SummaryThe NRT1/PTR family of proton-coupled transporters are responsible for nitrogen assimilation in eukaryotes and bacteria through the uptake of peptides. However, in the majority of plant species members of this family have evolved to transport nitrate as well as additional secondary metabolites and hormones. In response to falling nitrate levels, NRT1.1 is phosphorylated on an intracellular threonine that switches the transporter from a low to high affinity state. Here we present both the apo and nitrate bound crystal structures of Arabidopsis thaliana NRT1.1, which together with in vitro binding and transport data identify a key role for His356 in nitrate binding. Our data support a model whereby phosphorylation increases structural flexibility and in turn the rate of transport. Comparison with peptide transporters further reveals how the NRT1/PTR family has evolved to recognize diverse nitrogenous ligands, whilst maintaining elements of a conserved coupling mechanism within this superfamily of nutrient transporters.

SummaryThere has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interactions, and the functional implications of an enhanced understanding of the local membrane environment of each protein.

REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.Both prokaryotic and eukaryotic cells are endowed with multiple specialized DNA polymerases that are devoid of 3Ј35Ј proofreading exonuclease activity and replicate undamaged DNA in vitro with low fidelity and weak processivity (5). These specialized enzymes support DNA synthesis past a spectrum of template strand base damage by a process called translesion DNA synthesis (TLS), a mode of DNA damage tolerance that is fundamental to the survival of cells that suffer arrested DNA replication associated with damage to DNA.REV1 protein (which is confined to the eukaryotic kingdom) is a member of the Y family of DNA polymerases (14, 21). However, in vitro, the nucleotidyl transferase activity of REV1 is limited to the incorporation of just one or two dCMP moieties in a template-directed manner, regardless of the template nucleotide composition (19,30). This catalytic activity supports TLS past sites of base loss in vitro (19) and conceivably subserves this function in vivo. However, REV1 protein is also required for mutagenesis in both yeast and mammalian cells exposed to DNA-damaging agents that are not associated with the generation of sites of base loss, such as UV radiation (14). Remarkably, the dCMP transferase activity is dispensable for this function (1,14,18). Indeed, inactivation of the dCMP transferase activity in yeast does not result in defects in DNA damage-associated mutagenesis (9). Furthermore, a yeast mutant strain with a missense mutation in the N-terminal BRCT domain of REV1 retains dCMP transferase activity in vitro, even though it is deficient in TLS past sites of base loss and photoproducts (18).Several laboratories have demonstrated that the C-terminal ϳ100 amino acids of both mouse REV1 (mREV1) and human REV1 proteins can interact with multiple specialized DNA polymerases implicated in TLS (6,17,20,27). Additionally, different specialized DNA polymerases can compete with one another for binding to REV1 in vitro (6). Collectively, these observations suggest a presently unknown role(s) for REV1 in TLS that is unrelated to its dCMP transferase function.REV1 protein colocalizes with proliferating cell nuclear antigen (PCNA) in replication factories (27) and binds to other members of the Y family of DNA polymerases, to which it belongs, in...

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.

An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochemically distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.