Electrogenic Antiport Activities of the Gram-positive Tet Proteins Include a Na+(K+)/K+ Mode That Mediates Net K+ Uptake

Guffanti, Arthur A.; Cheng, Jianbo; Krulwich, Terry A.

doi:10.1074/jbc.273.41.26447

Cited by 32 publications

(57 citation statements)

References 34 publications

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“…Assays of 86 Rb + uptake by the P156C mutant of Tet(K) were carried out in comparison with wild type Tet(K) and the G155C Tet(K) mutant to assess whether mutations in Pro 156 result in an apparent K + leak as observed with Tet(L) mutants at this position. As shown in Figure 5, wild type Tet(K) exhibited a significantly greater rate of 86 Rb + uptake into RSO vesicles than Tet(L), as consistently observed before (40,42), and this uptake was dependent upon energization and intravesicular K + . By contrast, Tet(K) G155C exhibited no 86 Rb + uptake under any condition.…”

Section: Mutations In Gly 155 and Pro 156 Of Tet(k)mentioning

confidence: 85%

“…a Tet(L)-dependent, electron donor-dependent flux of 86 Rb + into RSO membrane vesicles in the absence of a pre-loaded efflux substrate (K + , Na + or Tc-Co 2+ ) inside the vesicles. This flux is thus not coupled to efflux of an intravesicular substrate, a coupling that is characteristic of antiport and is observed for such fluxes in wild type Tet(L) (40,42). In assays of Tet(L) Pro 156 RSO vesicles that were loaded with K + , enhancement of 86 Rb + uptake by the electron donor was observed with all three of the Pro 156 mutants, and was particularly evident in the P156A mutant in the early time points (compare data shown with closed triangles with open triangles in right panels of Figure 4).…”

Section: Discussionmentioning

confidence: 99%

“…In this assay, 86 Rb + uptake into the RSO vesicles by wild type Tet(L) depends upon both the addition of the electron donor and on the presence of an efflux substrate for Tet(L) inside the vesicles; no Tet(L)-dependent 86 Rb + uptake was observed in the absence of added D-lactate (the electron donor) and or intravesicular K + (the efflux substrate loaded inside the vesicles in this experiment) (Figure 4). Earlier work showed that the net Rb + -K + uptake in this assay is a mode of the antiport that is electrogenic and it could be supported by the presence of either K + or Tc-Co 2+ inside the vesicles (40,42). It was therefore notable that 86 Rb + uptake by the mutant Tet(L) P156A,G or C forms was as high or higher under conditions in which the electron donor was present but the intravesicular K + was absent.…”

Section: Mutants Of Pro 156 In the Motif C Gp Dipeptidementioning

confidence: 99%

“…RSO membrane vesicles were prepared by the lysozyme-method of Kaback (52) and were pre-loaded with either 100 μM choline chloride or KCl (42). Protein assays were conducted by the method of Lowry et al (53) using egg white lysozyme as the standard.…”

Section: Preparation Of Membrane Vesiclesmentioning

confidence: 99%

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Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

et al. 2005

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Proline and glycine residues are well represented among functionally important residues in hydrophobic domains of membrane transport proteins and several critical roles have been suggested for them. Here, the effects of mutational changes in membrane-embedded proline and glycine residues of Tet(L) were examined, with a focus on the conserved GP 155,156 dipeptide of Motif C, a putative "antiporter motif". Mutation of Gly 155 to cysteine resulted in a mutant Tet(L) that bound its tetracycline-divalent metal (Tc-Me 2+ ) substrate but did not catalyze efflux or exchange of TcMe 2+ or catalyze uptake or exchange of Rb + which was used to monitor the coupling ion. These results support suggestions that this region is involved in the conformational changes required for translocation. Mutations in Pro 156 resulted in reduction (P156G) or loss (P156A or C) of Tc-Me 2+ efflux capacity. All three Pro 156 mutants exhibited a K + leak (monitored by 86 Rb + fluxes) that was not observed in wild type Tet(L). A similar leak was observed in a mutant in a membrane-embedded proline residue elsewhere in the Tet(L) protein (P175C) as well as in a P156C mutant of related antiporter Tet(K). These findings are consistent with roles proposed for membrane-embedded prolines in tight helix packing. Patterns of Tc-resistance conferred by additional Tet(L) mutants indicate important roles for another GP dipeptide in transmembrane segment (TMS) X as well as for membrane-embedded glycine residues in TMS XIII.Almost all transport proteins have membrane-embedded proline residues in some of the multiple α-helices that are a general feature of transporters (1). These residues are preferentially paired with glycine residues that are also found in abundance in TMS 1 of transporters (2,3). Membrane-embedded proline and glycine residues of transporters are hypothesized to promote helix kinks and swivels that play crucial roles in the conformational flexibility required for transport mechanisms (2-6). They are further hypothesized to have roles in helix packing and association (7-9). These hypotheses have been stunningly supported for the largest family of membrane transport proteins, the MFS, a family of structurally related transporters of prokaryotes and eukaryotes that includes uniporters, antiporters and symporters. The MFS comprises about a quarter of all transport proteins (10,11). The first three high resolution

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Section: Mutations In Gly 155 and Pro 156 Of Tet(k)mentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Mutants Of Pro 156 In the Motif C Gp Dipeptidementioning

confidence: 99%

Section: Preparation Of Membrane Vesiclesmentioning

confidence: 99%

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Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

et al. 2005

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“…ϩ antiport by the secondary active multidrug transporter MdfA (37) and tetracyline transporter Tet(L) (38,39) in E. coli and of Na ϩ -Cl Ϫ -H ϩ symport by the multidrug ATP-binding cassette transporter LmrA in L. lactis (40 -42). These findings raise the paradox that multidrug transporters with a broad specificity for amphiphilic organic cations can, at the same time, be highly selective for certain inorganic ions.…”

Section: Discussionmentioning

confidence: 99%

The Multidrug Transporter LmrP Protein Mediates Selective Calcium Efflux

Schaedler¹,

Tong

Veen

2012

Journal of Biological Chemistry

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NhaK, a novel monovalent cation/H+ antiporter of Bacillus subtilis

et al. 2005

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Four Na+/H+ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na+/H+ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na+(Ca2+)/H+ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na+ and Li+. In addition, effects of yvgP expression on a K+ uptake-defective mutant of E. coli indicated that YvgP also supported K+ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na+ (K+, Li+, Rb+)/H+ antiport activity was demonstrated. Na+ (K+, Li+)/H+ activity was higher at pH 8.5 than at pH 7.5. Mg2+, Ca2+ and Mn2+ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase, increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of added K+ and Na+.

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Electrogenic Antiport Activities of the Gram-positive Tet Proteins Include a Na+(K+)/K+ Mode That Mediates Net K+ Uptake

Cited by 32 publications

References 34 publications

Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

The Multidrug Transporter LmrP Protein Mediates Selective Calcium Efflux

NhaK, a novel monovalent cation/H+ antiporter of Bacillus subtilis

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