
When the lactose-utilizing yeasts were grown on succinate or D-glucose, they transported
TMG much more slowly than when grown on lactose (Table 1). Uptake of TMG showed simple
Michaelis-Menten kinetics. Dixon (1953) plots of the inhibition of this uptake by lactose (Fig. 1)
showed that the transport system of Debaryomyces polymorphus had a much higher affinity for
the 6-D-galactopyranosidesthan that of Kluyveromycesfragilis, 46-fold for TMG and fourfold for
lactose (Table 1). The calculated maximum velocities were similar for each yeast, that is for
when the transport systems were saturated with TMG. For Debaryomyces polymorphus,
inhibition by lactose was non-competitive, consistent with the involvement of two sites on the
carrier (Dixon et al., 1979).
Aerobic uptake of TMG was clearly by active transport and, for Debaryomyces polymorphus,
was linear for at least 3 h. After 30 min, both yeasts had concentrated the exogenous 0.5 mMTMG > 50-fold, and this was >99% extractable with 60% (v/v) ethanol. Induced by lactose in
succinate-grown yeast, the rate of uptake of TMG synchronized with that of lactose (Fig. 2) and
P-D-galactosidase activity appeared simultaneously. TMG did not induce the P-D-galacto-
[Lactose] (mM)
ITMGl (mM)
Fig. 1. Kinetics of aerobic uptake of TMG by Debaryomyces polymorphus. Yeast was harvested in
exponential growth, at about 250 pg dry wt ml-', washed, resuspended in 0.1 M-KH,PO, at about 350
pg dry wt ml-l and re-incubated for 1 h to starve the yeast. Samples (10 ml) were filtered, and each
placed in 5 ml of incubation mixture with different concentrations of [14C]TMG, as indicated, and, in
(a) varying concentrations of lactose. Rates were based on incubations of 25 min at 25 "C; samples (1.0
ml) were filtered, washed and scintillation-counted. (a) Effect of different concentrations of lactose on
the rate of net uptake of TMG, plotted as described by Dixon (1953): u, nmol TMG taken up m1n-l (mg
dry wt yeast)-' ; 0 ,at 0.5 mM-TMG; @, at 1.0 mM-TMG. (b) TMG uptake at different concentrations
of TMG, plotted by the method of Hanes (1932): t'. nmol TMG taken up min-l (mg dry wt yeast)-' ;[S],
mM-TMG. The measured value for V 1was 0.1 1. The lines shown in this figure were calculated by
linear regression; their point of intersection was on the abscissa at -0.28.
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2306
J . A. BARNETT AND
A. P .
SIMS
Table 1. Characteristics of the aerobic lactose uptake system of two yeasts
For conditions, see Fig. 1.
K,, (mM-TMG)
Ki (mM-hCtOSe)
V [nmol TMG min-' (mg dry wt yeast)-']
V [nmol lactose min-' (mg dry wt yeast)-']
Rate of uptake from exogenous [TMG] = 1.0 mM
[nmol TMG min-' (mg dry wt yeast)-']
Lactose-grown yeast
D-Glucose-grown yeast
Succinate-grown yeast
Debaryomyces
polymorphus
Kluyveromyces
fragilis
0.27
0.28
9.1
12.5
3-7
0-05
Stimulated rate of proton uptake
[equiv. H+ min-' (mg dry wt yeast)-']
Lactose-grown yeast :
On adding lactose*
Stoicheiometryt
On adding TMG*
Stoicheiometryt
On adding melibiose or D-glucose*
D-Glucose-grown yeast :
On adding lactose, TMG, melibiose or D-glucose*
1.2
16.6
77.5
0.57
0.0 1
<0.01
53.4
0.87
9.07
1.18
0
0
* Lactose was added to give 4.6 mM and TMG 9.3 mM.
f Stoicheiometry was computed by dividing the total number of equivalents of extra protons absorbed by the
calculated number of equivalents of sugar absorbed.
pyranoside transport system or P-D-galactosidase. Further, stimulation of proton uptake on
adding lactose or TMG occurred only in lactose-grown and not in D-glucose-grown yeast. This
response was specific for the P-D-galactopyranosidesand was not observed on adding the cc-Dgalactopyranoside, melibiose (Table 1). Addition of D-glucose caused a sustained decrease in
pH, similar to that observed by Sigler et al. (1981).
The effects were studied of certain metabolic inhibitors on the active, aerobic transport of
TMG ; some results for Kluyveromyces marxianus and Kluyveromyces fragilis are given in Fig. 3.
Antimycin, diethylstilbestrol and DCCD each decreased the rate of transport, whilst proton
conductors, such as CCCP, completely abolished active transport (Fig. 3a). Growth in medium
containing antimycin, with D-glucose or lactose as a source of carbon, was preceded by a lag of
about 5 h, after which the yeasts grew at about half of the rate of those without antimycin (about
0.19 generations h-'). Although yeasts grown in this way took up TMG by active transport, they
did so more slowly than those from medium without antimycin and with reduced ability to
concentrate the TMG. The rate of oxygen uptake by these yeasts was reduced by about 85% by
antimycin. The residual oxygen uptake was sensitive to SHAM. Rotenone did not inhibit
oxygen uptake.
Characterization of anaerobic transport
Studies were made of the anaerobic uptake of TMG by the two non-lactose-fermenting yeasts,
Debaryomyces polymorphus and Kluyveromyces marxianus, and the lactose-fermenting Kluyueromyces fragilis. Kluyveromyces fragilis did not concentrate TMG anaerobically (Fig. 4). When
measured with a range of external concentrations of TMG, after equilibrium, each internal
concentration was the same as that externally (Fig. 5). In calculating the available cell-water, no
compartmentation was assumed. Addition of 0.5 mM-lactose to the incubation mixture allowed
continuous fermentation with the formation of ethanol, but did not promote active transport.
Comparable findings were made with anaerobic Klu~veromycesfragilis,grown in the presence of
antimycin. Furthermore, when a culture of Kluyueromyces fragilis, accumulating TMG aerobically, was divided into two parts, the part made anaerobic, or treated with antimycin, showed a
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Transport of sugars into yeasts
2307
Rate of TMG uptake
[nmol min-' (mg dry wt yeast)-' 1
Fig. 2. Association of the induction of lactose and TMG transport into Kluyueromycesfragilis. Cells
from an exponentially-growing culture of yeast, grown on succinate, were washed and transferred to Nbase 10 mM-lactose. At timed intervals of about 40 min, over a period of 5.5 h, samples were removed
for measuring uptake. For these measurements, the yeast was suspended in 0.1 M-KH,PO,, containing
either 5 m~-['~C]lactose
or 5 rn~-[l,c]TMG. For lactose uptake, the suspensions were sampled at 1.0
min intervals for 5 min, and for TMG at 2.0 min intervals for 10 min. Initial rates for lactose uptake
were calculated from tangents.
+
25
I
I
I
80
i
60
40
20
5
10
15
20
Time (min)
50
25
100
150
Time (min)
200
250
Fig. 3. Effects of inhibitors on the aerobic uptake of TMG by (a) Kluyveromyces marxianus and (b)
Kluyueromyces .fragilis. For each incubation, yeast growing exponentially on N-base
lactose was
filtered and washed three times with 0-1M-KH~PO,.At zero time, the yeast was resuspended to give the
equivalent of 400 pg dry wt ml-I in 10 ml of 0.1 M-KH,PO, with 1.0 rn~-[l,c]TMG and with or without
an inhibitor as indicated. Each such suspension was incubated aerobically at 25 "C; samples of 500 p1
were taken at recorded intervals, quickly filtered, washed twice with ice-cold 0.1 M-KH~PO,,and the
No addition; 0 , 500 p ~ - D C C D0
; ,
yeast and filter placed in scintillation fluid for counting. 0,
400 pM-diethylstilbestrol;
40 pM-antimycin; A, 50 ~M-CCCP.
.,
+
+
+
+
+
progressive net loss of TMG, whereas the unaltered, aerobic, control culture continued accumulating TMG. The non-lactose-fermenting yeasts also failed to concentrate TMG when
anaerobic.
The lack of any evidence of anaerobic active transport of TMG, even by a yeast that was
simultaneously fermenting lactose, prompted the investigation of uptake of the non-metabolizable amino acid, AIB. Amino acids are reputedly transported actively into yeasts under anaerobic conditions (e.g. Stoppani & Ramos, 1978) and, indeed, Fig. 6 shows that Kluyveromyces
fragilis, whilst fermenting D-glucose anaerobically, concentrated AIB. When made aerobic
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2308
J . A . BARNETT AND A . P. S I M S
8-
'
I
7120
-
7
2
-
I
I
n
5:
E
6-
," 5 -
100-
0
5
1 x
-
4-
-
60-
E*
1-
20
80 120 160 200
1
2
3
4
5
6
7
8
Time (rnin)
External [TMGI (mM)
Fig. 4
Fig. 5
Fig. 4. Effect of anaerobic conditions on the uptake of TMG by Kluyveromyces frugilis. 0,
Aerobic
incubation; 0 , anaerobic incubation. For conditions for measuring aerobic uptake, see Fig. 3; for
anaerobic uptake and monitoring of oxygen concentration, see Methods.
40
Fig. 5. Association between internal and external concentrations of TMG for Kluyveromyces fragilis,
after anaerobic equilibration for 10 min. For calculating the internal concentration of TMG, the waterspace was determined as 3.36 mg (mg dry wt yeast)-'. Oxygen concentration was monitored as
described in Methods.
15
-
7
I
h
5 10
E
W
20
40
Time (min)
Fig. 6
60
80
10
20 30 40
Time (min)
Fig. 7
50
Fig. 6 . Aerobic and anaerobic uptake of AIB by Kluyverornycesfragilis. The yeast was grown with Dglucose and glutamate as sources of carbon and nitrogen to the equivalent concentration of 0.8 mg dry
wt rnl-', washed three times with 0.1 M-KH,PO,, resuspended (at 0-4 mg dry wt ml-I) and starved in
0.1 M-KH,PO, for 1 h. At zero time, D-glucose and [ 14C]AIBwere added to give concentrations of 5 and
0.2 mM, respectively. Sampling for scintillation-counting and for measuring ethanol formation was
Aerobic incubation; 0 ,
done and oxygen concentration monitored as described in Methods. 0,
anaerobic incubation; 0,
ethanol formed during anaerobic incubation; the arrow indicates time at
which culture made aerobic.
Fig. 7. Ability to concentrate maltose of a mutant strain of Saccharomyces cerevisiae with enhanced
maltose uptake. Measurements of intracellular maltose were made as described in Methods. 0,
Aerobic uptake; 0 ,anaerobic uptake; ---, equilibrium conditions, when exogenous and endogenous
maltose concentrations are the same.
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Transport of sugars into yeasts
2309
again, there was only a slight increase in the rate of uptake. Maltose, too, has long been known to
enter Saccharomyces cerevisiae under aerobic conditions by active transport (e.g. Harris &
Thompson, 1961; Okada & Halvorson, 1964; Brocklehurst et al., 1977). This was confirmed
(Fig. 7), but for anaerobic incubations the results indicated an inability to concentrate the
exogenous maltose.
DISCUSSION
Energetics of aerobic transport
It is widely held that the active transport of sugars and amino acids often involves proton
symport. Such transport depends on the coupling of the movement of these metabolites up a
concentration gradient, to an associated movement of protons down a gradient. In yeasts, this
proton gradient is established by proton pumps in the plasmalemma, associated with ATPase
activity (e.g. Eddy, 1978; Dekk, 1978; Malpartida & Serrano, 1981). Both the proton gradient
across the plasmalemma and the membrane potential may contribute to the proton motive force
(for review, see Harold, 1977).
Evidence that the movement of sugars and amino acids into many yeasts is energized by
proton symport and ATPase activity depends chiefly on (i) the stoicheiometry of the metabolite
uptake and proton uptake (e.g. Eddy, 1978)and (ii) the action of specific inhibitors. Ramos et al.
(1980), for example, showed that the ATPase inhibitors, DCCD and diethylstilbestrol, each also
inhibit the transport of L-leucine into Saccharomyces cerevisiae. Proton-conducting uncouplers,
such as CCCP, and the lipophilic cation, TPP+ (tetraphenylphosphonium ion), which depolarized the membrane potential of Rhodotorula glutinis, also inhibited active uptake of D-glUCOSamine (Niemietz et al., 1981).
The findings described here, with lactose and TMG, show that (i) a single carrier subserves
both lactose and TMG transport and (ii) this carrier operates aerobically by active transport,
probably involving proton symport, energized by plasmalemma-bound ATPase and the mitochondrial electron transport chain.
A single carrier for fl-D-galactosides. Whilst yeasts grown on D-glucose or succinate took up
TMG only slowly, its transport occurred pre-eminently into lactose-grown cells (Table 1).
Secondly, the induction in succinate-grown yeast of the transport of lactose was synchronized
with that of TMG (Fig. 2). Thirdly, sugar-dependent proton uptake for lactose and TMG
occurred only in yeasts grown on lactose. Such yeasts gave a stoicheiometry between sugar and
proton uptake of about 1 : 1. Adding melibiose, an a-D-galactopyranoside, did not stimulate
proton uptake. However, since the inhibition of TMG uptake by lactose was non-competitive
(Fig. I), the two fi-D-galactopyranosideswere probably bound at different sites on the carrier.
Lactose was transported much faster than TMG : were lactose transported at the same maximum rate ( V ) as TMG (Table l), this would be insufficient to sustain the observed rates of
growth.
Proton symport. The results of using a number of inhibitors (Fig. 3) confirmed the observation
that entry of TMG involved proton symport, probably energized by means of a plasmalemmabound ATPase and the mitochondrial electron transport chain. First, the proton-conductor,
CCCP, completely eliminated the transport of TMG up a concentration gradient; secondly
antimycin, a specific inhibitor of mitochondrial electron transport that blocks the flow in the
span from cytochrome b to c, much reduced uptake of both TMG and oxygen, and to about the
same extent; thirdly, diethylstilbestrol and DCCD, inhibitors of ATPase and proton-pumping
(e.g. Serrano, 1980), also reduced the rates of uptake.
Active transport was observed, even in the presence of antimycin, and also with yeasts grown
with antimycin, which never totally eliminated aerobic respiration (cf. Morgan & Whittaker,
1978). The effects of antimycin on growth and oxygen uptake by the Kluyveromyces species were
similar to those already reported for Kluyveromyces lactis (Ferrero et al., 1981).
Two alternative antimycin-insensitive oxidase pathways have been described for a number of
yeasts (Lloyd, 1974; Goffeau & Crosby, 1978; Henry et al., 1978), and the observations with
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J . A . B A R N E T T AND A . P. SIMS
inhibitors, described in this paper, indicate that Kluyveromyces fragilis shows a similar oxidase
pathway to that reported for Kluyveromyces Zactis (Ferrero et al., 1981). Clearly, such alternative
pathways (here in lactose-grownyeast) can support active transport, although at a reduced rate,
and giving only about a 10-fold concentration of the exogenous substrate, compared with at least
50-fold for yeast free of antimycin.
Anaerobic transport
For the four yeasts investigated, with maltose as well as TMG, the sugars appeared to enter by
facilitated diffusion anaerobically, and not by active transport. At a range of concentrations, at
equilibrium, the internal concentration of substrate simply reached that provided externally
(Fig. 5). The time taken to reach this equilibrium was increased by the presence of exogenous
lactose. This was observed both with the non-lactose-fermenting Debaryomyces polymorphus and
the lactose-fermenting Kluyueromyces fiagilis. Even when the latter was fermenting lactose,
supplied exogenously, there was no evidence of active transport of TMG. The lactose had been
added at concentrations (i) sufficient for active glycolysis, as shown by high rates of ethanol
formation, but (ii) insufficient to reduce the rate of aerobic transport of TMG by more than
40 %.
Measurement of ethanol formation by Kluyveromyces fragilis (linear for >90 min) showed
that the anaerobic uptake of lactose could be sustained at least at 40 nmol min-l (mg dry wt
yeast)-l, and this value was > 50% of the maximum rate for aerobic active transport (see Table
1). A comparable value of about 50 nmol D-glucose min-l mg-l was calculated, from ethanol
formation, for anaerobic Saccharornyces cerevisiae utilizing maltose.
The findings reported here differed from those obtained initially with these yeasts and with
Torulopsis dattila (Sims & Barnett, 1978), for which ‘oxygen-free’nitrogen or unscrubbed argon
was used. Under these conditions, uptake was similar to that found in the presence of antimycin,
permitting about a five- or tenfold concentration of TMG. In view of the high affinity of some
yeasts for oxygen (e.g. K,,, = 0.4~ M - ;O
Wimpenny,
~
1969), concentrations of as little as 0.006%
of oxygen in the argon might allow aerobic processes to occur at as much as 15% of the
maximum rate, were the gas bubbled vigorously through the yeast suspension. After chemical
scrubbing, mass spectrometric analysis gave concentrations of <0.006% oxygen, and only
under these stringent conditions, namely, scrubbed argon bubbled slowly, was active transport
of TMG completely abolished.
Although anaerobic fermentative activity did not allow the active transport of TMG, it
sustained the active uptake of 2-aminoisobutyrate (Fig. 6). Clearly, the fermentation of lactose
does not rely on its active transport, but can be maintained at a high rate by its entry by means of
facilitated diffusion, as has been established for the aerobic utilization of D-glucose by Saccharomyces cerevisiae (for review, see Barnett, 1976). In this context, it is interesting that (like lactose
with Kluyverornyces spp.), although maltose enters Saccharomyces cerevisiae actively under
aerobic conditions, anaerobic uptake appears to be by facilitated diffusion (Fig. 7).
Amino acids have been found to be taken up actively by several yeasts, only if one of two
criteria are satisfied : either the yeasts must be aerobic or, if anaerobic, they must be in a state of
active fermentation (unpublished observations). Thus, there is a marked difference in the effects
of anaerobic conditions on the two kinds of uptake, namely, that for amino acids and that for
sugars. Whilst in fermenting yeasts under highly anaerobic conditions, amino acid uptake can
occur by active transport, sugars must enter anaerobic yeast cells by facilitated diffusion.
Anaerobic transport and the Kluyver efect
According to Kotyk & Hofer (1965), the yeast Rhodotorula glutinis (gracilis) transports Dglucose actively under aerobic conditions, but fails to take up that sugar anaerobically, even by
facilitated diffusion. The suggestion that such a total failure of the anaerobic transport of certain
sugars might explain the Kluyver effect (Sims & Barnett, 1978) was based mainly on experiments with metabolizable sugars. Now the present observations, with the non-metabolized
analogue of lactose, TMG, and under far more stringent conditions of anaerobiosis, refutes this
view ineluctably.
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Transport of sugars into yeasts
2311
A number of yeasts have now been examined in this context in the present and earlier
experiments (Sims & Barnett, 1978). Clearly, under anaerobic conditions, glycosides enter the
yeasts by facilitated diffusion and there is no evidence of deactivation of the relevant glycosidases. Furthermore, measuring carbon dioxide production had established that the responses to
changes between aerobic and almost anaerobic conditions were rapid and reversible, taking not
more than a few seconds. Hence, the control that underlies the Kluyver effect (i) cannot be
exerted at the early stages of catabolism, but could act on the pathway from pyruvate to ethanol,
and (ii) is not mediated by the slower processes involving induction or repression. The nature of
this regulation is currently under investigation.
A number of the preliminary experiments were done by vacation students, namely, Miss C. F. Taylor (University of Glasgow), Mr H. D. Schmitt, Mrs I. Breitenbach-Schmitt and Mr J. Heinisch (Technische Hochschule
Darmstadt). We thank them for their excellent and persistent work and Dr K.-D. Entian (Universitat Tubingen)
for the gift of the mutant strain of Saccharomyces cerevisiae.
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