The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (⌬por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37°C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of ⌬por1 yeast grown on glycerol at 37°C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in ⌬por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30°C, albeit more slowly than ⌬por1 single mutants. Like ⌬por1 single mutants, they could not grow on glycerol at 37°C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, ⌬por1 cells, ⌬por1 ⌬por2 cells, and ⌬por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in ⌬por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37°C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.

These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general.

We report here on the isolation and characterization of a serotonin (SHT) transporter from Drosophila melanogaster. A 3.1-kb complementary DNA clone (dSERT) was found to encode a protein of 622 amino acid residues with a predicted molecular mass of w69 kDa and a putative transmembrane topology characteristic of cloned members of the mammalian Na+/Cl-neurotransmitter cotransporter gene family. dSERT displays highest overall amino acid sequence identity with the mammalian 5HT (51%), norepinephrine (47%), and dopamine (47%) transporters and shares with all transporters 104 absolutely conserved amino acid residues.Upon tnsient expression in HeLa cells, dSERT exhibited saturabl, high-affinity, and sodium-dependent [3H5HT uptake with esimated K and V. values of w500 nM and 5.2 x 1o-i8 mol per cell per min, respectively. In marked contrast to the human SERT (hSERT), SHT-mediated transport by dSERT was not absolutely dependent on extracellular Cl-, while the sodium-dependent uptake of MHT was facilitatd by Increased extracellular Cl-concentrations. dSERT disys a pharmacological profile and rank order of potency consistent with, but not identical to, mammalian 5HT transporters. Comparison of the afnities of various compounds for the inhibiion of 5HT transport by both dSERT and hSERT revealed that antidepressants were 3-to 300-fold less potent on dSERT than on hSERT, while mazindol displayed w30-fold greater potency for dSERT. Both cocaine and RTI-55 inhibited 5HT uptake by dSERT with estmated inhibition constants of 500 nM, while high concentrations (>10 FM) of dopmin, norepinephrine, octopamine, tramine, and histamine failed to inhibit transport. In situ hybridization reveals the selective expression of dSERT mRNA to specific cell bodies in the ventral gagulon of the embryonic and larval Drosophila nervous system with a distribution pattern virtually identical to that of 5HT-containing neurons. The dSERT gene was mapped to position 60C on chromosome 2. The availability of the gene e ing the unique ion dependence and pharmacological characteristics of dSERT may allow for identification of those amino acid residues and structural motifs that confer the phannacologrc specificity and genetic regulation of the 5HT transport process. Serotonin (5HT) plays a vital role in modulation ofa variety of biochemical and physiological functions in the central nervous system, including sleep, appetite, and pain perception, and has been shown to regulate complex behavioral phenomena, such as learning and memory in invertebrates (1, 2). Numerous studies have provided evidence for production of SHT in Drosophila as well as its cell-specific localization in neurons within the central nervous system (3). Released from neuronal terminals into the synaptic cleft, 5HT is believed to mediate its effects in Drosophila via an interaction with specific multiple cell surface 5HT receptors (4, 5), which in turn are coupled to subtype-specific guanine nucleotide-binding proteins (see ref.6 and references therein) activating various membran...