Journal of Bacteriology, July 2002, p . 3756-3758, Vol . 184, No . 13

We have applied these techniques to the study of UhpT, the P_i-linked hexose phosphate antiport carrier of Escherichia coli (14, 15, 21) . In previous work, Asp³⁸⁸ and Lys³⁹¹, located in the 11th transmembrane segment (TM11) of this transporter, were identified as participants in an intrahelical salt bridge (7) . Study of this ion pair indicated that both positions 388 and 391 might lie on the UhpT translocation pathway since D388C and K391C single-cysteine variants are sensitive to the membrane impermeant, thiol-reactive probe p-chloromercuribenzosulfonate but are unaffected when treatment is in the presence of substrate (8) . Further analysis indicated that these two positions might also function as determinants of substrate specificity . Derivatives having an uncompensated cationic charge (the D388C and D388K/K391C variants) are gain-of-function mutants in which substrate preference is strongly biased in favor of phosphoenolpyruvate (PEP), a substrate that carries one more negative charge than sugar 6-phosphate (8) . Here, by using the lysine-reactive agent pyridoxal 5-phosphate (PLP), we provide evidence that the lysine introduced into these gain-of-function variants serves as a receptor for the reactive aldehyde of PLP, suggesting that this lysine is also poised to interact with the anionic carboxyl brought into the translocation pathway by PEP .

Strain RK5000 [araD139 (argF-lac)U169 relA1 rpsL150 thi gyrA219 non metE780 (ilvB-uhpABCT')2056 recA] served as the host for tests of function of plasmid-encoded UhpT (23) . Plasmid pTrc(HisC₀S₆) encodes the histidine-tagged, cysteineless UhpT that served as the parent for the D388C and D388K/K391C derivatives described in this study (7) . To assay transport function, overnight cultures were diluted 200-fold into either Luria-Bertani broth plus antibiotics (100 µg of streptomycin/ml, 100 µg of ampicillin/ml) or M63 minimal medium (17) (pH 7) containing thiamine (2 µg/ml), required amino acids (50 µg/ml), necessary antibiotics, and 0.2% glucose as the carbon source . Cells were grown at 37°C to a density of 2 x 10⁸ to 5 x 10⁸ cells/ml, harvested by centrifugation, and then washed twice and resuspended in buffer A (50 mM morpholinepropanesulfonic acid [MOPS]-K, 100 mM K₂SO₄, 1 mM MgSO₄, pH 7) at an optical density at 660 nm of 1.4, which is equivalent to about 2 x 10⁹ cells/ml . After equilibration at room temperature, tests of P_i transport ([³²P]KP_i) was obtained from Perkin Elmer Life Sciences) were initiated by adding a 1/20th volume of labeled substrate (final concentration, 100 µM) . At indicated times, aliquots were removed for filtration on Millipore filters (0.45-µm pore size) followed by two washes with 5 ml of buffer A lacking MgSO₄ . To monitor PLP inhibition of glucose-6-phosphate (G6P) and PEP transport, cells were first incubated with and without PLP (1 µM to 1 mM) for 10 min at room temperature to allow Schiff base formation with any exposed lysine residues (11) . Any Schiff base(s) formed was reduced by a subsequent 10-min exposure to 15 mM NaBH₄ to yield the chemically stable product N-phosphopyridoxal-lysine (11) . Aliquots were then filtered to remove additives, and after two washes with 5 ml of buffer A, tests of transport were initiated by overlaying cells (on the filter) with buffer A containing 50 µM radiolabeled G6P or PEP ([¹⁴C]G6P and [¹⁴C]PEP were obtained from Perkin Elmer Life Sciences and Amersham Pharmacia Biotech, respectively) . To assess substrate protection, the same protocol was used, except that cells were incubated with either 1 mM unlabeled substrate or 1 mM unlabeled substrate plus PLP before tests of transport .

As a cofactor, PLP functions by facilitating an array of mechanistically related reactions, almost always involving an interaction with a lysine residue during the course of catalysis (10, 22) . It is this selectivity that makes PLP a useful reagent for analyzing both the location and function of lysyl groups in membrane transport proteins (1-3, 6, 12, 18), including UhpT . However, since PLP resembles UhpT's natural substrates in overall size, shape, and charge, it was important to first determine whether this compound is transported by cysteineless UhpT and its D388C and D388K/K391C derivatives . Since these variants display normal levels of P_i self-exchange (8) (Fig . 1), we could address this issue by measuring the effectiveness with which added substrate (G6P, PEP, or PLP) caused the loss of P_i during heterologous exchange of internal substrate (Fig . 1) . As expected, the addition of G6P, but not of PEP, led to a marked loss of internal phosphate in cells carrying cysteineless UhpT, while PEP caused nearly equivalent P_i efflux from the D388C and D388K/K391C variants . By contrast, reduced P_i efflux occurred when a low-affinity substrate was added (G6P for the D388C and D388K/K391C gain-of-function mutants and PEP for cysteineless UhpT) . In these latter cases, we attribute residual P_i efflux to the use of these substrates at a concentration (1 mM) at or above their likely K_m values for transport (8) . PLP induced an even smaller efflux of P_i, suggesting that significant P_i/PLP exchange does not occur for the UhpT variants analyzed here . A low level of P_i efflux might be attributed to PLP functioning as an inhibitor of transport, and in the case of both the D388C and D388K/K391C mutants, this may be the case (see below) . However, PLP acts as a poor inhibitor of G6P transport by cysteineless UhpT (Table 1), indicating that the residual P_i efflux exhibited by this strain upon addition of PLP is likely due to this compound being, at best, a poor UhpT substrate (K_m >> 1 mM) .

 
Our next tests evaluated the response of each UhpT variant to the inhibition of G6P and PEP transport before and after covalent modification of accessible lysines by reduction of any Schiff base formed between PLP and its target(s) . As judged by G6P and PEP transport, exposure to reductant alone (NaBH₄) had no significant effect on UhpT function (Table 1) . We next examined the effect of exposure to PLP, with or without subsequent NaBH₄ treatment, including an estimation of the PLP concentration yielding 50% inhibition (K_0.5) . Those variants with an uncompensated lysine at either position 388 or 391 were strongly inhibited by PLP, showing K_0.5 values in the range of 10 to 40 µM (Fig . 2; Table 1) not dependent upon NaBH₄ reduction . By contrast, cysteineless UhpT, in which Lys³⁹¹ pairs with Asp³⁸⁸, responded poorly to PLP treatment (K_0.5 of about 1 to 2 mM) . For the PLP-responsive derivatives, we then determined whether protection was afforded by coincubation with PEP and G6P, using a probe concentration near its K_0.5 value . Both variants (D388C and D388K/K391C) benefited from the presence of substrate, showing an 80 to 90% retention of UhpT function (Table 1) . Collectively, these data imply that PEP and G6P, as well as PLP, interact with the 388/391 region of UhpT .

 
What conclusions can we draw from these experiments? First, even though PLP is not transported by UhpT, that PLP interacts at position 388 or 391 shows that it has a distribution within UhpT comparable to that of G6P and PEP . Second, these findings offer independent evidence in support of the hypothesis that both positions 388 and 391 are likely to be on the translocation pathway and, therefore, can serve as determinants of substrate selectivity . Thus, while the presence of an uncompensated lysine at either of these positions does not preclude PEP or G6P entrance onto the translocation pathway, it does bias transport preference toward the former and away from the latter . Finally, we speculate that our findings may let us address the orientation a substrate adopts as it moves through UhpT . We have previously shown that positions 388 and 391 lie deep within the hydrophobic core of UhpT (8) while those residues (Arg⁴⁶ and Arg²⁷⁵) required for recognition of the phosphoryl group at the substrate C-6 (or C-5) position are found near the periplasmic surface (5) . Here, we present evidence indicating that an uncompensated lysine at either position 388 or 391, in much the same way that it serves as a receptor for the carboxyl moiety of PEP, provides the amide component for Schiff base formation with the C-4 aldehyde of PLP . Together, these findings strongly suggest that substrates orient themselves within UhpT with their C-1 positions pointed toward the cytoplasm and phosphate groups toward the periplasm .

We thank Robert Kadner for the gift of strain RK5000 .

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