Enzymatic and biochemical probes of residues external to the translocation pathway of UhpT, the sugar phosphate carrier of Escherichia coli

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.