Three lines of evidence indicate that arginine-46 (R46) and arginine-275 (R275) are essential to the function of UhpT, the Pi-linked antiport protein of Escherichia coli. A role for arginine was initially suggested by the sensitivity of UhpT to inhibition by 2,3-butanedione, an arginine-directed probe. Since the presence of substrate protected against this inhibition, this work further suggested that arginine(s) may lie at or near the UhpT active site. In other work, each UhpT arginine was examined individually by using site-directed mutagenesis to generate a cysteine or a lysine derivative. With two exceptions (R46, R275), all arginines could be replaced by either cysteine (10 of 14 residues) or lysine (12 of 14) without loss of function, implicating R46 and R275 as essential to UhpT function. This idea was strengthened by examining a multiple alignment of the eleven known UhpT-related proteins (>/=30% identity). That alignment showed R46 and R275 were two of the only three arginines strongly conserved in this group of proteins. Considered together, these different approaches lead us to conclude that UhpT and its relatives have only two arginine residues (R46, R275) whose presence is essential to function. Prior biochemical work had placed R275 at the external entrance to the translocation pathway, and a symmetry argument emerging from the multiple alignment suggests a similar position for R46. Accordingly, by virtue of their locations at the entrance to this pathway, we speculate that R46 and R275 function in establishing substrate specificity.
Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp 388 or Lys 391 but not if both are replaced. This suggests that Asp 388 and Lys 391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys 391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of 32 P i by homologous P i /P i exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the P i /P i exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, P i /P i exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related P i -linked antiporter, which lacks the Asp 388 -Lys 391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP.In Escherichia coli, transport of hexose phosphates is mediated by the P i -linked antiport carrier, UhpT (1-3). This well characterized transporter is one of a class of secondary transport systems that together form the Major Facilitator Superfamily (4 -6), the largest known collection of related secondary transporters. Although members of the Major Facilitator Superfamily show great diversity in their substrate specificity and kinetic mechanism, they all share a common structural theme, one characterized by the presence of approximately 12 transmembrane segments thought to transverse the membrane in an ␣-helical conformation. Direct information as to the arrangement of these helices is limited, although several potential models have been formulated in specific cases (7-9).One approach to the analysis of helix packing in membrane proteins involves identification of interacting charged residues in transmembrane helices. Ordinarily, one expects the presence of an uncompensated electric charge to be highly destabilizing to such structures, due to the low dielectric of the hydrophobic environment. However, this circumstance can be mitigated if oppositely charged amino acids are brought together to form an ion pair or salt bridge (10, 11). The idea that such a salt bridge may stabilize secondary structure has both theoretical and experimental support (11-13). Moreover, intraand interhelical salt bridges have been i...
UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which rightside-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward-and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His 10 K291C/ K294N, in which the sole external tryptic cleavage site (Lys 294 ) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His 10 K291C/K294N led to loss of about 50% of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His 10 V284C derivative, in which a newly inserted external cysteine (Cys 284 ) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N-maleimidylpropionyl)biocytin treatment of proteoliposomes containing His 10 V284C gave about a 60% loss of activity, and immunodetection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (His 10 K291C/K294N) or ISO orientation (His 10 V284C) with respect to the kinetics of glucose 6-phosphate transport by phosphateloaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose 6-phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.In Escherichia coli, the uhp locus (for uptake of hexose phosphates) coordinates the expression of four proteins responsible for incorporation of external sugar phosphate. UhpA and UhpB together form a two-component regulatory system that activates expression of the transporter, UhpT, after extracellular G6P binds to the membrane receptor, UhpC (1). UhpT then acts to move sugar 6-phosphate inward in exchange for internal inorganic phosphate (2) in an electrically neutral antiport reaction (2, 3). Hydropathy analysis of the UhpT amino acid sequence (4), the properties of UhpT-PhoA fusions (5, 6), and comparisons with other members of the major facilitator superfamily (7, 8) all argue that UhpT has 12 transmembrane ␣-helices and that its N and C termini lie in the cytoplasm (see Fig. 1). Accordingly, the structure of UhpT (and related transporters) is strongly asymmetric along an axis perpendicular to the membrane surface. This fact, along with the biochemical asymmetry of the in vivo reaction, in which external sugar phosph...
Part of the substrate translocation pathway through UhpT, the Escherichia coli sugar phosphate carrier, has been assigned to a transmembrane helix extending between residues 260 and 282. To set limits on the external portion of the pathway, we identified nearby residues fully exposed to the periplasm. In one case, we used Western blots to evaluate cleavage by extracellular trypsin. The protease cleaved UhpT variants retaining lysine 294, but not those lacking lysine 294, indicating that trypsin acts at a single extracellular site, lysine 294. In other work we labeled single-cysteine variants with 3-(N-maleimidylpropionyl)biocytin and scored accessibility to extracellular streptavidin by shifts of SDS-polyacrylamide gel electrophoresis mobility. Positions 283 and 284 were fully exposed to the periplasm, since the modified residue was bound by streptavidin in the native protein; by contrast, although the biotin-linked probe modified position 276, streptavidin decoration was not achieved without protein denaturation. We conclude that a 12-residue stretch (283-294) of UhpT is sufficiently exposed to be accessible to large probes (trypsin, streptavidin), while position 276 and more proximal residues are more deeply buried or otherwise shielded from the external phase.
In Escherichia coli, the GlpT transporter, a member of the major facilitator superfamily, moves external glycerol 3-phosphate (G3P) into the cytoplasm in exchange for cytoplasmic phosphate. Study of intact cells showed that both GlpT and HisGlpT, a variant with an N-terminal six-histidine tag, are inhibited (50% inhibitory concentration Ϸ 35 M) by the hydrophilic thiol-specific agent p-mercurichlorobenzosulfonate (PCMBS) in a substrate-protectable fashion; by contrast, two other thiol-directed probes, N-maleimidylpropionylbiocytin (MPB) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), have no effect. Use of variants in which the HisGlpT native cysteines are replaced individually by serine or glycine implicates Cys-176, on transmembrane helix 5 (TM5), as the major target for PCMBS. The inhibitor sensitivity of purified and reconstituted HisGlpT or its cysteine substitution derivatives was found to be consistent with the findings with intact cells, except that a partial response to PCMBS was found for the C176G mutant, suggesting the presence of a mixed population of both right-side-out (RSO) (resistant) and inside-out (ISO) (sensitive) orientations after reconstitution. To clarify this issue, we studied a derivative (P290C) in which the RSO molecules can be blocked independently due to an MPB-responsive cysteine in an extracellular loop. In this derivative, comparisons of variants with (P290C) and without (P290C/C176G) Cys-176 indicated that this residue shows substrate-protectable inhibition by PCMBS in the ISO orientation in proteoliposomes. Since PCMBS gains access to Cys-176 from both periplasmic and cytoplasmic surfaces of the protein (in intact cells and in a reconstituted ISO orientation, respectively) and since access is unavailable when the substrate is present, we propose that Cys-176 is located on the transport pathway and that TM5 has a role in lining this pathway.
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