The major facilitator superfamily (MFS) is the largest family of secondary active transporters and is present in all life kingdoms. Detailed structural basis of the substrate transport and energycoupling mechanisms of these proteins remain to be elucidated. YajR is a putative proton-driven MFS transporter found in many Gram-negative bacteria. Here we report the crystal structure of Escherichia coli YajR at 3.15 Å resolution in an outward-facing conformation. In addition to having the 12 canonical transmembrane helices, the YajR structure includes a unique 65-residue C-terminal domain which is independently stable. The structure is unique in illustrating the functional role of "sequence motif A." This highly conserved element is seen to stabilize the outward conformation of YajR and suggests a general mechanism for the conformational change between the inward and outward states of the MFS transporters.T ransporters are a type of membrane protein essential for all living cells that actively up-take nutrition and export metabolic substances and toxic materials across cellular membranes. Transporters are divided into two major types based on their energy sources. Although primary active transporters directly consume energy from ATP hydrolysis to drive substrate transport, secondary active transporters use energy derived from the electrochemical potential across the cell membrane. The major facilitator superfamily (MFS) is the largest class of secondary transporters and is present in all life kingdoms (1). For example, 25% of prokaryotic transporters belong to the MFS family (2), and the human genome contains over 110 MFS proteins (3). Currently, 3D crystal structures of nine bacterial MFS transporters (4-13) and one from fungi (9) have been reported at medium-high resolution. These studies have shown that MFS proteins contain a 12-transmembrane (TM) helix core composed of two six-helix rigid domains forming a central TM channel, which transports substrates using a rocker-switch mechanism (5). In such a mechanism, MFS proteins are believed to switch between two major conformations, inward and outward, which differ by an ∼40°rotation of one domain relative to the other. Both conformations have been captured in MFS crystal structures. However, many questions remain to be addressed, particularly those related to energy coupling and functional roles of conserved motifs.YajR, a 49-kDa transporter of the MFS family, has putatively been classified as a drug efflux protein solely on the basis of amino acid sequence analysis (14). In Escherichia coli, YajR consists of 454 amino acid residues. Besides containing 12 TM helices, YajR is predicted to possess an extra domain of about 65 residues at the C-terminal. Of the MFS proteins with reported 3D structures, the TM core of YajR shares highest sequence homology (21% identity) with EmrD (SI Appendix, Fig. S1A), which belongs to the 12-TM drug-resistance H + -driven antiporter (DHA12) subfamily (15). The YajR gene is found in a number of Gram-negative bacteria, and it shows high ...

Plant-specific transcription factor NAC proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. In the NAC family, some members function as transcription activators while others act as repressors. In the present study we found that though the full-length GmNAC20 from soybean did not have transcriptional activation activity, the carboxy-terminal activation domain of GmNAC20 had high transcriptional activation activity in the yeast assay system. Deletion experiments revealed an active repression domain with 35 amino acids, named NARD (NAC Repression Domain), in the d subdomain of NAC DNA-binding domain. NARD can reduce the transcriptional activation ability of diverse transcription factors when fused to either the amino-terminal or the carboxy-terminal of the transcription factors. NARD-like sequences are also present in other NAC family members and they are functional repression domain when fused to VP16 in plant protoplast assay system. Mutation analysis of conserved amino acid residues in NARD showed that the hydrophobic LVFY motif may partially contribute to the repression function. It is hypothesized that the interactions between the repression domain NARD and the carboxy-terminal activation domain may finally determine the ability of NAC family proteins to regulate downstream gene expressions.