Carr, John W.

Knaff, David B.

Whetstone, Ricci

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The role of the membrane potential in active transport by the photosynthetic bacterium Chromatium vinosum 

Volume 99, number 2 FEBS LEll-ERS March 1979 
THE ROLE OF THE MEMBRANE POTENTIAL IN ACTIVE TRANSPORT BY THE 
PHOTOSYNTHETIC BACTERIUM CHROMATWM VhVOSUM 
David B. KNAFF, Ricci WHETSTONE and John W. CARR 
Department of Chemistry, Texas Tech. University, Lubbock, TX 79409, USA 
Received 10 January 1979 
1. ~troduction 
The photosynthetic purple sulfur bacterium 
Chromatium vinosum has been shown to accumulate 
a variety of metabolites in energy-dependent reac- 
tions [ 11. The observation that this metabolite accu- 
mulation was inhibited by uncouplers known to 
abolish transmembrane el ctrochemical proton gra- 
dients (A;@), suggested the possibility that the 
driving force for active transport in C. vinosum may 
be A;* [ l]. Our initial results uggested that both 
the pH gradient (ApH) and membrane potential 
(A$) components of A&+ may be involved in trans- 
port [I]. In this investigation, we have concentrated 
on the role of A$. Using the lipophilic cation TPP’, 
we have confirmed the fmding [2] that C vinosum 
cells generate A$ (exterior positive) in the light. We 
have also shown that C. vinosum maintains aAJ/ 
of similar polarity in the dark, apparently using 
energy from ATP hydrolysis. Evidence has been 
obtained that a A$, in the absence of other energy 
sources, is capable of supporting alanine uptake by 
C. vinosum cells. 
2. Materials and methods 
C’. vinosum was grown and alanine uptake assayed 
as in [ 11. Flow dialysis assays were performed under 
Abbreviations: TPP+, tetraphenylphosphonium; DCCD, 
N,A”dicyciohexylcarbodiimide; HOQNO, 2-heptyl_Q- 
hydroxyqu~o~ne-~-o~de; CCCP, carbonylcyanide-m- 
ch~orophenylhy~azone 
EisevierlfVbrth-Holland Biomedical Press 
anaerobic onditions as in [3,4]. C’. vino~m cells at a 
concentration equivalent to 175 I.~M bacteriochloro- 
phyll (estimated according to [5]) in 0.4 ml 50 mM 
potassium phosphate buffer (pH 6.6) were used with 
a 6.5 ml/mm flow rate. Fractions of -1.7 ml were 
collected. Illumination during flow dialysis was 
provided by a Photographic Research Organization 
high intensity movie lamp 30 cm from the sample 
compartment with 2% CuSO1, solution used as a 
heat filter and a 1 M NaN02 solution used as an 
ultraviolet fnter. E3H]TPPBr (6.3 mCi~mmo1) was 
prodded by the Isotope Synthesis Group of the 
Hofm~-troche Co. [ “C]Alanine (168 mCi/mmol) 
was purchased from New England Nuclear. CCCP, 
HOQNO, and DCCD were purchased from Calbio- 
them. Nigericin was a gift of Dr D. Berger. The last 
4 reagents were added as concentrated stock solu- 
tions in dimethylsulfoxide. 
3. Results and discus&on 
Uptake of several metabolites by C. vino~m cells 
has been shown to occur in the dark al~ou~ at lower 
rates than in the light [I]. This uptake in the dark 
was inhibited by DCCD [ I], a specific inhibitor of 
the membrane-bound ATPase in C. vinosum [2,6], 
suggesting that ATP hydrolysis provided the energy 
for uptake in the dark. If, as suggested by the uncou- 
pler sensitivity of transport, ATP hydrolysis produced 
an electrochemical proton gradient which in turn 
drove transport, it should be possible to demonstrate 
a DCCD-sensitive A$ in C. vinosum cells in the dark. 
Figure 1 shows that adding CCCP, an uncoupler known 
283 
Vohime 99, number 2 FEBS LETTERS March 1979 
I 1 I I 
20 40 60 80 
FRACTION NUMBER 
Fig. I. The effect of DCCD on [ “Ii]TPP* uptake by C. vinosum 
cells in the dark. Flow dialysis was conducted as in section 
2 with 0.8 mM TPP+ and 0.5 irM nigericin present. DCCD 
was present at 200 pM where indicated. CCCP was added, 
as indicated by the arrow, to give final cone. 20 PM. 
to eliminate A$, causes the release of f3H]TPP’ from 
C. vinosum cells kept in the dark. Release of this 
lipophilic cation on addition of CCCP implies that 
the cation had been pre-accumulated by C. vinosum 
in response to a A$ (interior negative). If C. vinosum 
was pre-incubated with DCCD, no release of TPP’ was 
observed on addition of CCCP. It, thus, appears that 
ATP hydrolysis, catalyzed by a DCCD-sensitive 
enzyme, results in a A$ (interior negative), detectable 
by the uptake of a lipoph~ic ation. 
Figure 2A shows that even in the presence of 
DCCD, an additional light-induced uptake of TPP’ 
can be detected. Adding CCCP causes release of the 
accumulated TPP’. The migration of a lipophilic 
cation, such as TPP’, into the C. vinosum cells is 
consistent with the formation of a light-dependent 
A$ (interior negative) and confirms our earlier esults 
using the light-induced carotenoid band-shift to 
monitor A$. Addition of HOQNO at concentrations 
known to inhibit cyclic electron flow [7] and active 
transport [I] in C. vino~m cells eliminated light- 
induced TPP’ uptake (data not shown), suggesting 
that the light-dependent A$ originates from cyclic 
electron flow. 
t , I t I 
20 40 60 a0 100 
FRACTION NUMBER 
Fig.2. The effect of CCCP and alanine on [ ‘H]TPP+ uptake 
by C. ~jnosu~ cells in the light. Conditions as in fig. 1 with 
200 PM DCcD present in both samples. CCCP (A) or 
alanine (B) was added, where indicated, to give fmal cone. 
20 pM and 200 MM, respectively. 
If A+ can indeed serve as a source of energy for 
transport in C. vinosum, use of this energy during 
metabolite uptake should lead to a decrease in the 
magnitude of A$. Figure 2B shows that A$ decreases 
when uptake is initiated by addition of the trans- 
portable metabolite, alanine. It should be pointed 
out that the experiments inboth frg.l,2 were per- 
formed in the presence of nigericin, an ionophore 
that will eliminate ApH but not AJ, [3,4,8]. This 
was done chiefly so that the relationship of A$ to 
active transport could be studied independently of
any contribution from ApH. However, it should be 
mentioned that the extent of TPP’ uptake was 
always much greater in the presence than in the 
absence of nigericin. In some preparations, no TPP’ 
uptake was detected unless nigericin was present. 
The reasons for this variability are under further 
investigation. It is worth noting that substantial 
increases in the size of A$ on nigericin addition 
have been observed with E. coli vesicles [3,4]. 
If A$ (exterior positive) does indeed provide the 
284 
Volume 99, number 2 FEBS LETTERS March 1979 
drivirtg force for a&nine uptake, the creation of a 
A$ of proper p&a&y should rest& in alanine uptake 
~~de~nde~t~y of any other energy input. Figure 3 
shows the results of an experiment in which a A$ 
across the C. vinosu~ membrane iscreated by a K* 
diffusion gradient in the dark and the presence of 
DCCD, C. hosum cells washed in buffer containing 
KC1 were placed in a K’-free medium aud the experi- 
ment was initiated by adding valinomycm to render 
the cells permeable to K’. As can be seen in fig.3, 
the A$ (exterior positive) produced by K* diffusion 
out af the cell in the presence of va~5rn~c~ results 
o* 
0 t 2 3 
TIME (MINUTESE 
Fig.3, K’-diiT’usion potential driven alanine uptake by C 
viurJsum cells. C. vinosum cells were washed twice with 
100 mM KCI/SO mM Tris-HCl (pH 7.3) and resuspended to 
final bacteriochlorophyli 150 PM in a reactian r &ure 
coat&&~ 250 gM DCCD, 20 pM [ ‘4c]aknine, SO mM 
Tris+BCi @W 7.3) and either llttf mM choline ctioride (a) 
or 100 mM Kc? (*). The experiment was initiated (t = f)) 
by adding v~omy~~ to &al EOlfC. 15 #I@. 
in substantial &nine uptake. No uptake of aianine 
was observed in the absence of K’ ~ad~ent. This can 
be seen from the exigent shown in fig& in which 
the cells were placed in a medium with a KCl concen- 
tration equal to that in the medium used to wash the 
cells. Similar uetabolite uptake driven by an art& 
cially created A$ has been observed in nonphoto 
synthetic bacteria [9-141 but has not been reported 
in an obligate phototroph. 
The abave results uggest that C. vinowm cells USI: 
energy (either from IQ&t-driven cyclic electron flow 
or ATP hydr5lysis catalyzed by a DCCD-sensitive 
ATPase) to mau~tain a membr~ potential (exterior 
positive). The ob~~t~~ that the size of AJI 
decreases a  energy is used to accurate a&mine 
and that A@ created by a K’-diffusion potential 
supports ahmine uptake both strongly suggest that 
AJ/ (exterior positive) can provide the energy f5r 
physiological &mine transport in C, vinosum. 
Acknowledgements 
The author would like to thank the Roche 
institute of molecular Biology and Dr H, R, Kaback 
for their holists in providing the facihtbs for 
some ofthese studies. The author is indebted to 
MS L&ha Pate1 for excellent echnical assistance 
and to Drs Sofia Ramos, Dan Robertson and Paul 
Strooband for extremely helpful discussions, This 
investigatiun was supported in part by grants (to 
D.B.K.) from the National Science Foundation 
(Grants PChl76-2413 1 and PCM78-17304) and the 
Robert A, Welch Foundation (D-l 70). 
Referances 
[ 2] Knaff$ D. B+ and Carr, J. W. (I 979) Arch. Biochem, 
Biophys. in press. 
[3] Kaback, H, R. (1976) J. Cell Physiol. 89,575~5% 
[4] Ramos, S,, Schuldiner, S. and Kaback, H. R. (1976) 
Proc. NotI. Acad. Sci USA 73,1892-1898. 
15 J Clayton, R, K. (1963) in: Bacterial Photosyntlmsie 
(Gest, H, st al. eds} p. 498, Ant&h Press, Yellow 
Sptiings. 
161 &pnst&, A. and Carmeli, C. (1977) Eur. J. Biochem. 
?4,463-469. 
Volume 99, number 2 FEBS LETTERS March 1979 
[7] Van Grondeil, R., Duysens, L. N. M., Van der Wel, J. A. 
and Van der Wal, H. N. (I 977) Biochim. Biophys. Acta 
461,188-201. 
[8] Pressman, B. C. (1976) Ann. Rev. Biochem. 45, 
501-530. 
[9] Kashket, E. R. and Wilson, T. H. (1972) Biochem. 
Biophys. Res. Commun. 49,615-620. 
[lo] Hirata, H., Altendorf, K. and Harold, F. M. (1973) 
Proc. Natl. Acad. Sci. USA 70, 1804-I 808. 
[ 111 Schuldiner, S. and Kaback, H. R. (1975) Biochemistry 
14,5451-5460. 
[ 121 Lanyi, J. K., Yearwood-Drayton, V. and MacDonald, 
R. E. (1976) Biochemistry 15,1595-1603. 
[ 131 MacDonald, R. E., Lanyi, J. K. and Greene, R. V. 
(1977) Proc. Natl. Acad. Sci. USA 74,3167-3170. 
[ 141 Tokuda, H. and Kaback, H. R. (1977) Biochemistry 
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286 


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The role of the membrane potential in active transport by the photosynthetic bacterium Chromatium vinosum

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