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J. Biol. Chem., Vol. 276, Issue 27, 25107-25113, July 6, 2001

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Transmembrane Segment 11 of UhpT, the Sugar Phosphate Carrier of Escherichia coli, Is an -Helix That Carries Determinants of Substrate Selectivity^*

Jason A. Hall and Peter C. Maloney^§

From the Department of Physiology, Johns Hopkins University Medical School, Baltimore, Maryland 21205

In Escherichia coli, transport of hexose 6-phosphates is mediated by the P_i-linked antiport carrier, UhpT, a member of the major facilitator superfamily. We showed earlier that Lys³⁹¹, a member of an intrahelical salt bridge (Asp³⁸⁸/Lys³⁹¹) in the eleventh transmembrane segment (TM11) of this transporter, can function as a determinant of substrate selectivity (Hall, J. A., Fann, M.-C., and Maloney, P. C. (1999) J. Biol. Chem. 274, 6148-6153). Here, we examine in detail the role of TM11 in setting substrate preference. Derivatives having an uncompensated cationic charge at either position 388 or 391 (the D388C, D388V, or D388K/K391C variants) are gain-of-function mutants in which phosphoenolpyruvate, not sugar 6-phosphate, is the preferred organic substrate. By contrast, when an uncompensated anionic charge is placed at position 388 (K391C), we observed behavior consistent with an increased preference for monovalent rather than divalent sugar 6-phosphate. Because positions 388 and 391 lie deep within the UhpT hydrophobic sector, these findings suggested that an extended length of TM11 may be accessible to external substrates and probes. To explore this issue, we used a panel of TM11 single cysteine variants to examine the transport of glucose 6-phosphate in the presence and absence of the membrane-impermeant, thiol-reactive agent p-chloromercuribenzosulfonate (PCMBS). Accessibility to PCMBS, together with the pattern of substrate protection against PCMBS inhibition, leads us to conclude that TM11 spans the membrane as an -helix, with approximately two-thirds of its surface lining a substrate translocation pathway. We suggest that this feature is a general property of carrier proteins in the major facilitator superfamily and that for this reason residues in TM11 will serve to carry determinants of substrate selectivity.

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