The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity - 14% or less – between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology / threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepTSt are most promising for further refinement.

The proton‐coupled folate transporter (PCFT, SLC46A1) transports folic acid across the plasma membrane, together with an excess of protons such that the net charge translocation is positive. We developed 3D structural models of PCFT threaded onto the X‐ray structures of major facilitator superfamily (MFS) members that were identified as close structural homologues. The model of PCFT threaded onto the glycerol‐3‐phosphate transporter (GlpT) structure is consistent with detailed accessibility studies in the absence of extracellular substrate and at pH 7.4 presented here, and additionally with a multitude of other mutagenesis and functional studies. Characteristic MFS structural features are preserved in this PCFT model, such as 12 transmembrane helices divided into two pseudosymmetric bundles, and a high density of positive charges on the periphery of the cytoplasmic site that allow interactions with negatively charged lipid head‐groups. Under the experimental conditions, PCFT predominantly samples the resting state, which in this case is inward‐open. Several positions lining the substrate cavity have been identified. Motif A, a helix‐turn‐helix motif that is a hallmark of MFS transporters between transmembrane segments II and III is oriented appropriately to interact with residues from transmembrane segments IV as well as XI upon conformational transition to the outward‐open state. A charge‐relay system between three charged residues as well as apposing glycines in two α‐helices, both contributed to by motif A, become engaged when PCFT is modeled on the outward‐open state of a putative proton‐driven transporter (YajR).

The evolutionary rates of functionally related genes often covary. We present a gene coevolution network inferred from examining nearly 3 million orthologous gene pairs from 332 budding yeast species spanning ~400 million years of evolution. Network modules provide insight into cellular and genomic structure and function. Examination of the phenotypic impact of network perturbation using deletion mutant data from the baker’s yeast Saccharomyces cerevisiae , which were obtained from previously published studies, suggests that fitness in diverse environments is affected by orthologous gene neighborhood and connectivity. Mapping the network onto the chromosomes of S. cerevisiae and Candida albicans revealed that coevolving orthologous genes are not physically clustered in either species; rather, they are often located on different chromosomes or far apart on the same chromosome. The coevolution network captures the hierarchy of cellular structure and function, provides a roadmap for genotype-to-phenotype discovery, and portrays the genome as a linked ensemble of genes.

Methylmercury (MeHg) is a potent bioaccumulative neurotoxin that is produced by certain anaerobic bacteria and archaea. Mercury (Hg) methylation has been linked to the gene pair hgcAB, which encodes a membrane-associated corrinoid protein and a ferredoxin. Although microbial Hg methylation has been characterized in vivo, the cellular biochemistry and the specific roles of the gene products HgcA and HgcB in Hg methylation are not well understood. Here, we report the kinetics of Hg methylation in cell lysates of Desulfovibrio desulfuricans ND132 at nanomolar Hg concentrations. The enzymatic Hg methylation mediated by HgcAB is highly oxygen sensitive, irreversible, and follows Michaelis-Menten kinetics, with an apparent K m of 3.2 nM and V max of 19.7 fmol · min Ϫ1 · mg Ϫ1 total protein for the substrate Hg(II). Although the abundance of HgcAB in the cell lysates is extremely low, Hg(II) was quantitatively converted to MeHg at subnanomolar substrate concentrations. Interestingly, increasing thiol/Hg(II) ratios did not impact Hg methylation rates, which suggests that HgcAB-mediated Hg methylation effectively competes with cellular thiols for Hg(II), consistent with the low apparent K m . Supplementation of 5-methyltetrahydrofolate or pyruvate did not enhance MeHg production, while both ATP and a nonhydrolyzable ATP analog decreased Hg methylation rates in cell lysates under the experimental conditions. These studies provide insights into the biomolecular processes associated with Hg methylation in anaerobic bacteria. IMPORTANCE The concentration of Hg in the biosphere has increased dramatically over the last century as a result of industrial activities. The microbial conversion of inorganic Hg to MeHg is a global public health concern due to bioaccumulation and biomagnification of MeHg in food webs. Exposure to neurotoxic MeHg through the consumption of fish represents a significant risk to human health and can result in neuropathies and developmental disorders. Anaerobic microbial communities in sediments and periphyton biofilms have been identified as sources of MeHg in aquatic systems, but the associated biomolecular mechanisms are not fully understood. In the present study, we investigate the biochemical mechanisms and kinetics of MeHg formation by HgcAB in sulfate-reducing bacteria. These findings advance our understanding of microbial MeHg production and may help inform strategies to limit the formation of MeHg in the environment.

The proton-coupled folate transporter (PCFT) provides an essential uptake route for the vitamin folic acid (B9) in mammals. In addition, it is currently of high interest for targeting chemotherapeutic agents to tumors due to the increased folic acid requirement of rapidly dividing tumor cells as well as the upregulated PCFT expression in several tumors. To understand its function, determination of its atomic structure and molecular mechanism of transport are essential goals that require large amounts of functional PCFT. Here, we present a high-level heterologous expression system for human PCFT using a recombinant baculovirus and Spodoptera frugiperda (Sf9) insect cells. We demonstrate folate transport functionality along the PCFT expression, isolation, and purification process. Importantly, purified PCFT transports folic acid after reconstitution. We thus succeeded in overcoming heterologous expression as a major bottleneck of PCFT research. The availability of an overexpression system for human PCFT provides the basis for future biochemical, biophysical and structural studies.