The lactose permease (LacY) catalyzes coupled stoichiometric symport of a galactoside and an H + . Crystal structures reveal 12, mostly irregular, transmembrane α-helices surrounding a cavity with sugar-and H + -binding sites at the apex, which is accessible from the cytoplasm and sealed on the periplasmic side (an inward-facing conformer). An outward-facing model has also been proposed based on biochemical and spectroscopic measurements, as well as the X-ray structure of a related symporter. Converging lines of evidence demonstrate that LacY functions by an alternating access mechanism. Here, we generate a model for an apo-intermediate of LacY based on crystallographic coordinates of LacY and the oligopeptide/H + symporter. The model exhibits a conformation with an occluded cavity inaccessible from either side of the membrane. Furthermore, kinetic considerations and double electron-electron resonance measurements suggest that another occluded conformer with bound sugar exists during turnover. An energy profile for symport is also presented. proteins represents the largest family of secondary transporters, with members from Archaea to Homo sapiens (1, 2). MFS proteins catalyze transport of a wide range of substrates, including amines, acids, amino acids, sugars, peptides, and antibiotics, in many instances, by transducing the energy stored in an H + electrochemical gradient into a concentration gradient of substrate. The lactose permease of Escherichia coli (LacY), which catalyzes the coupled transport of a galactopyranoside and an H + (galactoside/H + symport) (3-6), is arguably the most extensively studied member of the MFS (7,8). Essentially all 417 amino acyl side chains in LacY have been mutagenized (9), and functional analyses of the mutants reveals that fewer than 10 side chains play a central role in the symport mechanism ( Fig. 1 A and B): Glu126 (helix IV), Arg144 (helix V), and Trp151 (helix V) are directly involved in galactoside recognition and binding; Tyr236 (helix VII), Glu269 (helix VIII), and His322 (helix X) are involved in both H + translocation and affinity for sugar; and Arg302 (helix IX) and Glu325 (helix X) play important roles in H + translocation (7, 10). In the available inward-facing crystal structures of , these residues are located at the apex of a deep central hydrophilic cavity, which is open to the cytoplasm only ( Fig. 1 A and B). The cavity is formed by 12 mostly irregular transmembrane helices organized in two pseudosymmetrical 6-helix bundles (helices I-VI and helices VII-XII) (11)(12)(13)(14).A six-step kinetic model for lactose/H + symport has been proposed (15, 16) (Fig. 1C). The steps include (i) deprotonation of LacY, (ii) a conformational change allowing the galactoside-and H + -binding sites to become accessible to the other side of the membrane, (iii) protonation of LacY, (iv) substrate binding, (v) a global conformational change in which the protonated LacY with bound sugar returns to the initial inward-facing conformation, and (vi) dissociation of sugar. This seque...