Cation Transport in Escherichia coli
IX. Regulation of K Transport D A V I D B. R H O A D S and W O L F G A N G E P S T E I N From the Departments of Biochemistry and of Biophysics and Theoretical Biology,The University of Chicago, Chicago, Illinois 60637. Dr. Rhoads' present address is the Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
A B ST R AC T Kinetics of K exchange in the steady state and of net K uptake after osmotic upshock are reported for the four K transport systems of Escherichia coli: Kdp, TrkA, TrkD, and TrkF. Energy requirements for K exchange are reported for the Kdp and TrkA systems. For each system, kinetics of these two modes of K transport differ from those for net K uptake by K-depleted cells (Rhoads, D. B., F. B. Walters, and W. Epstein. 1976.J. Gen. Physiol. 67:325-341). The TrkA and TrkD systems are inhibited by high intracellular K, the TrkF system is stimulated by intracellular K, whereas the Kdp system is inhibited by external K when intracellular K is high. All four systems mediate net K uptake in response to osmotic upshock. Exchange by the Kdp and TrkA systems requires ATP but is not dependent on the protonmotive force. Energy requirements for the Kdp system are thus identical whether measured as net K uptake or K exchange, whereas the TrkA system differs in that it is dependent on the protonmotive force only for net K uptake. We suggest that in both the Kdp and TrkA systems formation of a phosphorylated intermediate is necessary for all K transport, although exchange transport may not consume energy. The protonmotive-force dependence of the TrkA system is interpreted as a regulatory influence, limiting this system to exchange except when the protonmotive force is high. Potassium t r a n s p o r t in Escherichia coli serves to maintain high intracellular K concentrations which are d e t e r m i n e d chiefly by the osmolarity o f the external m e d i u m (Epstein and Schultz, 1965, 1968). Maintenance o f a given K c o n t e n t d e t e r m i n e d by m e d i u m osmolarity but i n d e p e n d e n t o f m e d i u m K concentration implies the existence o f mechanisms that regulate K influx quite precisely. Some evidence for regulation was seen in the fact that K-depleted cells h a d high rates o f K uptake by a process that h a d a Km in the millimolar r a n g e (Schultz et al., 1963), whereas K/K e x c h a n g e in the steady state o c c u r r e d at a considerably lower rate a n d had a very high affinity for K (Epstein a n d Schultz, 1966). A n even m o r e striking regulatory response is p r o d u c e d by an osmotic upshock. Cells react to an u p s h o c k by taking u p K until they achieve a h i g h e r K content c o r r e s p o n d i n g to the new m e d i u m osmolarity (Epstein a n d Schultz, 1965). T h e s e studies were d o n e with wild-type cells, before it was k n o w n that E.
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T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 7 2 9 1 9 7 8
coli has f o u r distinct K t r a n s p o r t systems. T h e s e f o u r systems, whose properties
are s u m m a r i z e d in T a b l e I, have been characterized genetically (Epstein a n d Davies, 1970; Epstein a n d Kim, 1971) a n d on the basis o f the kinetics for net K u p t a k e by K - d e p l e t e d cells (Rhoads et al., 1976). O n e r e g u l a t o r y influence has been d o c u m e n t e d , n a m e l y , that the high affinity K d p system is repressible by growth in high K media, a n d is only d e r e p r e s s e d when o t h e r K t r a n s p o r t systems are not a d e q u a t e to satisfy the cells' needs for K (Rhoads et al., 1976). Because the o t h e r systems are constitutive, r e g u l a t o r y influences m u s t operate at the level o f the function r a t h e r than the level o f f o r m a t i o n o f the systems. I n this p a p e r we e x a m i n e two d i f f e r e n t m o d e s o f K t r a n s p o r t for each o f the f o u r systems: K/K e x c h a n g e in the steady state a n d net K u p t a k e p r o d u c e d in K-replete cells by osmotic upshock. In each case significant differences in the p r o p e r t i e s o f these two m o d e s o f K t r a n s p o r t are seen as c o m p a r e d with net K u p t a k e in K - d e p l e t e d cells. We suggest that these differences reflect regulatory TABLE
I
K TRANSPORT SYSTEMS IN E. coli* Characteristics of net K uptake Kinetic parameters System
Kdp TrkA TrkD TrkF
K=
Vmax*
mm
#mol[g/ min
0.002 1.5 0.5 >500
150 550 40 -
Energy requirements
ATP ATP, PMFw ATP, PMFw PMF
Genetics
Four linked kdp genes SingletrkA gene Single(?) trkD gene No mutations known
Other characteristics
Repressible by K Constitutive Constitutive Constitutive, low rate, linearly dependent on K concentration
* Genetic data from Epstein and Davies (1970), and Epstein and Kim (1971); kinetic data from Rhoads et al. (1976); energy requirements from Rhoads and Epstein (1977) and unpublished data. * Rates are at 37~ for Kdp and TrkA, at 30~ for TrkD. wProtonmotive force.
influences on the systems. In a t t e m p t i n g to d e t e r m i n e what forces could p r o d u c e such regulation, we e x a m i n e d e n e r g y r e q u i r e m e n t s for K/K e x c h a n g e by each o f the systems. This aspect o f the study was p r o m p t e d by o u r earlier finding that the T r k A system requires both A T P a n d the p r o t o n m o t i v e force in o r d e r to p e r f o r m net K u p t a k e (Rhoads a n d Epstein, 1977). This dual e n e r g y r e q u i r e m e n t is unique for bacterial t r a n s p o r t , a n d suggested to us that one o f these r e q u i r e m e n t s r e p r e s e n t s a r e g u l a t o r y influence, whereas the o t h e r provides e n e r g y . O u r results show that K/K e x c h a n g e by the T r k A system requires A T P but not the p r o t o n m o t i v e force, a n d are consistent with o u r m o d e l that the p r o t o n m o t i v e force exerts a r e g u l a t o r y influence on the T r k A system such that high values o f the p r o t o n m o t i v e stimulate, while low values inhibit, the T r k A system.
RHOADSANDEPSTEIN Regulation of K Transport in E. coli
285
METHODS
Bacterial Strains T h e strains used in this work, all Escherichia coli K-12 F-, are listed in Table II with the K transport systems present in each and the relevant genotypes.
Media and Growth o f Bacteria Phosphate-buffered media, described earlier (Epstein and Kim, 1971), are referred to by K concentration in millimolar, e.g., K l I 5 medium contains 115 mM K. K0 m e d i u m , to which no K is added, contains about 20/~M contaminating K. Cells were grown at 37~ with 11 mM glucose as the sole carbon source. Derepression of the Kdp system was accomplished by transferring growing cells to K0 medium at 37~ until the cells were K limited for 1 h.
Transport Studies Methods for transport measurements (Rhoads and Epstein, 1977), analysis of 42K uptake (Epstein and Schultz, 1966) and osmotic upshock (Epstein and Schuhz, 1965) have been described. T h r e e buffers were used; all were pH 7.0 and contained 0.15 mM chloroamTABLE
I1
BACTERIAL STRAINS
Strain
TK 1001 TK1110 TK405m TK509* TK510 TK1064 TK1097
K transport systems present
Relevant genotype
TrkA TrkF TrkD TrkF TrkF Kdp TrkF Kdp TrkF Kdp TrkA TrkF Kdp TrkA TrkF
kdpABC5 trkD1 kdpABC5 trkA405 kdpABC5 trkA405 trkD1 trkA405 trkD1 trkA405 trkD1 trkD1 trkD1 unc
Reference
Rhoads Rhoads Rhoads Rhoads
et al. (1976) et al. (1977) et al. (1977) and Epstein (1977)
Rhoads and Epstein (1977)
* This strain was called TAD 109 in Rhoads and Epstein (1977). phenicol. Buffer A contained 75 mM Na-PO4 and 0.4 mM MgSO4, buffer B contained 75 mM K-PO4 and 0.4 mM MgSO4, and buffer C contained 110 mM Mg-maleate. T r a n s p o r t assays were conducted at 30~ T o study a single K transport system in relative isolation, a combination of suitably chosen mutations, growth conditions, and assay conditions was used as previously described (Rhoads and Epstein, 1977). For instance, the TrkD system is assayed in strain T K l l l 0 which has both a kdp and a trkA mutation, and at K concentrations low e n o u g h that the T r k F system does not significantly contribute to the uptake. Uptake by the TrkD and T r k F systems was somewhat variable from experiment to experiment, so when a comparison was made between two modes of transport, both modes were measured on the same culture. Fluxes and cell K contents are expressed in units per gram dry weight, the latter determined from turbidity measurements at 610 n m with a Bausch and Lomb Spectronic 20 colorimeter (Bausch & Lomb Inc., Rochester, N. Y.) and a calibration curve.
Chemicals 42KCI and L-[U-14C]glutamine were purchased from New England Nuclear (Boston, Mass.). c-[U-14C]Proline was purchased from Amersham/Searle Corp. (Arlington Heights, Ill.).
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RESULTS
TrkA System S t e a d y - s t a t e K e x c h a n g e m e d i a t e d by t h e T r k A s y s t e m e x h i b i t s s a t u r a t i o n k i n e t i c s with r e s p e c t to t h e e x t e r n a l K c o n c e n t r a t i o n (Fig. I A ) . T h i s p r o c e s s has a Vmax o f l l0 /.tmol/g 9 m i n a n d a Km o f 0.5 m M K at 30~ Both kinetic .c_ 120 r /A
$
0
-k
,,,
1 80 | "~ ] S Upshock ~
Exchange
---0
8
r
16
~/[K]
/
I-9
|
[/ 0
%
~ I
2
EXTRACELLULAR 4
K+ CONCENTRATION (raM)
j'__. 2 r
_.,r"
0
3
,'o
6
TIME (rain)
FIGURE 1. K fluxes mediated by the T r k A system. (A) Exchange flux. K l l 5 grown strain TK1001 was suspended in buffer A containing 11 mM glucose and KC1 at the indicated K concentration. T h e cells were allowed to equilibrate for 30 min before addition o f the 4~K tracer to initiate the flux measurements. (B) Upshock-induced net uptake. Strain TK1001 was grown in KI15 medium diluted 1:1 with H20 and suspended in similarly diluted buffer A containing 11 mM glucose and the indicated a m o u n t o f KC1. After 30 rain net influx was initiated by the addition of 1/4 vol o f diluted buffer A containing the same K concentration plus 2.5 M glucose. Cell K was measured chemically. Units for the double reciprocal plots are millimolar -~ on the abscissa, and micromolar -~ per gram p e r minute for the ordinate. (C) Unidirectional K influx d u r i n g upshock. T h e e x p e r i m e n t was p e r f o r m e d as in B on K l l 5 - g r o w n strain TK1064 at 1.1 mM KCI. 42K tracer was a d d e d at zero time (O) and at the arrow an aliquot of the suspension was subjected to upshock (O). T h e latter counts have been corrected for dilution. Control 42K influx was 23 /~mol/g 9 min; after upshock, unidirectional 42K influx was 116 /~mol/g 9 min and net K influx was 92 p,mol/g 9 min.
p a r a m e t e r s a r e less t h a n t h o s e o b t a i n e d f o r n e t K u p t a k e by K - d e p l e t e d cells ( T a b l e I); t h e Vmax is r e d u c e d f i v e f o l d a n d t h e Km t h r e e f o l d . T e m p e r a t u r e e f f e c t s d o n o t a c c o u n t f o r this d i f f e r e n c e b e c a u s e t h e T r k A s y s t e m is r e l a t i v e l y t e m p e r a t u r e i n d e p e n d e n t b e t w e e n 30 ~ a n d 37~ I n a n e x p e r i m e n t at 30~ with s t r a i n T K I 0 0 1 , n e t u p t a k e h a d a Vmax o f 5 0 0 / ~ m o l / g 9 m i n a n d a K = o f 1.5 m M .
RHOADSANDEPSTEIN Regulation of K Transport in E. coli
287
In a n o t h e r e x p e r i m e n t with strain TK1001, the exchange rate was 5 0 / z m o l / g 9 rain h i g h e r at 114 than at 14 mM K. This increase is d u e in part to the contribution o f the T r k F system at h i g h e r K concentrations (see below), but mainly indicates that exchange by the T r k A system is not inhibited by high external K concentrations. I n a s m u c h as net K uptake was assayed in cells exposed to 2,4-dinitrophenol to deplete t h e m o f K, the possibility that t r e a t m e n t with d i n i t r o p h e n o l altered the T r k A system was ruled out by direct test. Cells o f strain TKI001 were either assayed for K/K exchange at 1 mM K or depleted o f K by d i n i t r o p h e n o l treatment. T h e K-depleted cells were divided into two portions. One portion was allowed to equilibrate with 1 mM K b e f o r e the addition o f a 42K tracer while the o t h e r was equilibrated in the absence o f K before addition o f 1 mM K containing a 42K tracer. T h e K-reloaded cells had a K influx o f 52 /~mol/g 9 min, close to the n o n - d i n i t r o p h e n o l - t r e a t e d control value o f 63 /~mol/g 9 min, while net K influx into the K-depleted cells was 152 k~mol/g 9 rain, about threefold higher. T h u s , K influx mediated by the T r k A system is r e d u c e d in K-replete cells regardless o f a prior d i n i t r o p h e n o l treatment. O n e line o f evidence that K is an o s m o r e g u l a t o r y solute in E. coli is that this bacterium is capable o f rapidly increasing its intracellular K concentration in response to an increase in m e d i u m osmolarity (Epstein and Schultz, 1965, 1968). T h e majority o f this response has been attributed to the T r k A system (Rhoads et al., 1976). Osmotic upshock-stimulated net uptake by the T r k A system is also a saturable function o f K concentration. T h e e x p e r i m e n t illustrated in Fig. IB shows a Km o f 0.4 mM and a Vma x o f 90 /~mol/g 9 min. T h r e e strains, in which both the T r k A and the lower rate T r k D systems (see below) were present, had upshock-stimulated t r a n s p o r t with K,, ranging f r o m 0.3 to 0.6 mM and Vma x f r o m 75 to 109 /~mol/g 9 min. T h e initial rate o f net K influx catalyzed by this system is i n d e p e n d e n t o f the d e g r e e o f upshock between osmolarity increases o f 0.2-0.6 osmol, but the extent o f K uptake increases with the change in osmolarity (data not shown). O u r findings that K t r a n s p o r t by K-replete cells, be it exchange or in response to upshock, is characterized by considerably lower K,, and Vmax than uptake by K-depleted cells, indicate that intracellular K inhibits the T r k A system in a way to lower both Km and Vma x. In the steady state, K influx and K efflux are by definition o f equal magnitude. When the steady state is disturbed by osmotic upshock, a net influx c o m p o n e n t emerges. Kepes et al. (1976) have shown that for wild-type E. coil, upshock stimulates influx but efflux remains u n c h a n g e d . This behavior was seen when the T r k A system was observed in isolation (Fig. IC). T h e control 42K uptake represents an exchange rate o f 23 # m o l / g 9 rain. Osmotic upshock induced a net u p t a k e rate o f 92 t~mol/g 9 rain while the unidirectional K influx rate increased to 116 /zmol/g 9 rain. T h e difference, 24 ~ m o l / g 9 min, is the efflux which is the same as that in the control. This result indicates that the major effect o f upshock on the T r k A system is to stimulate K influx. Kdp System
Steady-state K flux m e a s u r e m e n t s on the Kdp system yielded an u n e x p e c t e d result. This system mediates rapid K exchange at low external K concentrations
288
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PHYSIOLOGY
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9 1978
consistent with its high affinity, but the e x c h a n g e is inhibited in high external K (Fig. 2A). T h e flux is r e d u c e d m o r e than t h r e e f o l d when the external K is raised f r o m 30/.LM to 1 m M . No a t t e m p t was m a d e to m e a s u r e e x c h a n g e by this system n e a r its Km f o r net u p t a k e because large fluctuations in the external K would have been unavoidable. T h e K d p system r e s p o n d e d to an osmotic u p s h o c k at a low K c o n c e n t r a t i o n , but again, high e x t e r n a l K inhibited this process (Fig. 2B). At 2 m M K net influx was r e d u c e d to less t h a n h a l f that at 0.2 m M K. A b o v e 2 m M K the rate plateaus, but this is p r o b a b l y only a p p a r e n t since at 20 m M the T r k F system begins to m a k e a significant contribution (3.5 /~mol/g 9 rain; see
'~ 40
A
~
Exchange
120 ~-
B Net
0
E x
J 2O
I
h hi (.9 Z
,
Z m
0
Ihi
o
I I0 z OIJ. I O. I I 2 2.0 EXTRACELLULAR K § CONCENTRATION (raM) "-'2
I 20
4-
N~: ~-
Upshoc
";iL ~ K OE I~.
~
O0
I
I
1
I
6
I
I
12
TIME (rain)
FIGURE 2. K fluxes mediated by the Kdp system. Protocols were identical to those described in the legend to Fig. 1. (A) K exchange flux in K-limited strain TK510. (B) Upshock-induced net uptake in K-limited strain TK509. Note the logarithmic scales of the abscissae. (C) Unidirectional K influx during upshock of K-limited strain TK510 at 0.4 mM KCI. (9 Control; (O) upshock. Control 42K influx was 35 /~mol/g 9 min; after upshock, unidirectional 42K influx was 74/~mol/g 9 rain and net K influx was 38/~mol/g 9 min. Fig. 4B below). T h e s h a p e o f the curves o f Fig. 4A a n d B suggest that inhibition may be negligible at low external K concentrations close to the 2/~.M Km o f the K d p system. Inhibition by external K, evident when t r a n s p o r t is m e a s u r e d in Kreplete cells as e x c h a n g e or u p s h o c k - s t i m u l a t e d net u p t a k e , is not seen at concentrations u p to 10 m M when t r a n s p o r t is m e a s u r e d as net K u p t a k e in Kdepleted cells (Rhoads et al., 1976). We infer that the K d p system is inhibited by external K but only w h e n intracellular K is high. Unidirectional influx m e d i a t e d by the K d p system a f t e r osmotic u p s h o c k is shown in Fig. 2C. T h e control e x c h a n g e rate was 35/~mol/g 9 min whereas a f t e r
RHOADSANDEPSTEIN Regulation of K Transport in E. coli
289
u p s h o c k the net influx was 38/xmol/g 9 min and the unidirectional influx was 74 /xmol/g 9 min. Because the last quantity is equal to the sum o f the o t h e r two, we conclude that for the K d p system osmotic u p s h o c k stimulates influx a n d does not affect efflux. TrkD System
T h e m i n o r T r k D system exhibits an inhibition o f K influx by internal K similar to that shown by the T r k A system. I n a s m u c h as fluxes by the T r k D system are sometimes variable f r o m culture to culture, Fig. 3A shows the K-concentration d e p e n d e n c e o f both the steady-state e x c h a n g e flux a n d the net K influx into Kdepleted cells f r o m a single culture o f strain TK1110. For net K uptake the Vmax is 60/~mol/g 9 min a n d theKm is 0.4 mM; for exchange the values are 12/xmol/g 50
E ole
X ,_1 Z §v
25
(
lJ
I
I 2
3X ' 5 /
0
I I
~
IX. Regulation of K Transport D A V I D B. R H O A D S and W O L F G A N G E P S T E I N From the Departments of Biochemistry and of Biophysics and Theoretical Biology,The University of Chicago, Chicago, Illinois 60637. Dr. Rhoads' present address is the Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
A B ST R AC T Kinetics of K exchange in the steady state and of net K uptake after osmotic upshock are reported for the four K transport systems of Escherichia coli: Kdp, TrkA, TrkD, and TrkF. Energy requirements for K exchange are reported for the Kdp and TrkA systems. For each system, kinetics of these two modes of K transport differ from those for net K uptake by K-depleted cells (Rhoads, D. B., F. B. Walters, and W. Epstein. 1976.J. Gen. Physiol. 67:325-341). The TrkA and TrkD systems are inhibited by high intracellular K, the TrkF system is stimulated by intracellular K, whereas the Kdp system is inhibited by external K when intracellular K is high. All four systems mediate net K uptake in response to osmotic upshock. Exchange by the Kdp and TrkA systems requires ATP but is not dependent on the protonmotive force. Energy requirements for the Kdp system are thus identical whether measured as net K uptake or K exchange, whereas the TrkA system differs in that it is dependent on the protonmotive force only for net K uptake. We suggest that in both the Kdp and TrkA systems formation of a phosphorylated intermediate is necessary for all K transport, although exchange transport may not consume energy. The protonmotive-force dependence of the TrkA system is interpreted as a regulatory influence, limiting this system to exchange except when the protonmotive force is high. Potassium t r a n s p o r t in Escherichia coli serves to maintain high intracellular K concentrations which are d e t e r m i n e d chiefly by the osmolarity o f the external m e d i u m (Epstein and Schultz, 1965, 1968). Maintenance o f a given K c o n t e n t d e t e r m i n e d by m e d i u m osmolarity but i n d e p e n d e n t o f m e d i u m K concentration implies the existence o f mechanisms that regulate K influx quite precisely. Some evidence for regulation was seen in the fact that K-depleted cells h a d high rates o f K uptake by a process that h a d a Km in the millimolar r a n g e (Schultz et al., 1963), whereas K/K e x c h a n g e in the steady state o c c u r r e d at a considerably lower rate a n d had a very high affinity for K (Epstein a n d Schultz, 1966). A n even m o r e striking regulatory response is p r o d u c e d by an osmotic upshock. Cells react to an u p s h o c k by taking u p K until they achieve a h i g h e r K content c o r r e s p o n d i n g to the new m e d i u m osmolarity (Epstein a n d Schultz, 1965). T h e s e studies were d o n e with wild-type cells, before it was k n o w n that E.
j. GEN. PHYSIOL.9 The Rockefeller University Press 90022-1295/78/0901-028351.00
283
284
T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 7 2 9 1 9 7 8
coli has f o u r distinct K t r a n s p o r t systems. T h e s e f o u r systems, whose properties
are s u m m a r i z e d in T a b l e I, have been characterized genetically (Epstein a n d Davies, 1970; Epstein a n d Kim, 1971) a n d on the basis o f the kinetics for net K u p t a k e by K - d e p l e t e d cells (Rhoads et al., 1976). O n e r e g u l a t o r y influence has been d o c u m e n t e d , n a m e l y , that the high affinity K d p system is repressible by growth in high K media, a n d is only d e r e p r e s s e d when o t h e r K t r a n s p o r t systems are not a d e q u a t e to satisfy the cells' needs for K (Rhoads et al., 1976). Because the o t h e r systems are constitutive, r e g u l a t o r y influences m u s t operate at the level o f the function r a t h e r than the level o f f o r m a t i o n o f the systems. I n this p a p e r we e x a m i n e two d i f f e r e n t m o d e s o f K t r a n s p o r t for each o f the f o u r systems: K/K e x c h a n g e in the steady state a n d net K u p t a k e p r o d u c e d in K-replete cells by osmotic upshock. In each case significant differences in the p r o p e r t i e s o f these two m o d e s o f K t r a n s p o r t are seen as c o m p a r e d with net K u p t a k e in K - d e p l e t e d cells. We suggest that these differences reflect regulatory TABLE
I
K TRANSPORT SYSTEMS IN E. coli* Characteristics of net K uptake Kinetic parameters System
Kdp TrkA TrkD TrkF
K=
Vmax*
mm
#mol[g/ min
0.002 1.5 0.5 >500
150 550 40 -
Energy requirements
ATP ATP, PMFw ATP, PMFw PMF
Genetics
Four linked kdp genes SingletrkA gene Single(?) trkD gene No mutations known
Other characteristics
Repressible by K Constitutive Constitutive Constitutive, low rate, linearly dependent on K concentration
* Genetic data from Epstein and Davies (1970), and Epstein and Kim (1971); kinetic data from Rhoads et al. (1976); energy requirements from Rhoads and Epstein (1977) and unpublished data. * Rates are at 37~ for Kdp and TrkA, at 30~ for TrkD. wProtonmotive force.
influences on the systems. In a t t e m p t i n g to d e t e r m i n e what forces could p r o d u c e such regulation, we e x a m i n e d e n e r g y r e q u i r e m e n t s for K/K e x c h a n g e by each o f the systems. This aspect o f the study was p r o m p t e d by o u r earlier finding that the T r k A system requires both A T P a n d the p r o t o n m o t i v e force in o r d e r to p e r f o r m net K u p t a k e (Rhoads a n d Epstein, 1977). This dual e n e r g y r e q u i r e m e n t is unique for bacterial t r a n s p o r t , a n d suggested to us that one o f these r e q u i r e m e n t s r e p r e s e n t s a r e g u l a t o r y influence, whereas the o t h e r provides e n e r g y . O u r results show that K/K e x c h a n g e by the T r k A system requires A T P but not the p r o t o n m o t i v e force, a n d are consistent with o u r m o d e l that the p r o t o n m o t i v e force exerts a r e g u l a t o r y influence on the T r k A system such that high values o f the p r o t o n m o t i v e stimulate, while low values inhibit, the T r k A system.
RHOADSANDEPSTEIN Regulation of K Transport in E. coli
285
METHODS
Bacterial Strains T h e strains used in this work, all Escherichia coli K-12 F-, are listed in Table II with the K transport systems present in each and the relevant genotypes.
Media and Growth o f Bacteria Phosphate-buffered media, described earlier (Epstein and Kim, 1971), are referred to by K concentration in millimolar, e.g., K l I 5 medium contains 115 mM K. K0 m e d i u m , to which no K is added, contains about 20/~M contaminating K. Cells were grown at 37~ with 11 mM glucose as the sole carbon source. Derepression of the Kdp system was accomplished by transferring growing cells to K0 medium at 37~ until the cells were K limited for 1 h.
Transport Studies Methods for transport measurements (Rhoads and Epstein, 1977), analysis of 42K uptake (Epstein and Schultz, 1966) and osmotic upshock (Epstein and Schuhz, 1965) have been described. T h r e e buffers were used; all were pH 7.0 and contained 0.15 mM chloroamTABLE
I1
BACTERIAL STRAINS
Strain
TK 1001 TK1110 TK405m TK509* TK510 TK1064 TK1097
K transport systems present
Relevant genotype
TrkA TrkF TrkD TrkF TrkF Kdp TrkF Kdp TrkF Kdp TrkA TrkF Kdp TrkA TrkF
kdpABC5 trkD1 kdpABC5 trkA405 kdpABC5 trkA405 trkD1 trkA405 trkD1 trkA405 trkD1 trkD1 trkD1 unc
Reference
Rhoads Rhoads Rhoads Rhoads
et al. (1976) et al. (1977) et al. (1977) and Epstein (1977)
Rhoads and Epstein (1977)
* This strain was called TAD 109 in Rhoads and Epstein (1977). phenicol. Buffer A contained 75 mM Na-PO4 and 0.4 mM MgSO4, buffer B contained 75 mM K-PO4 and 0.4 mM MgSO4, and buffer C contained 110 mM Mg-maleate. T r a n s p o r t assays were conducted at 30~ T o study a single K transport system in relative isolation, a combination of suitably chosen mutations, growth conditions, and assay conditions was used as previously described (Rhoads and Epstein, 1977). For instance, the TrkD system is assayed in strain T K l l l 0 which has both a kdp and a trkA mutation, and at K concentrations low e n o u g h that the T r k F system does not significantly contribute to the uptake. Uptake by the TrkD and T r k F systems was somewhat variable from experiment to experiment, so when a comparison was made between two modes of transport, both modes were measured on the same culture. Fluxes and cell K contents are expressed in units per gram dry weight, the latter determined from turbidity measurements at 610 n m with a Bausch and Lomb Spectronic 20 colorimeter (Bausch & Lomb Inc., Rochester, N. Y.) and a calibration curve.
Chemicals 42KCI and L-[U-14C]glutamine were purchased from New England Nuclear (Boston, Mass.). c-[U-14C]Proline was purchased from Amersham/Searle Corp. (Arlington Heights, Ill.).
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RESULTS
TrkA System S t e a d y - s t a t e K e x c h a n g e m e d i a t e d by t h e T r k A s y s t e m e x h i b i t s s a t u r a t i o n k i n e t i c s with r e s p e c t to t h e e x t e r n a l K c o n c e n t r a t i o n (Fig. I A ) . T h i s p r o c e s s has a Vmax o f l l0 /.tmol/g 9 m i n a n d a Km o f 0.5 m M K at 30~ Both kinetic .c_ 120 r /A
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FIGURE 1. K fluxes mediated by the T r k A system. (A) Exchange flux. K l l 5 grown strain TK1001 was suspended in buffer A containing 11 mM glucose and KC1 at the indicated K concentration. T h e cells were allowed to equilibrate for 30 min before addition o f the 4~K tracer to initiate the flux measurements. (B) Upshock-induced net uptake. Strain TK1001 was grown in KI15 medium diluted 1:1 with H20 and suspended in similarly diluted buffer A containing 11 mM glucose and the indicated a m o u n t o f KC1. After 30 rain net influx was initiated by the addition of 1/4 vol o f diluted buffer A containing the same K concentration plus 2.5 M glucose. Cell K was measured chemically. Units for the double reciprocal plots are millimolar -~ on the abscissa, and micromolar -~ per gram p e r minute for the ordinate. (C) Unidirectional K influx d u r i n g upshock. T h e e x p e r i m e n t was p e r f o r m e d as in B on K l l 5 - g r o w n strain TK1064 at 1.1 mM KCI. 42K tracer was a d d e d at zero time (O) and at the arrow an aliquot of the suspension was subjected to upshock (O). T h e latter counts have been corrected for dilution. Control 42K influx was 23 /~mol/g 9 min; after upshock, unidirectional 42K influx was 116 /~mol/g 9 min and net K influx was 92 p,mol/g 9 min.
p a r a m e t e r s a r e less t h a n t h o s e o b t a i n e d f o r n e t K u p t a k e by K - d e p l e t e d cells ( T a b l e I); t h e Vmax is r e d u c e d f i v e f o l d a n d t h e Km t h r e e f o l d . T e m p e r a t u r e e f f e c t s d o n o t a c c o u n t f o r this d i f f e r e n c e b e c a u s e t h e T r k A s y s t e m is r e l a t i v e l y t e m p e r a t u r e i n d e p e n d e n t b e t w e e n 30 ~ a n d 37~ I n a n e x p e r i m e n t at 30~ with s t r a i n T K I 0 0 1 , n e t u p t a k e h a d a Vmax o f 5 0 0 / ~ m o l / g 9 m i n a n d a K = o f 1.5 m M .
RHOADSANDEPSTEIN Regulation of K Transport in E. coli
287
In a n o t h e r e x p e r i m e n t with strain TK1001, the exchange rate was 5 0 / z m o l / g 9 rain h i g h e r at 114 than at 14 mM K. This increase is d u e in part to the contribution o f the T r k F system at h i g h e r K concentrations (see below), but mainly indicates that exchange by the T r k A system is not inhibited by high external K concentrations. I n a s m u c h as net K uptake was assayed in cells exposed to 2,4-dinitrophenol to deplete t h e m o f K, the possibility that t r e a t m e n t with d i n i t r o p h e n o l altered the T r k A system was ruled out by direct test. Cells o f strain TKI001 were either assayed for K/K exchange at 1 mM K or depleted o f K by d i n i t r o p h e n o l treatment. T h e K-depleted cells were divided into two portions. One portion was allowed to equilibrate with 1 mM K b e f o r e the addition o f a 42K tracer while the o t h e r was equilibrated in the absence o f K before addition o f 1 mM K containing a 42K tracer. T h e K-reloaded cells had a K influx o f 52 /~mol/g 9 min, close to the n o n - d i n i t r o p h e n o l - t r e a t e d control value o f 63 /~mol/g 9 min, while net K influx into the K-depleted cells was 152 k~mol/g 9 rain, about threefold higher. T h u s , K influx mediated by the T r k A system is r e d u c e d in K-replete cells regardless o f a prior d i n i t r o p h e n o l treatment. O n e line o f evidence that K is an o s m o r e g u l a t o r y solute in E. coli is that this bacterium is capable o f rapidly increasing its intracellular K concentration in response to an increase in m e d i u m osmolarity (Epstein and Schultz, 1965, 1968). T h e majority o f this response has been attributed to the T r k A system (Rhoads et al., 1976). Osmotic upshock-stimulated net uptake by the T r k A system is also a saturable function o f K concentration. T h e e x p e r i m e n t illustrated in Fig. IB shows a Km o f 0.4 mM and a Vma x o f 90 /~mol/g 9 min. T h r e e strains, in which both the T r k A and the lower rate T r k D systems (see below) were present, had upshock-stimulated t r a n s p o r t with K,, ranging f r o m 0.3 to 0.6 mM and Vma x f r o m 75 to 109 /~mol/g 9 min. T h e initial rate o f net K influx catalyzed by this system is i n d e p e n d e n t o f the d e g r e e o f upshock between osmolarity increases o f 0.2-0.6 osmol, but the extent o f K uptake increases with the change in osmolarity (data not shown). O u r findings that K t r a n s p o r t by K-replete cells, be it exchange or in response to upshock, is characterized by considerably lower K,, and Vmax than uptake by K-depleted cells, indicate that intracellular K inhibits the T r k A system in a way to lower both Km and Vma x. In the steady state, K influx and K efflux are by definition o f equal magnitude. When the steady state is disturbed by osmotic upshock, a net influx c o m p o n e n t emerges. Kepes et al. (1976) have shown that for wild-type E. coil, upshock stimulates influx but efflux remains u n c h a n g e d . This behavior was seen when the T r k A system was observed in isolation (Fig. IC). T h e control 42K uptake represents an exchange rate o f 23 # m o l / g 9 rain. Osmotic upshock induced a net u p t a k e rate o f 92 t~mol/g 9 rain while the unidirectional K influx rate increased to 116 /zmol/g 9 rain. T h e difference, 24 ~ m o l / g 9 min, is the efflux which is the same as that in the control. This result indicates that the major effect o f upshock on the T r k A system is to stimulate K influx. Kdp System
Steady-state K flux m e a s u r e m e n t s on the Kdp system yielded an u n e x p e c t e d result. This system mediates rapid K exchange at low external K concentrations
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consistent with its high affinity, but the e x c h a n g e is inhibited in high external K (Fig. 2A). T h e flux is r e d u c e d m o r e than t h r e e f o l d when the external K is raised f r o m 30/.LM to 1 m M . No a t t e m p t was m a d e to m e a s u r e e x c h a n g e by this system n e a r its Km f o r net u p t a k e because large fluctuations in the external K would have been unavoidable. T h e K d p system r e s p o n d e d to an osmotic u p s h o c k at a low K c o n c e n t r a t i o n , but again, high e x t e r n a l K inhibited this process (Fig. 2B). At 2 m M K net influx was r e d u c e d to less t h a n h a l f that at 0.2 m M K. A b o v e 2 m M K the rate plateaus, but this is p r o b a b l y only a p p a r e n t since at 20 m M the T r k F system begins to m a k e a significant contribution (3.5 /~mol/g 9 rain; see
'~ 40
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FIGURE 2. K fluxes mediated by the Kdp system. Protocols were identical to those described in the legend to Fig. 1. (A) K exchange flux in K-limited strain TK510. (B) Upshock-induced net uptake in K-limited strain TK509. Note the logarithmic scales of the abscissae. (C) Unidirectional K influx during upshock of K-limited strain TK510 at 0.4 mM KCI. (9 Control; (O) upshock. Control 42K influx was 35 /~mol/g 9 min; after upshock, unidirectional 42K influx was 74/~mol/g 9 rain and net K influx was 38/~mol/g 9 min. Fig. 4B below). T h e s h a p e o f the curves o f Fig. 4A a n d B suggest that inhibition may be negligible at low external K concentrations close to the 2/~.M Km o f the K d p system. Inhibition by external K, evident when t r a n s p o r t is m e a s u r e d in Kreplete cells as e x c h a n g e or u p s h o c k - s t i m u l a t e d net u p t a k e , is not seen at concentrations u p to 10 m M when t r a n s p o r t is m e a s u r e d as net K u p t a k e in Kdepleted cells (Rhoads et al., 1976). We infer that the K d p system is inhibited by external K but only w h e n intracellular K is high. Unidirectional influx m e d i a t e d by the K d p system a f t e r osmotic u p s h o c k is shown in Fig. 2C. T h e control e x c h a n g e rate was 35/~mol/g 9 min whereas a f t e r
RHOADSANDEPSTEIN Regulation of K Transport in E. coli
289
u p s h o c k the net influx was 38/xmol/g 9 min and the unidirectional influx was 74 /xmol/g 9 min. Because the last quantity is equal to the sum o f the o t h e r two, we conclude that for the K d p system osmotic u p s h o c k stimulates influx a n d does not affect efflux. TrkD System
T h e m i n o r T r k D system exhibits an inhibition o f K influx by internal K similar to that shown by the T r k A system. I n a s m u c h as fluxes by the T r k D system are sometimes variable f r o m culture to culture, Fig. 3A shows the K-concentration d e p e n d e n c e o f both the steady-state e x c h a n g e flux a n d the net K influx into Kdepleted cells f r o m a single culture o f strain TK1110. For net K uptake the Vmax is 60/~mol/g 9 min a n d theKm is 0.4 mM; for exchange the values are 12/xmol/g 50
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