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Uphill transport of sugars in the yeast Rhodotorula gracilis
410
BIOCHIMICA ET BIOPHYSICA ACTA
UBA 45200 UPHILL TRANSPORT
OF SUGARS IN THE YEAST
RHODOTORULA GRACILIS A R N O ~ T K O T Y K AND M I L A N H(I~FER
Laboratory for Cell Melabolism, Dzstitute of .,>Iicrobiology, Czechoslovak .4 cademy of Sciences, Prague (Czechoslovakia) ( R e c e i v e d J a n u a r y 26th, 1965)
SUMMARY
T h e lipid-forming y e a s t Rhodotorula gracilis was found to t r a n s p o r t b o t h met a b o l i z a b l e a n d n o n - m e t a b o l i z a b l e sugars against a c o n c e n t r a t i o n gradient. The process a p p e a r s to require m e t a b o l i c energy b u t o p e r a t e s to a l i m i t e d e x t e n t even a n a e r o b i c a l l y when no gas exchange a n d s u b s t r a t e u t i l i z a t i o n are d e t e c t a b l e in the cells. The a c c u m u l a t e d sugar is present i n t r a c e l l u l a r l y in an osmotically active s t a t e a n d is r e a d i l y e x c h a n g e a b l e for e x t e r n a l sugar. The t r a n s p o r t is p H - d e p e n d e n t with an o p t i m u m near p H 5 b u t it is N a +- a n d K+-independent. A carrier s y s t e m a p p e a r s to be i n v o l v e d which t r a n s p o r t s I sugar molecule at a t i m e a n d possesses different effective affinities for s u b s t r a t e s at the two sides of the membrane. Of all t h e sugars t e s t e d only D-glucose does not p e n e t r a t e the cell a n a e r o b i c a l l y a l t h o u g h it p r e v e n t s the t r a n s p o r t of o t h e r sugars.
INTRODUCTION
KLEINZELLER AND SLECHTA1 a n d l a t e r LITCHFIELD AND ORDAL2 found t h a t the lipid-forming r e d y e a s t Rhodotorula gracilis only m e t a b o l i z e d sugars aerobically a n d the m i c r o o r g a n i s m was therefore e x a m i n e d to see w h e t h e r this is due to a m e t a b o l i c deficiency or to a block in the m e m b r a n e w i t h respect to sugar t r a n s p o r t . W h i l e it now can be said conclusively t h a t the t r a n s p o r t step is not limiting, either aerobically or anaerobically, j u d g m e n t will be reserved for t h e future to decide on the n a t u r e of the m e t a b o l i c block causing the i n a b i l i t y of R. gracilis to utilize sugars anaerobically. The t r a n s p o r t of sugars, however, w-as f o u n d to possess an u n e x p e c t e d feature which, a p a r t from its heuristic value, m i g h t be m a d e use of for t e s t i n g some recent h y p o t h e s e s of sugar t r a n s p o r t 3-5, viz. an intrinsic a s y m m e t r y causing sugars to be t r a n s p o r t e d uphill into the cell. The p r e s e n t p a p e r deals with the kinetics of the process, a n d is to be followed b y a s t u d y of its m e t a b o l i c coupling a n d of sugar m e t a b o l i s m in R. graciIis as a whole. Biochim. Biophys..4cta, lO2 (1965) 41o-422
UPHILL
TRANSPORT
OF SUGARS
411
MATERIALS AND METHODS
A collection strain of R. gracilis (taxonomically R. glutinis 5/Fres/Harrison) was grown at 3 °0 on a reciprocal shaker in the following medium: 4° g glucose, 0.66 g NH4N03, I.O g K2HPO 4, 0.5 g NaC1, I.O g MgSO4.7H~O, 0.25 g CaC12, 0.05 g FeCls.6H~O, in a total volume of IOOO ml. The pH was adjusted to 5-5 with HC1 after sterilization and a trace of yeast extract added before cultivation. Cells were harvested after 24 h, washed several times with distilled water and shaken for 2 h in suspension as it was found that subsequently they were much easier to filter. Cell suspensions were prepared in distilled water or in o.I M KH~PO~ (no difference in behaviour being found in the two media) and incubated at 28 ° either in 0 2 or in highly purified N 2 with the appropriate sugar and inhibitor concentrations. The density of the yeast suspension varied between 4 and IO mg dry wt./ml. Samples were removed at regular intervals to be filtered through a Millipore HA filter. After washing the pellet twice with ice-cold water it was suspended in boiling water to extract cell solutes and the volume made up to a mark. The aqueous extract was deproteinized with ZnSO 4 and Ba(OH)~ and sugars estimated by standard methods: hexoses according to SOMOGYI6 and NELSON7, pentoses according to ME1JBAUM (see ref. 8), radioactive sugars in a methane-flow ~'rieseke-Hoepfner counter after drying on aluminium planchets or in a Tracerlab liquid scintillation counter. Cell water content was determined by accurate hematocrit measurements, using serum albumin to assay the extracellular space. Serum albumin was estimated according to the method of LOWRY et al. 9. It deserves mentioning here that application of inulin, methylene blue or erythrosine B yielded excessively high extracellular space values, apparently due to surface adsorption of the compounds on R. gracilis cells. It was found that the intraceflular water volume corresponds to 69-72 % of the total when suspended in distilled water (cf. Fig. 3)The intracellular pH was estimated by using the weakly acid dye, bromophenol blue, as described earlier 1°. Non-labelled D-glucose, D-arabinose, D-galactose, D-fructose, D-mannose and D-ribose were products of Spofa, Czechoslovakia; D-xylose, L-xylose and L-rhamnose of Hoffmann-LaRoche, Switzerland; D-E14C]xylose was obtained from the Radiochemical Centre, Amersham, Great Britain and L-[14C]rhamnose was very kindly prepared by Dr. J. MAJER of this Institute. All other reagents were commercial products of analytical purity. RESULTS
Uptake of sugars The yeast strain used was found to take up all the sugars tested, viz. D-glucose, D-fructose, D-mannose, D-galactose, D-arabinose, D-xylose, L-xylose, D-ribose and L-rhamnose. Of these, D-glucose, D-fructose, D-mannose, D-galactose and D-xylose were aerobically metabolized with Qo~ at 28 ° ranging from 28 to 40/A 0 2 per mg dry wt. per h, this being only slightly in excess of endogenous metabolism where Qo2 values between I6 and 20 are generally found. D-Arabinose produced a Qo2 of 20 Biochim. Biophys. Acta, lO2 (1965) 4 1 o - 4 2 2
412
A. KOTYK, M, HOFER
as compared with 16.5 in the endogenous control. It appears that endogenous metabolism proceeds undiminished in the presence of external substrate (cf. K L E I N Z E L L E R 11, and measurements of sugar utilization from the medium with concomitant Qo2 and Qco2 values) so that the Qo~ attributable to external sugar utilization varied between 4 and 24.
Uphill transport The kinetic parameters of uptake, the apparent K m , l n , Win as well as the maximum ratio of internal to external substrate concentration (Sl/So), in the steady state attainable at very low concentrations, were measured with D-arabinose, I)-xylose, L-rhamnose and D-glucose, the results being summarized inTable I and in Figs. I and 2. It m a y be seen that the apparent Kin,In and Vln vary for different sugars but that the change from aerobic to anaerobic conditions does not bring about any drastic changes in the constants, the apparent affinity being actually either higher or lower in N 2 than in 0 2 (glucose behaves anomalously in that it is not transported anaerobically at all). On the other hand, the maximum attainable ratio of Sl/So not only TABLE I TRANSPORT
PARAMI~TERS
OF
Sugar
VARIOUS
SUGARS
Gaseous phase
n-Xylose D- Arab inose L-Rhamnose D-Glucose
R.
gracilis
Appare~t Kin,in (M)
Vln
(rag per g dry wt. per min)
02 No 02 N2 Oo N2 02
2" 10 -3 8- l o -4 4.1. l o 2 1.3" Io -1 3-4" lo-3 4-4" lo-3 5-5" Io ~
3.21 2.82 4.o3 4.27 0.55 o.19 2.98
N 2
--
--
_____.__--c.-----
100
IN
Maximum St~So
IOOO 3oo 4o 20 90 Io i .8 o
_____..__---<>-
"5 80
60
40
E P
O0
10
20
30
(So)(mglmu) Fig. I. R e l a t i o n s h i p b e t w e e n e x t e r n a l (So) a n d i n t r a c e l l u l a r (St) c o n c e n t r a t i o n s of D-xylose a f t e r r e a c h i n g s t e a d y s t a t e a e r o b i c a l l y ( O - - O ) or a n a e r o b i c a l l y ( O - - O ) a t 28 °. T h e b r o k e n s t r a i g h t line c o r r e s p o n d s to St/So = I, as w o u l d o b t a i n in a diffusion e q u i l i b r i u m .
Biochim. Biophys. dcta, lO2 (1965) 4 i o - 4 2 2
413
UPHILL TRANSPORT OF SUGARS
strikingly varies from sugar to sugar but is always significantly lower anaerobically, apparently since less energy is expendable for its maintenance under anaerobic conditions. This is most likely reflected in the parameters for sugar efflux, which is unfortunately not readily amenable to investigation on account of its rapidity and the difficulty of attaining sufficiently low intracellular steady-state concentrations. It should be noted that even with D-xylose, which is metabolized under aerobic conditions, a considerable concentration gradient is built up intracellularly.
"C
6O
b 4O
P
~
~o
E
0 0
i
i
i
10
20
30
(So)(mg/ml) Fig. 2. The Sl/So relationship for o-arabinose after aerobic ( O - - O ) or anaerobic ( O - - O ) incubation. Other details as for Fig. i. 18
7O
%] ol o c
~o
10
30
t_
L D
6
..o-
lo _E
2 0 0
i Q1
I Q3
i
I
i
Q5
07
0.9
NQCI (M)
Fig. 3. Correlation between the intracellular amount of L-rbamnose ( O - - - O ) and the intracellular water content ( O - - 0 ) after a 3-h aerobic incubation with 0.5 % L-rhamnose in different concentrations of NaC1 at 28 °.
The relationship between membrane carrier and intracellular adsorption Since u n e q u a l d i s t r i b u t i o n of s u b s t r a t e b e t w e e n cell a n d m e d i u m c o u l d b e acc o u n t e d for e i t h e r b y i n t r a c e l l u l a r a d s o r p t i o n or b y a s y m m e t r i c m e m b r a n e p r o p e r t i e s , t h e s t e a d y - s t a t e i n t r a c e l l u l a r a m o u n t s of L - r h a m n o s e w e r e e s t i m a t e d u s i n g d i f f e r e n t c o n c e n t r a t i o n s of NaC1 in t h e m e d i u m , t h u s b r i n g i n g a b o u t d i f f e r e n t d e g r e e s of cell v o l u m e s h r i n k a g e . F i g . 3 s h o w s t h a t t h e r e is g o o d a g r e e m e n t b e t w e e n t h e a m o u n t of i n t r a c e l l u l a r w a t e r a n d t h e a m o u n t of s u g a r p r e s e n t in t h e cell. T h e f i n d i n g t h u s
Biochim. Biophys. Acta, lO2 (1965) 41o-422
414
A. KOTYK, M. HOFER
strongly supports the view that the sugar inside the cell is in an osmotically active form and that the asymmetric distribution is caused by properties of the cell membrane.
Exchangeability of intracellular sugar Since WILKINS AND O'IKANEa observed in Streptococcus faecalis that, once accumulated, sugar is not freely exchangeable with sugar added to the medium, cells 3~0
--Q
2
b f
2000
E
o
i 0
0
i 30
-
w
i 60
i 90
o
-
i 120
Time (rain)
Fig. 4. Efflux of D-[14C]xylose from R . gracilis. The cells were p r e - i n c u b a t e d aerobically at 28 ° for 3 h w i t h t w o different c o n c e n t r a t i o n s of labelled D-xylose (curve 1: o.O4~o, curve 2: 0. 4 ~o), centrifuged and at zero t i m e resuspended in w a t e r ( O - - O ) or non-labelled D-xylose ( @ - - @ ) at a c o n c e n t r a t i o n corresponding to the intracellular one.
25
I_
=
20 D
2
~5
o 21
5
0
L 0 Time
i
J
I
20
40
60
(min)
Fig. 5. Effect of D-glucose on efflux of L-xylose from R . gracilis. Cells were i n c u b a t e d w i t h 0. 5 ~o L-xylose for 5 h aerobically or anaerobically. At zero t i m e t h e centrifuged pellet w a s resuspended in w a t e r ( O , G) or o.I ~o D-glucose (@, ~k).
were preloaded with labelled D-p*C]xylose and, after centrifuging, resuspended in water or in D-[l=C]xylose or D-[12C]glucose. The results are shown in Figs. 4 and 5The rapid drop observed on suspending in water corresponds to a practically unidirectional flux of D-[14C]xylose out of the cells until a new steady state is reached. This is rather high since cells accumulate practically all sugar then available: that retained by cells and also that from the intercellular space after centrifugation. The somewhat slower decrease observed on suspending cells in unlabelled xylose is due Biochim.
Biophys. Acta, lO2 (1965) 4IO-422
415
UPHILL TRANSPORT OF SUGARS
to a higher saturation of the carrier when the attainment of equilibrium is slower but the drop is greater because unlabelled xylose replaces most of the labelled xylose in cells and medium and only labelled xylose is being determined. The lack of effect of glucose on anaerobic transport out of cells seems to indicate that glucose acts only on the outside reaction of the carrier with sugar. The ready availability of sugar in the cell is further demonstrated by changing from aerobic to anaerobic conditions during the steady state, when a rapid escape of sugar from cells takes place until a new steady state corresponding to anaerobiosis is reached (Fig. 6). [
xJ
2000
.~-~ 1500
E iooo
50O
01
I
;
0
0 Time
i
I
60
90
l 120
I 150
(min)
Fig. 6. Change of intracellular level of sugar on changing from aerobic to anaerobic conditions. Cells were incubated aerobically at 28 ° w i t h o . I % ( • - - • ) or o.4 % ( O - - O ) D- [14C]xylose for 3 h and then at zero time the 02 supply w a s cut off and replaced b y a powerful stream of N 2. "6
--o
B --,o
8OOO
--o
2 20
>~ 6000
S c
2
E 4000
--o
25
---A
o
o
&
2 2000
-A
10
L
t
20
30
t 40
//
// -'T-t
0 360
0
10
20
30
40
360
Tirne(min)
Fig. 7. Competition of sugars for entry into R. gracilis ceils. For Fig. 7 A, the suspension w a s incubated w i t h 0.45 % D-arabinose aerobically (curves I, 2) or anaerobically (curves 3, 4) w i t h o u t (curves i, 3) or after (curves 2, 4) a Io-min pre-incubation with o . i % D-glucose. For Fig. 7]3, t h e suspension w a s incubated with o.I % D-[14C]xylose aerobically (curves i, 2) or anaerobically (curves 3, 4) w i t h o u t (curves I, 3) or w i t h (curves 2, 4) simultaneous addition of 1% L-xylose. T h e dotted lines in both parts of the figure correspond to a diffusion equilibrium. I t is n o t e w o r t h y t h a t in the presence of D-glucose anaerobically the level of D-arabinose does not even reach t h e equilibrium ratio St~So = i. Biochim. Biophys. Acta, lO2 (1965) 4 1 o - 4 2 2
416
A. KOTYK, M. H O F E R
Con@etition between sugars It was shown that, e.g., D-xylose and L-xylose compete for the same carrier and that o-glucose competes with D-arabinose for uptake (Fig. 7). Hence it is concluded that the sugars tested here share one carrier although the existence of other additional carriers is not hereby excluded.
Counter-transport One of the features of transport mediated by a mobile carrier is the existence of counter-transport of a substrate in equilibrium with cells on adding another substrate to the medium. R. gracilis cells were therefore incubated with labelled m-xylose
• /
10000 ~
~
~
~1
o
,:,
o
n
~ 7500
P 5000 o 2500
I
0
310
610
910
I
I
120
150
Tirne(min)
F i g . 8. A b s e n c e of c o u n t e r - t r a n s p o r t i n R. gracilis. Cells w e r e p r e - i n c u b a t e d a e r o b i c a l l y a t 28 ° w i t h o . i % D-[14C]xylose a n d a f t e r 2 h (at zero t i m e ) o n e - t e n t h of t h e v o l u m e of w a t e r ( c u r v e i), 2 °/o n o n - l a b e l l e d D - x y l o s e ( c u r v e 2) or 2 ~o D - g l u c o s e ( c u r v e 3) w a s a d d e d .
1.8
15
................
....
~ 1.2 -J
Q9
06 -2A
i -2:1
i -1.9
F i g . 9. D e m o n s t r a t i o n
r -1.6
i -1.3
i -1D
i -07
Log(S) of the number of sugar molecules attached to the carrier. Using suitable
v a l u e s of t h e d i s s o c i a t i o n c o n s t a n t s b e t w e e n c a r r i e r a n d s u g a r , t h e o r e t i c a l c u r v e s w e r e o b t a i n e d for t h e t r a n s p o r t of a s i n g l e m o l e c u l e a t t a c h e d t o t h e c a r r i e r ( d a s h e d c u r v e i) or of 2 m o l e c u l e s a t t a c h e d a t a t i m e ( d o t t e d c u r v e 2) ; cf. ref. 12. T h e s o l i d c i r c l e s a r e e x p e r i m e n t a l v a l u e s o b t a i n e d w i t h L - r h a m n o s e a f t e r p r e - i n c u b a t i n g a cell s u s p e n s i o n a e r o b i c a l l y for 3 h w i t h d i f f e r e n t c o n c e n t r a t i o n s of n o n - l a b e l l e d L - r h a m n o s e , t h e n a d d i n g a n a n a l y t i c a l l y n e g l i g i b l e a m o u n t of L-[14C] r h a m n o s e a n d e s t i m a t i n g t h e h a l f - t i m e of u p t a k e of t h e l a t t e r . L o g S r e f e r s t o t h e c o n c e n t r a t i o n of n o n - l a b e l l e d s u g a r , log vR t o t h e r a t e of u p t a k e of t h e l a b e l l e d one.
Biochim. Biophys. ~Icta, I o 2 (1965) 4IO 422
UPHILL
TRANSPORT
417
OF SUGARS
in concentrations ranging from o.o5 % to 4 % until steady state was reached (3-6 h) and non-labelled D-xylose or D-glucose was then added to a final concentration of 1- 4 %, both aerobically and anaerobically. The content of the label in cells was then examined as shown by sample curves in Fig. 8. The intracellular label invariably dropped to a lower level, as one would expect from the following consideration: On adding non-labelled sugar to the steady-state mixture the total concentration of sugar increases and the SI/So ratio in the final steady state is lower, as expected (cf. Figs. i and 2). Hence the level of labelled sugar must also decrease to a correspondingly lower value. However, no unambiguous minimum in the internal sugar level could be observed in the numerous experiments, contrary to observations where a pronounced counter-transport takes place (e.g. ref. 12).
Number of sugar molecules attached per carrier A test described by WILBRANDTAND KOTYK 12, to distinguish between transport where one or two substrate molecules are attached to the carrier, was applied to the transport of L-rhamnose. The results shown in Fig. 9 indicate an exclusive or strongly predominant mono-complex transport (cf. ref. 13).
Effect of metabolic inhibitors Of the various kinetic constants examined here the steady-state ratio of intracellular to extracellular sugar appeared to be most straightforward in connection with the use of metabolic blocking agents. Table I I shows the results obtained with TABLE
II
EFFECT OF METABOLIC INHIBITORS ON STEADY-STATE RATIO OF L-XYLOSE Incubation
w a s c a r r i e d o u t a t 28 ° f o r 6 h w i t h 0 . 2 5 % L - x y l o s e .
Inhibitor
Final concentration
(M)
None Iodoacetamide
5" i o 4
2,4-Dinitrophenol
5" l O - 4
Sodium azide Iodoacetamide Oligomycin
lO -3 plus 2,4-dinitrophenol
5" IO-4 _[_ 5 ' l O - 4 I. 5 t t g / m l
Gaseous phase
SI/So
0.2
6.0 4.2 3.1 0.72 0.3 0.45 0.34 o.I o o 4-3
N2 Oz N2 O2 N~ O2 N2 02 N2 02
L-xylose. It will be seen that none of the inhibitors reduced the ratio to I as would be expected in an equilibrating transport when all energy sources are cut off. The effect is rather varied, indicating that the inhibitors may interact directly with one or more carriers involved and/or that different types of metabolic coupling are in operation--apparently no metabolic pump is simply superimposed here over equilibrating carrier transport. Biochim. Biophys. Acta, lO2 (1965) 4 1 o - 4 2 2
418
A. KOTYK, M. HOFER
Effect of pH Cells were incubated in o.15 M citrate-phosphate or Tris-HC1 buffers in the pH range 3 to 8 with D-arabinose. The apparent Km,ln, Vln, Si/So ratio and metabolic parameters were measured. Fig. IO shows that pH 5 is near the optimum for the accumulation ratio, both aerobically and anaerobically. The apparent K i n , i n w a s found to be lowest (corresponding to highest effective affinity) at pH 5 while the Vin was highest also at pH 5. However, the metabolic optimum seems to lie near pH 4. This might suggest that the carrier itself is an enzyme with dissociable protons or can undergo allosteric configurational change linked with the attachment of H+ ions.
4o _=
~
40
r
! J
3oi
3O
c~
o ~5 d q0
E
I
1
r
I
I
I
3
4
5
6
7
8
0
pFI Fig. IO. T h e effect of p H on m e t a b o l i c and t r a n s p o r t parameters. 0. 5 % D-arabinose was used for e s t i m a t i n g t h e s t e a d y - s t a t e SI/So ratio ( A - - ~ k ) , as welt as t h e 002 ( O - - O ) and Qco2 ( O - - O ) q u o t i e n t s as d e t e r m i n e d b y t h e c o n v e n t i o n a l W a r b u r g m a n o m e t r i c t e c h n i q u e (expressed as ~1 gas per m g d r y wt. per h). TABLE III EFFECT
OF
ALKALINE
IONS
ON
THE
STEADY-STATE
RATIO
OF
L-XYLOSE
I n c u b a t i o n w a s carried o u t aerobically at 28 ° for 6 h w i t h o.I % L-xylose. The higher v a l u e obt a i n e d in w a t e r is a p p a r e n t l y due to o s m o t i c swelling of cells.
Medium composition
St/So
Water o.I M K H 2 P O 4 33 mM N a H 2 P O 4 plus 66 mM K H 2 P O a 66 mM N a H 2 P O 4 plus 33 mM K H 2 P O 4 o.i M N a H 2 P O 4
5-45 5.25 5.15 5.9o
7.2
It should be noted here that the intracellular pH of R. gracilis cells, both in the presence and absence of glucose, was found to be 6.o6-6.1o, i.e. higher than either the metabolic or the transport optimum. The pH of the external medium during this Biochim. Biophys. Acta, lO2 (1965) 41o-422
UPHILL TRANSPORT OF SUGARS
419
measurement was 4-5-4-7 su that it appears that the intracellular milieu maintains a pH independent of that of the outside medium (cf. ref. IO).
Effect of alkaline ions Since examples of uphill transport have been described which require either Na + or Na + and K + for function (cf. refs. I4, 15) it was tested whether the transport observed here exhibits such a dependence. Table 111 shows that Na + and K+ are devoid of any influence on the St/So ratio of L-xylose. The rates of uptake, too, were completely identical irrespective of the ion composition of the medium. DISCUSSION The mechanism of the uphill transport observed in R. gracilis poses numerous problems as it possesses features not previously found to be present together in any other organism. The sugar is accumulated against a considerable concentration gradient but it is in free solution inside the cell (cf. Figs. 3 and 6). Hence it must be the membrane that accounts for the final asymmetry of distribution. Fundamentally, the membrane can transport substrates either by transient attachment to a mobile carrier or by movement of substrate molecules along an array of adsorption sites 1~ (if we disregard simple diffusion and pinocytosis which certainly are of no importance in sugar transport). However, the adsorption-site mechanism Call hardly be envisaged to mediate uphill transport. Higher intracellular substrate concentration would be possible only if the adsorption sites opening toward the cell interior had a lower affinity for substrate than the external ones, and the question then arises why external substrate should move toward the interior at all. The only remaining possibility hitherto considered in the literature is a mobile carrier functioning generally on the principle described b y ROSENBERG AND WILBRANDT3 (the permease concept would also fall into this category). Here the mobile carrier can exist in two forms, e.g. C and Z, these forms having different affinities for substrate and being interconvertible. Moreover, metabolic reaction coupling is required for at least one of the conversion reactions, C -+ Z or Z -+ C. If, for instance, Z has a lower affinity for substrate and if it predominates over C on the internal side of the membrane, in the steady state there will be more substrate intracellularly than there is on the outside. Apart from the steady-state St/So ratio which is different from I, the system would be expected to possess all the features of equilibrating carrier transport, one of the more important being counter-transport. R O S E N B E R G AND WILBRANDT3 predict counter-transport to occur in the case where the final Si/So is independent of concentration. If, however, the St/So ratio decreases with rising S (as is actually observed experimentally), due to insufficient capacity of the metabolic coupling reactions to replenish the required form of carrier, the conditions for the occurrence of counter-transport are more complex as discussed in the following. Let Ro and Rt be the concentrations of a labelled sugar permitted to reach steady state and So and St the concentrations of non-labelled sugar added in steady state of R; let Kz be the dissociation constant of carrier form Z with either S or R; Kc, that for the carrier form C ; q, the ratio Kz/Kc; ~, the apparent Michaelis constant of transport; A and B, constants associated with the interconversion of carrier forms Biochim. Biophys. Acta, lO2 (1965) 41o-422
420
A. KOTYK, M. HOFER
Z 1 ~ C 1 a n d w i t h their diffusion (cf. ref. 3) a n d V the a p p a r e n t m a x i m u m r a t e of t r a n s p o r t (when mobilities of b o t h carriers, free a n d loaded, are considered equal). The r a t e of m o v e m e n t of R will t h e n be given b y dR V[ R0 d--t- = vn = )?o + So + Kz/q(ARo + ASo + B)
Ri ] Ri + St + Oi
a n d t h e r a t e of m o v e m e n t of S b y dS dl -- v,s
V[
So So + Ro + Kz/q(ASo + ARo + B)
Si ] Si @ Ri 47, Oi
I t can be easily shown t h a t vR = vs = o after t h e o r e t i c a l l y infinite time, when b o t h s u b s t r a t e s reach a r a t i o corresponding to Ri/Ro : Si/So :
0i Kz/q(ARo + ASo + B)
This is lower t h a n the r a t i o RI/Ro prior to a d d i t i o n of S b y an a m o u n t r e l a t e d to i n t r o d u c i n g ASo into the d e n o m i n a t o r of t h e foregoing expression. I t was t h e n t e s t e d w h e t h e r t h e r e is a t i m e o t h e r t h a n infinite t i m e where vn = o (but vs # o), this c o r r e s p o n d i n g to a m i n i m u m in t h e curve of Rl a n d indic a t i n g the presence of c o u n t e r - t r a n s p o r t , even u n d e r the conditions where carrier i n t e r c o n v e r s i o n is limiting at higher s u b s t r a t e concentration. The a b o v e differential e q u a t i o n s were therefore solved a n a l y t i c a l l y a n d the solution checked on a D i a n a analogue c o m p u t e r , using a v a r i e t y of values for the different c o n s t a n t s (Kz, q, A, B a n d 0i) as well as different Ro a n d So. F o r all sets of values yielding Rl/Ro ratios b o t h before a d d i n g S a n d in the final s t e a d y - s t a t e condition such as f o u n d experim e n t a l l y , the function of RI d i s p l a y s a m i n i m u m before reaching its final value. The m i n i m u m is i n d e e d not as p r o n o u n c e d as in an e q u i l i b r a t i n g system. I n the best case it lies a b o u t 2o % below the final level which, moreover, it t a k e s v e r y long to reach, so t h a t the rise of the Ri curve after its m i n i m u m is v e r y gentle. Hence it is questionable w h e t h e r the a c c u r a c y of the e m p l o y e d a n a l y t i c a l m e t h o d s was sufficient to observe the r a t h e r u n p r o n o u n c e d m i n i m u m p r e d i c t e d b y theory. The n u m b e r of e x p e r i m e n t s p e r f o r m e d (14) a n d the absence of a n y i n d i c a t i o n of a c o u n t e r - t r a n s p o r t m i n i m u m would, however, f a v o u r an a l t e r n a t i v e e x p l a n a t i o n , according to which the c o u n t e r - t r a n s p o r t m i n i m u m would be lost c o m p l e t e l y a n d which w o u l d even b e t t e r explain some of t h e a b o v e - r e p o r t e d findings. Let us assume t h e existence of two discrete carriers in the m e m b r a n e , mobile p r o b a b l y b y v i r t u e of r o t a t i o n w i t h i n the m e m b r a n e , each of which w o u l d be capable of t r a n s p o r t i n g in v i r t u a l l y one direction only. The affinity of the " i n w a r d " carrier (I) for s u b s t r a t e would be g r e a t e r t h a n t h a t of t h e " o u t w a r d " one (II) so t h a t K I
421
U P H I L L T R A N S P O R T OF S U G A R S
petitive and governed by the same affinity constant as the reaction with substrate to be transported ( K s z K i , s = K ) , the rate of movement of substrate S is given by vs = V1 So/KI + Si/K~ + ~ - - ~ ~
~/K~
+ So/KI~ +
It may be calculated that the outward movement of S occurring on adding another competing substrate in steady state of S will be very slow and will produce no observable minimum. In the latter steady state S l # S o but rather Si =
S o ( ~ - - VII) - - K I V I I + ~/(So(V1 - - VII) - - KIVII) 2 + 4ViVii(82o + SoKH) 2 VH
Furthermore, this mechanism permits a calculation to show that at higher substrate concentrations the final SI/So ratio will be smaller than at lower ones (cf. Figs. I and 2), tending toward unity at very high concentrations of VI = VII without the necessity of invoking metabolic limitation at the higher concentrations. This hypothesis may also account for the anomalous behaviour of glucose anaerobically by assuming that glucose is bound to the inward carrier and immobilizes it without affecting the outward one (cf. Fig. 5). Differences in aerobic and anaerobic concentration ratios, which are doubtless due to decreased resources in metabolic energy anaerobically, are apparently brought about by the fact that at least one of the carriers (probably the one transporting outwards since the "inward" constants are very similar both in 0 2 and N2; cf. Table I) must be activated to raise or lower its affinity for substrate. Similarly, the effect of various metabolic inhibitors can be envisaged to take place at this activating reaction (or reactions) and/or at the carrier molecule itself, thereby changing either the maximum rate of transport or the apparent effective affinity or both. Since this effect may be quantitatively or even qualitatively different on each of the two carriers it is not surprising that a ratio of SI/So ---- I is not naturally attained, as the case is in Escherichia coliiT, is, when energy sources are cut off. The "two-carrier" hypothesis advanced here is readily reconcilable with the general asymmetry of the membrane 19 in which movement in one direction with substrate and in the other direction without it can take place, especially if metabolic coupling is involved. It would be of interest to establish whether the proposed mechanism would apply in S . f a e c a l i s as described by WILKINS AND O'KANE4. It seems to the present authors that the findings made with S. faecalis can all be accounted for by the "twocarrier" mechanism although differences in affinities and inhibition constants doubtless exist. It will have to be seen in the future whether a rigorously selective use of inhibitors will provide evidence for or against this hypothesis. There are indications that the carrier(s) may be de-repressed (anomalous S-shaped curves of uptake are occasionally observed, particularly after prolonged anaerobiosis) but no studies in this direction have been undertaken so far (cf. ref. 20). A "glucose effect" in this connection could account for some of the anomalies of glucose-inhibited sugar uptake. The chemical character of the carrier(s) has not been explored but there is evidence that at least for the inward flux a single site with sugar affinity is present Biochim. Biophys. Acta, lO2 (1965) 41o-422
422
A. KOTYK, M. HOFER
and that the molecule interacts directly with H + ions and some of the inhibitors used (NAN3, iodoacetamide). ACKNOWLEDGEMENTS
The authors are indebted to Professor A. KLEINZELLER for stimulating comments and frequent consultations, and to Mrs. E. HOROV£ and Mrs. L. RiHOV£ for skilled technical help. The differential equations were solved and programmed for the analogue computer by Dr. 0. SCHMIDT of the College of Chemical Technology and his help in this respect was of fundamental importance. ~EFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21
A. KLEINZELLER AND L. ~LECHTA,Chem. Listy, 48 (1954) 602. J. H. LITCHFIELD AND Z. J. ORDAL, Can. J. Microbiol., 4 (1958) 205TH. ROSENBERG AND W. WILBRANDT, J. Theoret. Biol., 5 (1963) 288. P. O. WILKINS AND D. J. O'KANE, J. Gen. Microbiol., 34 (1964) 389. J. VAN STEVENINCK AND H. L. BOOIJ, J. Gen. Physiol., 48 (1964) 43. M. SOMOGYI, J. Biol. Chem., 195 (1952) 19. M. NELSON, J. Biol. Chem., 153 (1944) 375. W . W . UMBREIT, 1:~. H. BURRIS AND J. P. STAUFFER, Manometric Techniques, Burgess, Mi nne a polis, 1957, p. 274. H. O. LOWRY, •. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . A. IXOTYK, Folia Microbiol. Prague, 8 (1963) 27. A. I~LEINZELLER, :~Ianometriekd 2letody, SZN, P r a h a , 1954; Manometrische Methoden, G. Fischer-Verlag, Jena, 1966, in t h e press. W. WILBRANOT AND A. I~OTYK, Naunyn-Schmiedebergs Arch. Exptl. Pathol. Pharmakol., 249 (1964) 279. A. I~OTYK, Folia Microbiol. Prague, i o (1965) 3 o. R. K. CRANE, Physiol. Rev., 4 ° (196o) 789. A. I~LEINZELLER AND A. KOTYK, Biochim. Biophys. Acta, 54 (1961) 367. TH. ROSENBERG AND W. WILBRANDT, J. Gen. Physiol., 41 (1958) 289. A. KEPES, Biochim. Biophys. Acta, 4 ° (196o) 7 o. A. L. KOCH, Biochim. Biophys. Acta, 79 (1964) 177. P. MITCHELL, in A. KLEINZELLER AND A. KOTYK, Membrane Transport and Metabolism, Acad elnic Press, L o n d o n , 1961, p. 22. H. OKADA AND H. O. HALVORSON, Biochim. Biophys. Acta, 82 (1964) 538. H. OKADA AND H. O. HALVORSON, Biochim. Biophys. Acta, 82 (1964) 547.
Biochim. Biophys. Aeta, lO2 (1965) 41o 422
BIOCHIMICA ET BIOPHYSICA ACTA
UBA 45200 UPHILL TRANSPORT
OF SUGARS IN THE YEAST
RHODOTORULA GRACILIS A R N O ~ T K O T Y K AND M I L A N H(I~FER
Laboratory for Cell Melabolism, Dzstitute of .,>Iicrobiology, Czechoslovak .4 cademy of Sciences, Prague (Czechoslovakia) ( R e c e i v e d J a n u a r y 26th, 1965)
SUMMARY
T h e lipid-forming y e a s t Rhodotorula gracilis was found to t r a n s p o r t b o t h met a b o l i z a b l e a n d n o n - m e t a b o l i z a b l e sugars against a c o n c e n t r a t i o n gradient. The process a p p e a r s to require m e t a b o l i c energy b u t o p e r a t e s to a l i m i t e d e x t e n t even a n a e r o b i c a l l y when no gas exchange a n d s u b s t r a t e u t i l i z a t i o n are d e t e c t a b l e in the cells. The a c c u m u l a t e d sugar is present i n t r a c e l l u l a r l y in an osmotically active s t a t e a n d is r e a d i l y e x c h a n g e a b l e for e x t e r n a l sugar. The t r a n s p o r t is p H - d e p e n d e n t with an o p t i m u m near p H 5 b u t it is N a +- a n d K+-independent. A carrier s y s t e m a p p e a r s to be i n v o l v e d which t r a n s p o r t s I sugar molecule at a t i m e a n d possesses different effective affinities for s u b s t r a t e s at the two sides of the membrane. Of all t h e sugars t e s t e d only D-glucose does not p e n e t r a t e the cell a n a e r o b i c a l l y a l t h o u g h it p r e v e n t s the t r a n s p o r t of o t h e r sugars.
INTRODUCTION
KLEINZELLER AND SLECHTA1 a n d l a t e r LITCHFIELD AND ORDAL2 found t h a t the lipid-forming r e d y e a s t Rhodotorula gracilis only m e t a b o l i z e d sugars aerobically a n d the m i c r o o r g a n i s m was therefore e x a m i n e d to see w h e t h e r this is due to a m e t a b o l i c deficiency or to a block in the m e m b r a n e w i t h respect to sugar t r a n s p o r t . W h i l e it now can be said conclusively t h a t the t r a n s p o r t step is not limiting, either aerobically or anaerobically, j u d g m e n t will be reserved for t h e future to decide on the n a t u r e of the m e t a b o l i c block causing the i n a b i l i t y of R. gracilis to utilize sugars anaerobically. The t r a n s p o r t of sugars, however, w-as f o u n d to possess an u n e x p e c t e d feature which, a p a r t from its heuristic value, m i g h t be m a d e use of for t e s t i n g some recent h y p o t h e s e s of sugar t r a n s p o r t 3-5, viz. an intrinsic a s y m m e t r y causing sugars to be t r a n s p o r t e d uphill into the cell. The p r e s e n t p a p e r deals with the kinetics of the process, a n d is to be followed b y a s t u d y of its m e t a b o l i c coupling a n d of sugar m e t a b o l i s m in R. graciIis as a whole. Biochim. Biophys..4cta, lO2 (1965) 41o-422
UPHILL
TRANSPORT
OF SUGARS
411
MATERIALS AND METHODS
A collection strain of R. gracilis (taxonomically R. glutinis 5/Fres/Harrison) was grown at 3 °0 on a reciprocal shaker in the following medium: 4° g glucose, 0.66 g NH4N03, I.O g K2HPO 4, 0.5 g NaC1, I.O g MgSO4.7H~O, 0.25 g CaC12, 0.05 g FeCls.6H~O, in a total volume of IOOO ml. The pH was adjusted to 5-5 with HC1 after sterilization and a trace of yeast extract added before cultivation. Cells were harvested after 24 h, washed several times with distilled water and shaken for 2 h in suspension as it was found that subsequently they were much easier to filter. Cell suspensions were prepared in distilled water or in o.I M KH~PO~ (no difference in behaviour being found in the two media) and incubated at 28 ° either in 0 2 or in highly purified N 2 with the appropriate sugar and inhibitor concentrations. The density of the yeast suspension varied between 4 and IO mg dry wt./ml. Samples were removed at regular intervals to be filtered through a Millipore HA filter. After washing the pellet twice with ice-cold water it was suspended in boiling water to extract cell solutes and the volume made up to a mark. The aqueous extract was deproteinized with ZnSO 4 and Ba(OH)~ and sugars estimated by standard methods: hexoses according to SOMOGYI6 and NELSON7, pentoses according to ME1JBAUM (see ref. 8), radioactive sugars in a methane-flow ~'rieseke-Hoepfner counter after drying on aluminium planchets or in a Tracerlab liquid scintillation counter. Cell water content was determined by accurate hematocrit measurements, using serum albumin to assay the extracellular space. Serum albumin was estimated according to the method of LOWRY et al. 9. It deserves mentioning here that application of inulin, methylene blue or erythrosine B yielded excessively high extracellular space values, apparently due to surface adsorption of the compounds on R. gracilis cells. It was found that the intraceflular water volume corresponds to 69-72 % of the total when suspended in distilled water (cf. Fig. 3)The intracellular pH was estimated by using the weakly acid dye, bromophenol blue, as described earlier 1°. Non-labelled D-glucose, D-arabinose, D-galactose, D-fructose, D-mannose and D-ribose were products of Spofa, Czechoslovakia; D-xylose, L-xylose and L-rhamnose of Hoffmann-LaRoche, Switzerland; D-E14C]xylose was obtained from the Radiochemical Centre, Amersham, Great Britain and L-[14C]rhamnose was very kindly prepared by Dr. J. MAJER of this Institute. All other reagents were commercial products of analytical purity. RESULTS
Uptake of sugars The yeast strain used was found to take up all the sugars tested, viz. D-glucose, D-fructose, D-mannose, D-galactose, D-arabinose, D-xylose, L-xylose, D-ribose and L-rhamnose. Of these, D-glucose, D-fructose, D-mannose, D-galactose and D-xylose were aerobically metabolized with Qo~ at 28 ° ranging from 28 to 40/A 0 2 per mg dry wt. per h, this being only slightly in excess of endogenous metabolism where Qo2 values between I6 and 20 are generally found. D-Arabinose produced a Qo2 of 20 Biochim. Biophys. Acta, lO2 (1965) 4 1 o - 4 2 2
412
A. KOTYK, M, HOFER
as compared with 16.5 in the endogenous control. It appears that endogenous metabolism proceeds undiminished in the presence of external substrate (cf. K L E I N Z E L L E R 11, and measurements of sugar utilization from the medium with concomitant Qo2 and Qco2 values) so that the Qo~ attributable to external sugar utilization varied between 4 and 24.
Uphill transport The kinetic parameters of uptake, the apparent K m , l n , Win as well as the maximum ratio of internal to external substrate concentration (Sl/So), in the steady state attainable at very low concentrations, were measured with D-arabinose, I)-xylose, L-rhamnose and D-glucose, the results being summarized inTable I and in Figs. I and 2. It m a y be seen that the apparent Kin,In and Vln vary for different sugars but that the change from aerobic to anaerobic conditions does not bring about any drastic changes in the constants, the apparent affinity being actually either higher or lower in N 2 than in 0 2 (glucose behaves anomalously in that it is not transported anaerobically at all). On the other hand, the maximum attainable ratio of Sl/So not only TABLE I TRANSPORT
PARAMI~TERS
OF
Sugar
VARIOUS
SUGARS
Gaseous phase
n-Xylose D- Arab inose L-Rhamnose D-Glucose
R.
gracilis
Appare~t Kin,in (M)
Vln
(rag per g dry wt. per min)
02 No 02 N2 Oo N2 02
2" 10 -3 8- l o -4 4.1. l o 2 1.3" Io -1 3-4" lo-3 4-4" lo-3 5-5" Io ~
3.21 2.82 4.o3 4.27 0.55 o.19 2.98
N 2
--
--
_____.__--c.-----
100
IN
Maximum St~So
IOOO 3oo 4o 20 90 Io i .8 o
_____..__---<>-
"5 80
60
40
E P
O0
10
20
30
(So)(mglmu) Fig. I. R e l a t i o n s h i p b e t w e e n e x t e r n a l (So) a n d i n t r a c e l l u l a r (St) c o n c e n t r a t i o n s of D-xylose a f t e r r e a c h i n g s t e a d y s t a t e a e r o b i c a l l y ( O - - O ) or a n a e r o b i c a l l y ( O - - O ) a t 28 °. T h e b r o k e n s t r a i g h t line c o r r e s p o n d s to St/So = I, as w o u l d o b t a i n in a diffusion e q u i l i b r i u m .
Biochim. Biophys. dcta, lO2 (1965) 4 i o - 4 2 2
413
UPHILL TRANSPORT OF SUGARS
strikingly varies from sugar to sugar but is always significantly lower anaerobically, apparently since less energy is expendable for its maintenance under anaerobic conditions. This is most likely reflected in the parameters for sugar efflux, which is unfortunately not readily amenable to investigation on account of its rapidity and the difficulty of attaining sufficiently low intracellular steady-state concentrations. It should be noted that even with D-xylose, which is metabolized under aerobic conditions, a considerable concentration gradient is built up intracellularly.
"C
6O
b 4O
P
~
~o
E
0 0
i
i
i
10
20
30
(So)(mg/ml) Fig. 2. The Sl/So relationship for o-arabinose after aerobic ( O - - O ) or anaerobic ( O - - O ) incubation. Other details as for Fig. i. 18
7O
%] ol o c
~o
10
30
t_
L D
6
..o-
lo _E
2 0 0
i Q1
I Q3
i
I
i
Q5
07
0.9
NQCI (M)
Fig. 3. Correlation between the intracellular amount of L-rbamnose ( O - - - O ) and the intracellular water content ( O - - 0 ) after a 3-h aerobic incubation with 0.5 % L-rhamnose in different concentrations of NaC1 at 28 °.
The relationship between membrane carrier and intracellular adsorption Since u n e q u a l d i s t r i b u t i o n of s u b s t r a t e b e t w e e n cell a n d m e d i u m c o u l d b e acc o u n t e d for e i t h e r b y i n t r a c e l l u l a r a d s o r p t i o n or b y a s y m m e t r i c m e m b r a n e p r o p e r t i e s , t h e s t e a d y - s t a t e i n t r a c e l l u l a r a m o u n t s of L - r h a m n o s e w e r e e s t i m a t e d u s i n g d i f f e r e n t c o n c e n t r a t i o n s of NaC1 in t h e m e d i u m , t h u s b r i n g i n g a b o u t d i f f e r e n t d e g r e e s of cell v o l u m e s h r i n k a g e . F i g . 3 s h o w s t h a t t h e r e is g o o d a g r e e m e n t b e t w e e n t h e a m o u n t of i n t r a c e l l u l a r w a t e r a n d t h e a m o u n t of s u g a r p r e s e n t in t h e cell. T h e f i n d i n g t h u s
Biochim. Biophys. Acta, lO2 (1965) 41o-422
414
A. KOTYK, M. HOFER
strongly supports the view that the sugar inside the cell is in an osmotically active form and that the asymmetric distribution is caused by properties of the cell membrane.
Exchangeability of intracellular sugar Since WILKINS AND O'IKANEa observed in Streptococcus faecalis that, once accumulated, sugar is not freely exchangeable with sugar added to the medium, cells 3~0
--Q
2
b f
2000
E
o
i 0
0
i 30
-
w
i 60
i 90
o
-
i 120
Time (rain)
Fig. 4. Efflux of D-[14C]xylose from R . gracilis. The cells were p r e - i n c u b a t e d aerobically at 28 ° for 3 h w i t h t w o different c o n c e n t r a t i o n s of labelled D-xylose (curve 1: o.O4~o, curve 2: 0. 4 ~o), centrifuged and at zero t i m e resuspended in w a t e r ( O - - O ) or non-labelled D-xylose ( @ - - @ ) at a c o n c e n t r a t i o n corresponding to the intracellular one.
25
I_
=
20 D
2
~5
o 21
5
0
L 0 Time
i
J
I
20
40
60
(min)
Fig. 5. Effect of D-glucose on efflux of L-xylose from R . gracilis. Cells were i n c u b a t e d w i t h 0. 5 ~o L-xylose for 5 h aerobically or anaerobically. At zero t i m e t h e centrifuged pellet w a s resuspended in w a t e r ( O , G) or o.I ~o D-glucose (@, ~k).
were preloaded with labelled D-p*C]xylose and, after centrifuging, resuspended in water or in D-[l=C]xylose or D-[12C]glucose. The results are shown in Figs. 4 and 5The rapid drop observed on suspending in water corresponds to a practically unidirectional flux of D-[14C]xylose out of the cells until a new steady state is reached. This is rather high since cells accumulate practically all sugar then available: that retained by cells and also that from the intercellular space after centrifugation. The somewhat slower decrease observed on suspending cells in unlabelled xylose is due Biochim.
Biophys. Acta, lO2 (1965) 4IO-422
415
UPHILL TRANSPORT OF SUGARS
to a higher saturation of the carrier when the attainment of equilibrium is slower but the drop is greater because unlabelled xylose replaces most of the labelled xylose in cells and medium and only labelled xylose is being determined. The lack of effect of glucose on anaerobic transport out of cells seems to indicate that glucose acts only on the outside reaction of the carrier with sugar. The ready availability of sugar in the cell is further demonstrated by changing from aerobic to anaerobic conditions during the steady state, when a rapid escape of sugar from cells takes place until a new steady state corresponding to anaerobiosis is reached (Fig. 6). [
xJ
2000
.~-~ 1500
E iooo
50O
01
I
;
0
0 Time
i
I
60
90
l 120
I 150
(min)
Fig. 6. Change of intracellular level of sugar on changing from aerobic to anaerobic conditions. Cells were incubated aerobically at 28 ° w i t h o . I % ( • - - • ) or o.4 % ( O - - O ) D- [14C]xylose for 3 h and then at zero time the 02 supply w a s cut off and replaced b y a powerful stream of N 2. "6
--o
B --,o
8OOO
--o
2 20
>~ 6000
S c
2
E 4000
--o
25
---A
o
o
&
2 2000
-A
10
L
t
20
30
t 40
//
// -'T-t
0 360
0
10
20
30
40
360
Tirne(min)
Fig. 7. Competition of sugars for entry into R. gracilis ceils. For Fig. 7 A, the suspension w a s incubated w i t h 0.45 % D-arabinose aerobically (curves I, 2) or anaerobically (curves 3, 4) w i t h o u t (curves i, 3) or after (curves 2, 4) a Io-min pre-incubation with o . i % D-glucose. For Fig. 7]3, t h e suspension w a s incubated with o.I % D-[14C]xylose aerobically (curves i, 2) or anaerobically (curves 3, 4) w i t h o u t (curves I, 3) or w i t h (curves 2, 4) simultaneous addition of 1% L-xylose. T h e dotted lines in both parts of the figure correspond to a diffusion equilibrium. I t is n o t e w o r t h y t h a t in the presence of D-glucose anaerobically the level of D-arabinose does not even reach t h e equilibrium ratio St~So = i. Biochim. Biophys. Acta, lO2 (1965) 4 1 o - 4 2 2
416
A. KOTYK, M. H O F E R
Con@etition between sugars It was shown that, e.g., D-xylose and L-xylose compete for the same carrier and that o-glucose competes with D-arabinose for uptake (Fig. 7). Hence it is concluded that the sugars tested here share one carrier although the existence of other additional carriers is not hereby excluded.
Counter-transport One of the features of transport mediated by a mobile carrier is the existence of counter-transport of a substrate in equilibrium with cells on adding another substrate to the medium. R. gracilis cells were therefore incubated with labelled m-xylose
• /
10000 ~
~
~
~1
o
,:,
o
n
~ 7500
P 5000 o 2500
I
0
310
610
910
I
I
120
150
Tirne(min)
F i g . 8. A b s e n c e of c o u n t e r - t r a n s p o r t i n R. gracilis. Cells w e r e p r e - i n c u b a t e d a e r o b i c a l l y a t 28 ° w i t h o . i % D-[14C]xylose a n d a f t e r 2 h (at zero t i m e ) o n e - t e n t h of t h e v o l u m e of w a t e r ( c u r v e i), 2 °/o n o n - l a b e l l e d D - x y l o s e ( c u r v e 2) or 2 ~o D - g l u c o s e ( c u r v e 3) w a s a d d e d .
1.8
15
................
....
~ 1.2 -J
Q9
06 -2A
i -2:1
i -1.9
F i g . 9. D e m o n s t r a t i o n
r -1.6
i -1.3
i -1D
i -07
Log(S) of the number of sugar molecules attached to the carrier. Using suitable
v a l u e s of t h e d i s s o c i a t i o n c o n s t a n t s b e t w e e n c a r r i e r a n d s u g a r , t h e o r e t i c a l c u r v e s w e r e o b t a i n e d for t h e t r a n s p o r t of a s i n g l e m o l e c u l e a t t a c h e d t o t h e c a r r i e r ( d a s h e d c u r v e i) or of 2 m o l e c u l e s a t t a c h e d a t a t i m e ( d o t t e d c u r v e 2) ; cf. ref. 12. T h e s o l i d c i r c l e s a r e e x p e r i m e n t a l v a l u e s o b t a i n e d w i t h L - r h a m n o s e a f t e r p r e - i n c u b a t i n g a cell s u s p e n s i o n a e r o b i c a l l y for 3 h w i t h d i f f e r e n t c o n c e n t r a t i o n s of n o n - l a b e l l e d L - r h a m n o s e , t h e n a d d i n g a n a n a l y t i c a l l y n e g l i g i b l e a m o u n t of L-[14C] r h a m n o s e a n d e s t i m a t i n g t h e h a l f - t i m e of u p t a k e of t h e l a t t e r . L o g S r e f e r s t o t h e c o n c e n t r a t i o n of n o n - l a b e l l e d s u g a r , log vR t o t h e r a t e of u p t a k e of t h e l a b e l l e d one.
Biochim. Biophys. ~Icta, I o 2 (1965) 4IO 422
UPHILL
TRANSPORT
417
OF SUGARS
in concentrations ranging from o.o5 % to 4 % until steady state was reached (3-6 h) and non-labelled D-xylose or D-glucose was then added to a final concentration of 1- 4 %, both aerobically and anaerobically. The content of the label in cells was then examined as shown by sample curves in Fig. 8. The intracellular label invariably dropped to a lower level, as one would expect from the following consideration: On adding non-labelled sugar to the steady-state mixture the total concentration of sugar increases and the SI/So ratio in the final steady state is lower, as expected (cf. Figs. i and 2). Hence the level of labelled sugar must also decrease to a correspondingly lower value. However, no unambiguous minimum in the internal sugar level could be observed in the numerous experiments, contrary to observations where a pronounced counter-transport takes place (e.g. ref. 12).
Number of sugar molecules attached per carrier A test described by WILBRANDTAND KOTYK 12, to distinguish between transport where one or two substrate molecules are attached to the carrier, was applied to the transport of L-rhamnose. The results shown in Fig. 9 indicate an exclusive or strongly predominant mono-complex transport (cf. ref. 13).
Effect of metabolic inhibitors Of the various kinetic constants examined here the steady-state ratio of intracellular to extracellular sugar appeared to be most straightforward in connection with the use of metabolic blocking agents. Table I I shows the results obtained with TABLE
II
EFFECT OF METABOLIC INHIBITORS ON STEADY-STATE RATIO OF L-XYLOSE Incubation
w a s c a r r i e d o u t a t 28 ° f o r 6 h w i t h 0 . 2 5 % L - x y l o s e .
Inhibitor
Final concentration
(M)
None Iodoacetamide
5" i o 4
2,4-Dinitrophenol
5" l O - 4
Sodium azide Iodoacetamide Oligomycin
lO -3 plus 2,4-dinitrophenol
5" IO-4 _[_ 5 ' l O - 4 I. 5 t t g / m l
Gaseous phase
SI/So
0.2
6.0 4.2 3.1 0.72 0.3 0.45 0.34 o.I o o 4-3
N2 Oz N2 O2 N~ O2 N2 02 N2 02
L-xylose. It will be seen that none of the inhibitors reduced the ratio to I as would be expected in an equilibrating transport when all energy sources are cut off. The effect is rather varied, indicating that the inhibitors may interact directly with one or more carriers involved and/or that different types of metabolic coupling are in operation--apparently no metabolic pump is simply superimposed here over equilibrating carrier transport. Biochim. Biophys. Acta, lO2 (1965) 4 1 o - 4 2 2
418
A. KOTYK, M. HOFER
Effect of pH Cells were incubated in o.15 M citrate-phosphate or Tris-HC1 buffers in the pH range 3 to 8 with D-arabinose. The apparent Km,ln, Vln, Si/So ratio and metabolic parameters were measured. Fig. IO shows that pH 5 is near the optimum for the accumulation ratio, both aerobically and anaerobically. The apparent K i n , i n w a s found to be lowest (corresponding to highest effective affinity) at pH 5 while the Vin was highest also at pH 5. However, the metabolic optimum seems to lie near pH 4. This might suggest that the carrier itself is an enzyme with dissociable protons or can undergo allosteric configurational change linked with the attachment of H+ ions.
4o _=
~
40
r
! J
3oi
3O
c~
o ~5 d q0
E
I
1
r
I
I
I
3
4
5
6
7
8
0
pFI Fig. IO. T h e effect of p H on m e t a b o l i c and t r a n s p o r t parameters. 0. 5 % D-arabinose was used for e s t i m a t i n g t h e s t e a d y - s t a t e SI/So ratio ( A - - ~ k ) , as welt as t h e 002 ( O - - O ) and Qco2 ( O - - O ) q u o t i e n t s as d e t e r m i n e d b y t h e c o n v e n t i o n a l W a r b u r g m a n o m e t r i c t e c h n i q u e (expressed as ~1 gas per m g d r y wt. per h). TABLE III EFFECT
OF
ALKALINE
IONS
ON
THE
STEADY-STATE
RATIO
OF
L-XYLOSE
I n c u b a t i o n w a s carried o u t aerobically at 28 ° for 6 h w i t h o.I % L-xylose. The higher v a l u e obt a i n e d in w a t e r is a p p a r e n t l y due to o s m o t i c swelling of cells.
Medium composition
St/So
Water o.I M K H 2 P O 4 33 mM N a H 2 P O 4 plus 66 mM K H 2 P O a 66 mM N a H 2 P O 4 plus 33 mM K H 2 P O 4 o.i M N a H 2 P O 4
5-45 5.25 5.15 5.9o
7.2
It should be noted here that the intracellular pH of R. gracilis cells, both in the presence and absence of glucose, was found to be 6.o6-6.1o, i.e. higher than either the metabolic or the transport optimum. The pH of the external medium during this Biochim. Biophys. Acta, lO2 (1965) 41o-422
UPHILL TRANSPORT OF SUGARS
419
measurement was 4-5-4-7 su that it appears that the intracellular milieu maintains a pH independent of that of the outside medium (cf. ref. IO).
Effect of alkaline ions Since examples of uphill transport have been described which require either Na + or Na + and K + for function (cf. refs. I4, 15) it was tested whether the transport observed here exhibits such a dependence. Table 111 shows that Na + and K+ are devoid of any influence on the St/So ratio of L-xylose. The rates of uptake, too, were completely identical irrespective of the ion composition of the medium. DISCUSSION The mechanism of the uphill transport observed in R. gracilis poses numerous problems as it possesses features not previously found to be present together in any other organism. The sugar is accumulated against a considerable concentration gradient but it is in free solution inside the cell (cf. Figs. 3 and 6). Hence it must be the membrane that accounts for the final asymmetry of distribution. Fundamentally, the membrane can transport substrates either by transient attachment to a mobile carrier or by movement of substrate molecules along an array of adsorption sites 1~ (if we disregard simple diffusion and pinocytosis which certainly are of no importance in sugar transport). However, the adsorption-site mechanism Call hardly be envisaged to mediate uphill transport. Higher intracellular substrate concentration would be possible only if the adsorption sites opening toward the cell interior had a lower affinity for substrate than the external ones, and the question then arises why external substrate should move toward the interior at all. The only remaining possibility hitherto considered in the literature is a mobile carrier functioning generally on the principle described b y ROSENBERG AND WILBRANDT3 (the permease concept would also fall into this category). Here the mobile carrier can exist in two forms, e.g. C and Z, these forms having different affinities for substrate and being interconvertible. Moreover, metabolic reaction coupling is required for at least one of the conversion reactions, C -+ Z or Z -+ C. If, for instance, Z has a lower affinity for substrate and if it predominates over C on the internal side of the membrane, in the steady state there will be more substrate intracellularly than there is on the outside. Apart from the steady-state St/So ratio which is different from I, the system would be expected to possess all the features of equilibrating carrier transport, one of the more important being counter-transport. R O S E N B E R G AND WILBRANDT3 predict counter-transport to occur in the case where the final Si/So is independent of concentration. If, however, the St/So ratio decreases with rising S (as is actually observed experimentally), due to insufficient capacity of the metabolic coupling reactions to replenish the required form of carrier, the conditions for the occurrence of counter-transport are more complex as discussed in the following. Let Ro and Rt be the concentrations of a labelled sugar permitted to reach steady state and So and St the concentrations of non-labelled sugar added in steady state of R; let Kz be the dissociation constant of carrier form Z with either S or R; Kc, that for the carrier form C ; q, the ratio Kz/Kc; ~, the apparent Michaelis constant of transport; A and B, constants associated with the interconversion of carrier forms Biochim. Biophys. Acta, lO2 (1965) 41o-422
420
A. KOTYK, M. HOFER
Z 1 ~ C 1 a n d w i t h their diffusion (cf. ref. 3) a n d V the a p p a r e n t m a x i m u m r a t e of t r a n s p o r t (when mobilities of b o t h carriers, free a n d loaded, are considered equal). The r a t e of m o v e m e n t of R will t h e n be given b y dR V[ R0 d--t- = vn = )?o + So + Kz/q(ARo + ASo + B)
Ri ] Ri + St + Oi
a n d t h e r a t e of m o v e m e n t of S b y dS dl -- v,s
V[
So So + Ro + Kz/q(ASo + ARo + B)
Si ] Si @ Ri 47, Oi
I t can be easily shown t h a t vR = vs = o after t h e o r e t i c a l l y infinite time, when b o t h s u b s t r a t e s reach a r a t i o corresponding to Ri/Ro : Si/So :
0i Kz/q(ARo + ASo + B)
This is lower t h a n the r a t i o RI/Ro prior to a d d i t i o n of S b y an a m o u n t r e l a t e d to i n t r o d u c i n g ASo into the d e n o m i n a t o r of t h e foregoing expression. I t was t h e n t e s t e d w h e t h e r t h e r e is a t i m e o t h e r t h a n infinite t i m e where vn = o (but vs # o), this c o r r e s p o n d i n g to a m i n i m u m in t h e curve of Rl a n d indic a t i n g the presence of c o u n t e r - t r a n s p o r t , even u n d e r the conditions where carrier i n t e r c o n v e r s i o n is limiting at higher s u b s t r a t e concentration. The a b o v e differential e q u a t i o n s were therefore solved a n a l y t i c a l l y a n d the solution checked on a D i a n a analogue c o m p u t e r , using a v a r i e t y of values for the different c o n s t a n t s (Kz, q, A, B a n d 0i) as well as different Ro a n d So. F o r all sets of values yielding Rl/Ro ratios b o t h before a d d i n g S a n d in the final s t e a d y - s t a t e condition such as f o u n d experim e n t a l l y , the function of RI d i s p l a y s a m i n i m u m before reaching its final value. The m i n i m u m is i n d e e d not as p r o n o u n c e d as in an e q u i l i b r a t i n g system. I n the best case it lies a b o u t 2o % below the final level which, moreover, it t a k e s v e r y long to reach, so t h a t the rise of the Ri curve after its m i n i m u m is v e r y gentle. Hence it is questionable w h e t h e r the a c c u r a c y of the e m p l o y e d a n a l y t i c a l m e t h o d s was sufficient to observe the r a t h e r u n p r o n o u n c e d m i n i m u m p r e d i c t e d b y theory. The n u m b e r of e x p e r i m e n t s p e r f o r m e d (14) a n d the absence of a n y i n d i c a t i o n of a c o u n t e r - t r a n s p o r t m i n i m u m would, however, f a v o u r an a l t e r n a t i v e e x p l a n a t i o n , according to which the c o u n t e r - t r a n s p o r t m i n i m u m would be lost c o m p l e t e l y a n d which w o u l d even b e t t e r explain some of t h e a b o v e - r e p o r t e d findings. Let us assume t h e existence of two discrete carriers in the m e m b r a n e , mobile p r o b a b l y b y v i r t u e of r o t a t i o n w i t h i n the m e m b r a n e , each of which w o u l d be capable of t r a n s p o r t i n g in v i r t u a l l y one direction only. The affinity of the " i n w a r d " carrier (I) for s u b s t r a t e would be g r e a t e r t h a n t h a t of t h e " o u t w a r d " one (II) so t h a t K I
421
U P H I L L T R A N S P O R T OF S U G A R S
petitive and governed by the same affinity constant as the reaction with substrate to be transported ( K s z K i , s = K ) , the rate of movement of substrate S is given by vs = V1 So/KI + Si/K~ + ~ - - ~ ~
~/K~
+ So/KI~ +
It may be calculated that the outward movement of S occurring on adding another competing substrate in steady state of S will be very slow and will produce no observable minimum. In the latter steady state S l # S o but rather Si =
S o ( ~ - - VII) - - K I V I I + ~/(So(V1 - - VII) - - KIVII) 2 + 4ViVii(82o + SoKH) 2 VH
Furthermore, this mechanism permits a calculation to show that at higher substrate concentrations the final SI/So ratio will be smaller than at lower ones (cf. Figs. I and 2), tending toward unity at very high concentrations of VI = VII without the necessity of invoking metabolic limitation at the higher concentrations. This hypothesis may also account for the anomalous behaviour of glucose anaerobically by assuming that glucose is bound to the inward carrier and immobilizes it without affecting the outward one (cf. Fig. 5). Differences in aerobic and anaerobic concentration ratios, which are doubtless due to decreased resources in metabolic energy anaerobically, are apparently brought about by the fact that at least one of the carriers (probably the one transporting outwards since the "inward" constants are very similar both in 0 2 and N2; cf. Table I) must be activated to raise or lower its affinity for substrate. Similarly, the effect of various metabolic inhibitors can be envisaged to take place at this activating reaction (or reactions) and/or at the carrier molecule itself, thereby changing either the maximum rate of transport or the apparent effective affinity or both. Since this effect may be quantitatively or even qualitatively different on each of the two carriers it is not surprising that a ratio of SI/So ---- I is not naturally attained, as the case is in Escherichia coliiT, is, when energy sources are cut off. The "two-carrier" hypothesis advanced here is readily reconcilable with the general asymmetry of the membrane 19 in which movement in one direction with substrate and in the other direction without it can take place, especially if metabolic coupling is involved. It would be of interest to establish whether the proposed mechanism would apply in S . f a e c a l i s as described by WILKINS AND O'KANE4. It seems to the present authors that the findings made with S. faecalis can all be accounted for by the "twocarrier" mechanism although differences in affinities and inhibition constants doubtless exist. It will have to be seen in the future whether a rigorously selective use of inhibitors will provide evidence for or against this hypothesis. There are indications that the carrier(s) may be de-repressed (anomalous S-shaped curves of uptake are occasionally observed, particularly after prolonged anaerobiosis) but no studies in this direction have been undertaken so far (cf. ref. 20). A "glucose effect" in this connection could account for some of the anomalies of glucose-inhibited sugar uptake. The chemical character of the carrier(s) has not been explored but there is evidence that at least for the inward flux a single site with sugar affinity is present Biochim. Biophys. Acta, lO2 (1965) 41o-422
422
A. KOTYK, M. HOFER
and that the molecule interacts directly with H + ions and some of the inhibitors used (NAN3, iodoacetamide). ACKNOWLEDGEMENTS
The authors are indebted to Professor A. KLEINZELLER for stimulating comments and frequent consultations, and to Mrs. E. HOROV£ and Mrs. L. RiHOV£ for skilled technical help. The differential equations were solved and programmed for the analogue computer by Dr. 0. SCHMIDT of the College of Chemical Technology and his help in this respect was of fundamental importance. ~EFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 20 21
A. KLEINZELLER AND L. ~LECHTA,Chem. Listy, 48 (1954) 602. J. H. LITCHFIELD AND Z. J. ORDAL, Can. J. Microbiol., 4 (1958) 205TH. ROSENBERG AND W. WILBRANDT, J. Theoret. Biol., 5 (1963) 288. P. O. WILKINS AND D. J. O'KANE, J. Gen. Microbiol., 34 (1964) 389. J. VAN STEVENINCK AND H. L. BOOIJ, J. Gen. Physiol., 48 (1964) 43. M. SOMOGYI, J. Biol. Chem., 195 (1952) 19. M. NELSON, J. Biol. Chem., 153 (1944) 375. W . W . UMBREIT, 1:~. H. BURRIS AND J. P. STAUFFER, Manometric Techniques, Burgess, Mi nne a polis, 1957, p. 274. H. O. LOWRY, •. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . A. IXOTYK, Folia Microbiol. Prague, 8 (1963) 27. A. I~LEINZELLER, :~Ianometriekd 2letody, SZN, P r a h a , 1954; Manometrische Methoden, G. Fischer-Verlag, Jena, 1966, in t h e press. W. WILBRANOT AND A. I~OTYK, Naunyn-Schmiedebergs Arch. Exptl. Pathol. Pharmakol., 249 (1964) 279. A. I~OTYK, Folia Microbiol. Prague, i o (1965) 3 o. R. K. CRANE, Physiol. Rev., 4 ° (196o) 789. A. I~LEINZELLER AND A. KOTYK, Biochim. Biophys. Acta, 54 (1961) 367. TH. ROSENBERG AND W. WILBRANDT, J. Gen. Physiol., 41 (1958) 289. A. KEPES, Biochim. Biophys. Acta, 4 ° (196o) 7 o. A. L. KOCH, Biochim. Biophys. Acta, 79 (1964) 177. P. MITCHELL, in A. KLEINZELLER AND A. KOTYK, Membrane Transport and Metabolism, Acad elnic Press, L o n d o n , 1961, p. 22. H. OKADA AND H. O. HALVORSON, Biochim. Biophys. Acta, 82 (1964) 538. H. OKADA AND H. O. HALVORSON, Biochim. Biophys. Acta, 82 (1964) 547.
Biochim. Biophys. Aeta, lO2 (1965) 41o 422