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The major facilitator superfamily represents the largest group of secondary membrane transporters in the cell. Here we report the 3.3 angstrom resolution structure of a member of this superfamily, GlpT, which transports glycerol-3-phosphate into the cytoplasm and inorganic phosphate into the periplasm. The amino- and carboxyl-terminal halves of the protein exhibit a pseudo two-fold symmetry. Closed off to the periplasm, a centrally located substrate-translocation pore contains two arginines at its closed end, which comprise the substrate-binding site. Upon substrate binding, the protein adopts a more compact conformation. We propose that GlpT operates by a single-binding site, alternating-access mechanism through a rocker-switch type of movement.
Membrane transport in cells is a fundamental biological process that is mediated by various channel and transporter proteins. A major type of such proteins is secondary active membrane transporters, which use a solute gradient to drive the translocation of other substrates (J). The largest secondary transporter protein family known so far is the major facilitator superfamily (MFS) (2-4), with more than 1000 members identified to date (5). These proteins transport ions, sugars, sugar-phosphates, drugs, neurotransmitters, nucleosides, amino acids, peptides, and other hydrophilic solutes. Members of this superfamily are ubiquitous in all three kingdoms of living organisms, and many have medical or pharmacological relevance. For example, the mammalian glucose transporter Glut4 from muscle and adipose cells is responsible for their glucose uptake, a process that is impaired in type II diabetes (6). Mutations in a related transporter, Glut1 from erythrocyte and brain-blood barrier, cause glucose transporter 1 deficiency syndrome, a disease whose symptoms include infantile seizures and developmental delay (7). Similarly, mutations in human glucose-6-phosphate transporter (G6PT) cause glycogen storage disease type 1b (8). In bacteria, MFS proteins function principally for nutrient uptake [like E. coli lactose permease (LacY) (9)], but some act as drug-efflux pumps that confer antibiotic resistance (10).
MFS proteins are typically 400 to 600 amino acids long and share transmembrane topology similarities and signature sequences in two cytosolic loops. Hydropathy sequence analysis and reporter-fusion experiments indicate that most MFS proteins have 12 transmembrane [alpha] helices, with both the N- and C-termini located in the cytosol (4). The two six-helix halves of an MFS protein, connected by a long central loop, are related by weak sequence similarity (11)....