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H+ antiporter (KefC) of Escherichia coli
FEMSMicrobiologyLetters94 (1992)161-164 © 1992Federationof EuropeanMicrobiologicalSocieties0378-1097/92/$05.00 Publishedby Elsevier
161
FEMSLE04943
The putative Na+/H + antiporter (NapA) of Enterococcus hirae is homologous to the putative K+/H ÷ antiporter (KefC) of Escherichia coli Jonathan Reizer, Aiala Reizer and Milton H. Saier Jr. Department of Biology, Unit'ersityof California. San Diego. La dolla. California, USA
Recei",'d 17April 1992 Accepted24 April1992 Key words: Homology; Cation/H + antiporter; Sodium ions; Potassium ions; Enterococcus hirae; Escherichia coli
1. SUMMARY Two monovalent ion porters, the putative Na+/H ÷ antiporter (NapA) of Enterococcus hirae and the putative K+/H ÷ antiporter (KefC)of Escherichia coli, are similar in sequence throughout their hydrophobic domains. These two proteins, which comprise a novel family of transporters unrelated to the previously characterized Na÷/H + exchangers of E. coil (NhaA and NhaB) are proposed to function by essentially the same mechanism.
2. INTRODUCTION Homeostatic intracellular ionic conditions are controlled largely by transmembrane transport proteins. K + and Na ÷ which are respectively
Correspondence to: J. Reizer,Departmentof Biology,University of California,San Diego,La Jolla, CA 92093-0f16, USA.
accumulated and expelled from cells are transported by a variety of mechanistically distinct transport systems including ATP-driven porters, chemio-osmotic driven porters, and those driven by the chemical gradients of protons. Recently, two putative porters of the latter class were sequenced, a glutathione-regulated K + efflux system (KefC) of Escherichia coli [1,2], and a putative Na +/H + antiporter (NapA) of Enterococcus hirae [31. Both systems were reported to be unique, exhibiting no homology with other sequenced porters. In this note, we report our observation that these two proteins are sufficiently similar in sequence to establish a common evolutionary origin.
3, RESULTS AND DISCUSSION The sequence alignment of the KefC and NapA proteins is shown in Fig. I. The two sequences are 27% identical in an overlapping stretch of 373 residues with five gaps of one or two residues.
162 10
NapA(Eh)
20 •
KofC(Ec)
.
.
.
.
40 .
o
o
50
o
oo
70
60
o
,
.oo,
o
,
80
ooo
o
o
90
•
o
oo
MDSHTLI~AL~YLG~AAL~PiA~RLGLG~VLG¥L~AGCI~GPWGLRLVTDAE~ILHFAEiGVVLMLF~GLE~QRLWK-LRAAVFG~GALQ ~o
Sap^(Eh)
30
~EFIGILCD~L~ATTI~HISRRFG~PAVIGQLL~GVLLGQAGLGwVHPN~LVHDFSEIG~ILLMFLAGLE~DLSLLKK¥FKPGM~--VALL
~oo
2o
~o
11o
t2o
~o
5o
~3o
6o
,o
~o
,o
~o
~7o
,s~
,o
~8o
G~LFPv~FGwLTGE~FQv~N~A~FrGIILA~T~v~svEvLKELmmNTKEGsT~LGAsW~D~LW-LvL~F~L~FLTGK~T~"L~LPLLLL .
.
,
.
.
.
.
.
:
:
|
|
|:
::
:
::
:
:
Napk(Eh) ~cG~LLG-LF~LLGI~`RwQvA~L~G"T~Lss~AI~QA~R~v~RsAP^vLLrQD~A~pL~M~PLLA?$~A$TT~GAF~L$^L 1oo
zzo
190
NapA(Eh)
200
no
no
210
220
z4o
zso
230
1~o
240
tso
260
270
EQLF~FLF~LLVKW~A~LN~LAEKI¥A~.SA~I~MSLV~LG~¥LADLIGL~GAFFAGI~SQ~KV~HE~VEALG¥~vHPvFFV~ . . . . . . . . . . . . .. KVAGALVLWLLG~¥VTRPALRF1~ARS~LREVFSA~ALFL~FGFGLLLEEVGLSMAMGAFLAGVLLASSE¥P~ALESDIE~FK~GLLLGLFF~G •
KefC(Ec)
zTo
250
~o
280
~oo
290
~o
300
220
310
~o
320
.
~o
330
o
~o
340
~o
350
~o
360
NapA(Eh)
~GLEVDF~KF~E~ILF~LILTL~A~LTKLIGG~GAK~F~N~AI~4VGAGM~RGEMAL~L~S~L~ENH¥¥~PL~WLL~TL
KefC(Ec)
VGMSID;~TLLENP~.RIV;LLLGFL~IK~AMLWLI~PLQ~PNKQRRWFAVLLGQGSEFAF~FG~Q~VLEPE~SLT~;ALS~TFI 280
290
300
310
320
330
340
350
360
370
~':PL 370
380
NapA(Eh)
ILKYFTKKV
KefC(Ec)
LLVILNRLE 38O
Fig. l. Atignmentofthe putative Na + / H + antiporter(NapA)of E, hirae(Eh)with the K+/H + antiporter(KefC)of E. co~ (Ec). Residuescommonto thetwo proteins arcindicated with colons.
The comparison score, using the RDF2 program [4] was 25 standard deviations, establishing that these two proteins are homologous. The results presented in Fig. 2 show that KefC and NapA exhibit almost superimposable hydropathy profiles and that in the regions of overlap the two proteins are strikingly hydrophobic with 11-12
4530
~ -15-30 -45
I 100
I 200
b 300
I 400
T 500
600
Residue Number
Fig. 2. Hydropathy plots of the E. hirae NapA (broken line) and the E. coli KefC (solid line) putative cation/H + antiporters. Positive hydropathy values indicate hydrophobic regions. Vatues were determined for a window of 15 residues using the algorithm of Kyte and Doolittle [11].
putative transmembrane helical segments. The length of the E. coil K + efflux protein is 620 residues while that of the E. hirae Na+/H + antiporter of is 383 residues. Munro et al. [2] noted that part of the hydrophobic domain of the KefC protein exhibited limited sequence similarity to the cation-translocating ATPase of Leishmania donowni [5] as well as the A subunit of the Kdp K+-translocating ATPase of E. coil [6]. We found that these two proteins gave optimised comparison scores of 2 and 1 standard deviations with the KefC protein, respectively. Consequently, homology cannot be established on the basis of the observed sequence similarity. It can be seen (Fig. 2) that KefC and NaoA differ with respect to a large hydrophilic domain at the C-terminus of the E. coil KefC protein which is lacking in the E. hirae NapA protein. This hydrophilic domain presumably mediates glutathione regulation which has not been observed for the E. hirae antiporter. As reported by Munro et al. [2], this hydrophilic domain exhibits limited sequence similarity with a putative glutathione binding site of two bacterial enzymes that utilize glutathione, glyoxalase and dehaloge-
163 nase. Optimized binary comparison scores for these proteins with KefC gave values as follows: glyoxalase of Pseudomonas putida [7], 1 standard deviation; and dehalogenase of Methylobacterium sp. [8] 0.2 standard deviations. Although homology of KefC with glyoxalase and dehalogenase cannot be established, the results do not refute the possibility that the regions of sequence similarity have a common function. In summary, our findings establish that two bacterial monovalent ion porters, one specific for K +, the other for Na +, are similar in sequence throughout their hydrophobic domains. We propose that these two proteins, which comprise a novel family of transporters unrelated to the previously characterized N a + / H + exchangers of E. coil (NhaA and NhaB; [9,10]) function by essentially the same mechanism. Further studies will be required to establish the mechanistic details.
ACKNOWLEDGEMENTS This work was supported by Public Health Service Grants 5ROIAI 21702 and 2RO1AI 14176
from the National Institute of Allergy and Infectious Diseases.
REFERENCES [ll Smith, D.R. and Cairo, J.M, (1980) 8, 2~5-2274, [2] Munro, A.W., Ritchie, G.Y., Lamb, A.J., Douglas, R.M. and Booth, R, (1991) Mol. Microbiol.5, 607-616. [3] Waser, M., Hess-Bienz, D., Davies, K. and Solioz, M. 0992) ]. Biol. Chem. 2t~7,5396-5400. [4] Pearson, W.R. and Lipman,DJ. (t988) Proc, Nail, Acad. Sci. USA 85, 2444-2448, [5] Meade, J.C., Shaw, J., Lemaster, S., Gallagher, G. and Stringer, J.R. (1987) Mo]. Cell. Biol. 7, 3937-3946. [6] Hesse, J.E.+ Weiczorek, L., Altendoff, K., Rejoin, A.S., Dorus, E. and Epstein, W. (1984) Proc. Natl. Acad. ¢~¢i. USA 81, 4746-4750. 171 Rhee, H.I., Sato, N., Marata, K. and Kimura, A. {1988) Aerie. Biol. Chem. 52, 2243-2246, [81 La Roche, S,D. and Leisinger,T. (1990)J. Bacteriol. 172, 164-171. 1.9] Karpel, R.. Olami, Y., Taglieht, D., Schuldiner, S. and Padan, E. (1988) J. Biol, Chem. 263, 10408-10414, [10l Pinner, E., Padan, E. and Schuldiner. S. 0992) J. Biol. Chem., in press. Ill] Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132.
161
FEMSLE04943
The putative Na+/H + antiporter (NapA) of Enterococcus hirae is homologous to the putative K+/H ÷ antiporter (KefC) of Escherichia coli Jonathan Reizer, Aiala Reizer and Milton H. Saier Jr. Department of Biology, Unit'ersityof California. San Diego. La dolla. California, USA
Recei",'d 17April 1992 Accepted24 April1992 Key words: Homology; Cation/H + antiporter; Sodium ions; Potassium ions; Enterococcus hirae; Escherichia coli
1. SUMMARY Two monovalent ion porters, the putative Na+/H ÷ antiporter (NapA) of Enterococcus hirae and the putative K+/H ÷ antiporter (KefC)of Escherichia coli, are similar in sequence throughout their hydrophobic domains. These two proteins, which comprise a novel family of transporters unrelated to the previously characterized Na÷/H + exchangers of E. coil (NhaA and NhaB) are proposed to function by essentially the same mechanism.
2. INTRODUCTION Homeostatic intracellular ionic conditions are controlled largely by transmembrane transport proteins. K + and Na ÷ which are respectively
Correspondence to: J. Reizer,Departmentof Biology,University of California,San Diego,La Jolla, CA 92093-0f16, USA.
accumulated and expelled from cells are transported by a variety of mechanistically distinct transport systems including ATP-driven porters, chemio-osmotic driven porters, and those driven by the chemical gradients of protons. Recently, two putative porters of the latter class were sequenced, a glutathione-regulated K + efflux system (KefC) of Escherichia coli [1,2], and a putative Na +/H + antiporter (NapA) of Enterococcus hirae [31. Both systems were reported to be unique, exhibiting no homology with other sequenced porters. In this note, we report our observation that these two proteins are sufficiently similar in sequence to establish a common evolutionary origin.
3, RESULTS AND DISCUSSION The sequence alignment of the KefC and NapA proteins is shown in Fig. I. The two sequences are 27% identical in an overlapping stretch of 373 residues with five gaps of one or two residues.
162 10
NapA(Eh)
20 •
KofC(Ec)
.
.
.
.
40 .
o
o
50
o
oo
70
60
o
,
.oo,
o
,
80
ooo
o
o
90
•
o
oo
MDSHTLI~AL~YLG~AAL~PiA~RLGLG~VLG¥L~AGCI~GPWGLRLVTDAE~ILHFAEiGVVLMLF~GLE~QRLWK-LRAAVFG~GALQ ~o
Sap^(Eh)
30
~EFIGILCD~L~ATTI~HISRRFG~PAVIGQLL~GVLLGQAGLGwVHPN~LVHDFSEIG~ILLMFLAGLE~DLSLLKK¥FKPGM~--VALL
~oo
2o
~o
11o
t2o
~o
5o
~3o
6o
,o
~o
,o
~o
~7o
,s~
,o
~8o
G~LFPv~FGwLTGE~FQv~N~A~FrGIILA~T~v~svEvLKELmmNTKEGsT~LGAsW~D~LW-LvL~F~L~FLTGK~T~"L~LPLLLL .
.
,
.
.
.
.
.
:
:
|
|
|:
::
:
::
:
:
Napk(Eh) ~cG~LLG-LF~LLGI~`RwQvA~L~G"T~Lss~AI~QA~R~v~RsAP^vLLrQD~A~pL~M~PLLA?$~A$TT~GAF~L$^L 1oo
zzo
190
NapA(Eh)
200
no
no
210
220
z4o
zso
230
1~o
240
tso
260
270
EQLF~FLF~LLVKW~A~LN~LAEKI¥A~.SA~I~MSLV~LG~¥LADLIGL~GAFFAGI~SQ~KV~HE~VEALG¥~vHPvFFV~ . . . . . . . . . . . . .. KVAGALVLWLLG~¥VTRPALRF1~ARS~LREVFSA~ALFL~FGFGLLLEEVGLSMAMGAFLAGVLLASSE¥P~ALESDIE~FK~GLLLGLFF~G •
KefC(Ec)
zTo
250
~o
280
~oo
290
~o
300
220
310
~o
320
.
~o
330
o
~o
340
~o
350
~o
360
NapA(Eh)
~GLEVDF~KF~E~ILF~LILTL~A~LTKLIGG~GAK~F~N~AI~4VGAGM~RGEMAL~L~S~L~ENH¥¥~PL~WLL~TL
KefC(Ec)
VGMSID;~TLLENP~.RIV;LLLGFL~IK~AMLWLI~PLQ~PNKQRRWFAVLLGQGSEFAF~FG~Q~VLEPE~SLT~;ALS~TFI 280
290
300
310
320
330
340
350
360
370
~':PL 370
380
NapA(Eh)
ILKYFTKKV
KefC(Ec)
LLVILNRLE 38O
Fig. l. Atignmentofthe putative Na + / H + antiporter(NapA)of E, hirae(Eh)with the K+/H + antiporter(KefC)of E. co~ (Ec). Residuescommonto thetwo proteins arcindicated with colons.
The comparison score, using the RDF2 program [4] was 25 standard deviations, establishing that these two proteins are homologous. The results presented in Fig. 2 show that KefC and NapA exhibit almost superimposable hydropathy profiles and that in the regions of overlap the two proteins are strikingly hydrophobic with 11-12
4530
~ -15-30 -45
I 100
I 200
b 300
I 400
T 500
600
Residue Number
Fig. 2. Hydropathy plots of the E. hirae NapA (broken line) and the E. coli KefC (solid line) putative cation/H + antiporters. Positive hydropathy values indicate hydrophobic regions. Vatues were determined for a window of 15 residues using the algorithm of Kyte and Doolittle [11].
putative transmembrane helical segments. The length of the E. coil K + efflux protein is 620 residues while that of the E. hirae Na+/H + antiporter of is 383 residues. Munro et al. [2] noted that part of the hydrophobic domain of the KefC protein exhibited limited sequence similarity to the cation-translocating ATPase of Leishmania donowni [5] as well as the A subunit of the Kdp K+-translocating ATPase of E. coil [6]. We found that these two proteins gave optimised comparison scores of 2 and 1 standard deviations with the KefC protein, respectively. Consequently, homology cannot be established on the basis of the observed sequence similarity. It can be seen (Fig. 2) that KefC and NaoA differ with respect to a large hydrophilic domain at the C-terminus of the E. coil KefC protein which is lacking in the E. hirae NapA protein. This hydrophilic domain presumably mediates glutathione regulation which has not been observed for the E. hirae antiporter. As reported by Munro et al. [2], this hydrophilic domain exhibits limited sequence similarity with a putative glutathione binding site of two bacterial enzymes that utilize glutathione, glyoxalase and dehaloge-
163 nase. Optimized binary comparison scores for these proteins with KefC gave values as follows: glyoxalase of Pseudomonas putida [7], 1 standard deviation; and dehalogenase of Methylobacterium sp. [8] 0.2 standard deviations. Although homology of KefC with glyoxalase and dehalogenase cannot be established, the results do not refute the possibility that the regions of sequence similarity have a common function. In summary, our findings establish that two bacterial monovalent ion porters, one specific for K +, the other for Na +, are similar in sequence throughout their hydrophobic domains. We propose that these two proteins, which comprise a novel family of transporters unrelated to the previously characterized N a + / H + exchangers of E. coil (NhaA and NhaB; [9,10]) function by essentially the same mechanism. Further studies will be required to establish the mechanistic details.
ACKNOWLEDGEMENTS This work was supported by Public Health Service Grants 5ROIAI 21702 and 2RO1AI 14176
from the National Institute of Allergy and Infectious Diseases.
REFERENCES [ll Smith, D.R. and Cairo, J.M, (1980) 8, 2~5-2274, [2] Munro, A.W., Ritchie, G.Y., Lamb, A.J., Douglas, R.M. and Booth, R, (1991) Mol. Microbiol.5, 607-616. [3] Waser, M., Hess-Bienz, D., Davies, K. and Solioz, M. 0992) ]. Biol. Chem. 2t~7,5396-5400. [4] Pearson, W.R. and Lipman,DJ. (t988) Proc, Nail, Acad. Sci. USA 85, 2444-2448, [5] Meade, J.C., Shaw, J., Lemaster, S., Gallagher, G. and Stringer, J.R. (1987) Mo]. Cell. Biol. 7, 3937-3946. [6] Hesse, J.E.+ Weiczorek, L., Altendoff, K., Rejoin, A.S., Dorus, E. and Epstein, W. (1984) Proc. Natl. Acad. ¢~¢i. USA 81, 4746-4750. 171 Rhee, H.I., Sato, N., Marata, K. and Kimura, A. {1988) Aerie. Biol. Chem. 52, 2243-2246, [81 La Roche, S,D. and Leisinger,T. (1990)J. Bacteriol. 172, 164-171. 1.9] Karpel, R.. Olami, Y., Taglieht, D., Schuldiner, S. and Padan, E. (1988) J. Biol, Chem. 263, 10408-10414, [10l Pinner, E., Padan, E. and Schuldiner. S. 0992) J. Biol. Chem., in press. Ill] Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132.