A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

The main function of vacuolar H+-ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membranes of the vacuolar system. The enzyme is composed of a catalytic sector with five subunits (A-E) and a membrane sector containing at least two subunits (a and c). We disrupted two genes of this enzyme, in yeast cells, one encoding a subunit of the membrane sector (subunit c) and another encoding a subunit of the catalytic sector (subunit B). The resulting mutants did not grow in medium with a pH value higher than 6.5 and grew well only within a narrow pH range around 5.5. Transformation of the mutants with plasmids containing the corresponding genes repaired the mutations. Thus failure to lower the pH in the vacuolar system of yeast, and probably other eukaryotic cells, is lethal and the mutants may survive only if a low external pH allows for this acidification by fluid-phase endocytosis.The pH of various compartments in eukaryotic cells is maintained by carriers and ion pumps. The limited number of primary ion pumps found in nature suggests that each type may well perform a number of processes in a wide variety of organelles (1, 2). H+-ATPases are such ion pumps. They generate protonmotive force at the expense of energy stored as ATP and also form ATP from protonmotive force generated by photosynthetic and respiratory electron transport. Proton pumps can be classified into three families: plasmamembrane type (P-ATPase), eubacterial type (F-ATPase), and vacuolar type (V-ATPase) (3, 4). The P-ATPases are evolutionarily distinct from the F-and V-type ATPases, which have been shown to be related and have probably evolved from a common ancestral enzyme (4-6). F-ATPases function mainly in ATP synthesis and are present in eubacterial as well as in chloroplasts and mitochondria. V-ATPases are present in archaebacteria, where they function both in ATP synthesis and hydrolysis, and the vacuolar system of eukaryotes, where they function solely as ATP-dependent proton pumps (5-7). A wide variety of organelles in the vacuolar system, including lysosomes, plants and fungal vacuoles, synaptic vesicles, clathrin-coated vesicles, and the trans-Golgi, are energized by the protonmotive force generated by V-ATPases (8-10). Moreover, V-ATPases have been found to be highly conserved, and it appears that in all these organelles a similar, if not identical, proton pump is operating (10-12). Like the F-ATPases, V-ATPases are multisubunit enzymes with distinct catalytic and membrane sectors. The catalytic sector is composed of at least five polypeptides denoted as subunits A-E, in the order of decreasing molecular masses (6, 10, 11). The membrane sector is comprised of at least two hydrophobic subunits designated a and c (proteolipid). The genes encoding the B subunit (57 kDa) of the catalytic sector and the proteolipid or c subunit (16 kDa) in yeast have been cloned and sequenced, and both genes were shown to exist as single copies in the yeast genome (12, 13).This feature allowed a convenient inte...

A novel Saccharomyces cerevisiae mutant, unable to grow in the presence of 12.5 mM EGTA, was isolated by replica plating. The phenotype of the mutant is caused by a single amino acid change (Gly'49 to Arg) leu2, his3, ade2, trpl, ura3), leu2, his3, ade2, trpl, ura3), E20 (MA Ta, masi), and CRB1-5A (MATa, leu2, his3, his4-4Q1, trpl, ura3-52, HOLJ-1, masl-ts) were used. EGTA-sensitive mutants were generated by exposing yeast cells to ethyl methanesulfonate (EMS) as described (14) and replica plating on YPD plates (pH 6.0) containing 50 mM Mes and 12.5 mM EGTA. The mutants denoted as csp (chelator-sensitive phenotype) were back-crossed to the wild-type strain as described elsewhere (15)

A taurine/fl-alanine transporter was cloned from a mouse brain cDNA library by screening with a partial cDNA probe of the glycine transporter at low stringency. The deduced amino acid sequence predicts 590 amino acids with typical characteristics of the sodium-dependent neurotransmitter transporters such as sequence homology and membrane topography. However, the calculated isoelectric point of the taurine/fi-alanine transporter is more acidic (pI = 5.98) than those (pI > 8.0) of other cloned neurotransmitter transporters. Xenopus oocytes Injected with cRNA of the cloned transporter expressed uptake activities with K. = 4.5 FM for taurine and K. = 56 FM for (3-alanine. Northern hybridization showed a single transcript of 7.5 kil9bases that was highly enriched in kidney and distributed evenly in various parts of the brain. In situ hybridization showed the mRNA of the taurine/(3-alanine transporter to be localized in the corpus callosum, striatum, and anterior commisure. Specific localization ofthe taurine/palanine transporter in mouse brain suggests a-potential function for taurine and (3-alanine as neurotransmitters.

Vacuolar H+-ATPases function in generating protonmotive force across the membranes of organelies connected with the vacuolar system of eukaryotic cells. This family of H+-ATPases is distinct from the two other families of H+-ATPases, the plasma membrane-type and the eubacterialtype. One of the subunits of the vacuolar H+-ATPase binds N,N'-dicyclohexylcarbodiimide (DCCD) and has been implicated in the proton-conducting activity of these enzymes. We have cloned and sequenced the gene encoding the DCCDbinding protein (proteolipid) of the H+-ATPase of bovine chromaffim granules. The gene encodes a highly hydrophobic protein of 15,849 Da. Hydropathy plots revealed four transmembrane segments, one of which contains a glutamic residue that is the likely candidate for the DCCD binding site. Sequence homology with the vacuolar proteolipid and with the proteolipids of eubacterial-type H+-ATPases was detected. The proteolipids from Escherichia coli, spinach chloroplasts, and yeast mitochondria matched better to the NH2-terminal part of the vacuolar protein. The proteolipids of bovine mitochondria and Neurospora mitochondria matched better to the COOHterminal end of the vacuolar proteolipid. These findings suggest that the proteolipids of the vacuolar H+-ATPases were evolved in parallel with the eubacterial proteolipid, from a common ancestral gene that underwent gene duplication.Proton-transporting ATPases (H+-ATPases) play a crucial role in biological energy transduction (1). These ion pumps can be classified into three main families: plasma membranetype, eubacterial-type, and vacuolar-type enzymes (2-5). The plasma membrane-type enzyme is present in the plasma membrane ofplants, fungi, and acid-secreting gastric vesicles (6, 7). The catalysis of these enzymes involves a phosphoenzyme intermediate. The gene coding for this 100-kDa protein in yeast and Neurospora crassa has been cloned and sequenced (8)(9)(10).The eubacterial-type enzyme occurs in chloroplasts, mitochondria and bacteria and .operates without a phosphoenzyme intermediate (11)(12)(13). These enzymes are composed of two distinct structures, a membrane sector, which is hydrophobic, and a catalytic sector, which is hydrophilic in nature. The function of the membrane sector is to conduct protons across the membrane. This sector is composed of three or more polypeptides, one of which is an N,N'-dicyclohexylcarbodiimide (DCCD)-binding protein (proteolipid of about 8 kDa) that is involved in the proton conduction (14-16). The catalytic sector, which is the site of the ATPase reaction, can be readily separated from the membrane by EDTA treatment or by applying mechanical force (17).The vacuolar-type enzyme is present in organelles connected with the vacuolar system of eukaryotic cells and pumps protons without the involvement of a phosphoenzyme intermediate (2-5). These H+-ATPases are composed of several polypeptides, and to our knowledge none of these have been sequenced. A 16-kDa subunit resembles the eubacterial proteolipids in that it binds DCCD and is s...

Yeast mutants in which genes encoding subunits of the vacuolar H+-ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities. The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions. Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled. Labeling with N,N'-["4Cdicyclohexylcarbodi-imide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted. No labeling was detected in the mutant in which the gene encoding the proteolipid was interrupted. We conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of-the other subunits. Site-specific mutations were generated in the gene encoding the proteolipid. All of the drastic changes and replacements gave inactive proteins. About half of the single amino acid replacements gave active proteins. Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme. Other amino acids that may function in proton conductance were changed. It was found that glycine residues may replace amino acids with exchangeable protons.The vacuolar H+-ATPase (V-ATPase) is a multisubunit protein complex containing catalytic and membrane sectors (1-5). The catalytic sector of the enzyme from a variety of sources contains five different polypeptides (5-9). In contrast, the precise subunit composition of the membrane sector is not yet defined (3-6). However, an N,N'-dicyclohexylcarbodiimide (DCCD)-binding protein (proteolipid) of about 16 kDa has been identified in all eukaryotic V-ATPases that have been characterized (10-16). Moreover, it was shown in several systems that DCCD caused inhibition of ATPase and protonpumping activities, leading to conclusions that the vacuolar proteolipid functions in proton conductance across the membrane (14)(15)(16) Most of the available shuttle vectors are larger than 7 kilobase pairs (kb), making it difficult to clone large genes into them. Therefore, we designed a plasmid carrying an inducible promoter suitable for efficient transformation. The plasmid was derived from YRP17, which was digested by the restriction enzymes Nde I and Cla I. The DNA fragment between nucleotides 4939 (Nde I site) and 1487 (Cla I site) was isolated and ligated with two oligonucleotides to generate a polylinker with restriction sites in the following order: Cla I (on the 5' end), EcoRI, BamHI, Sma I, Pst I, Sph I, Kpn I, and Nde I (on the 3' end). The 0.8-kb GAL] promoter having EcoRI and BamHI ends (20) was cloned into the respective restriction sites in the polylinker. The EcoRI site was then abolished by cutting the plasmid with EcoRI, followed by S1 nuclease digestion and religation in the presence of EcoRI. The resulting plasmid was digested with BamHI and Sph I and a new polylinker...

Metal ions are important for all living cells. In man, metal ion deficiency leads to anemia (1), whereas metal ion overload is toxic and leads to hemochromatosis (3), Menkes' disease (2), Wilson's disease (4), and neurodegenerative diseases (5-7). Metal ions such as iron, manganese, zinc, and cobalt are involved in many catalytic reactions, gene regulation, and signal transduction pathways (8 -10). An adequate supply of metal ions to cells is important and is provided by specialized transporters.The recently cloned mammalian metal ion transporter DCT1 (11, 12), originally named Nramp2 (natural resistance-associated macrophage protein 2) (13-15), is present in both plasma membranes and endosomal vesicles for translocation, via transferrin-dependent and -independent pathways, of metal ions into the cytoplasm of cells and for maintenance of systemic metal ion homeostasis. It has been found that a mutation in DCT1 at position 185 (G185R) causes microcytic anemia in mk Ϫ/Ϫ mice and Belgrade rats (12). This mutation was subsequently shown to result in loss of Fe 2ϩ transport ability (16). DCT1-mediated iron absorption in the intestine depends on the body iron status, which is regulated in part by the hemochromatosis gene HFE, a major histocompatibility complex gene (17, 18). A single point mutation in HFE (C282Y) results in iron overload in hemochromatosis patients (3). SMF1, SMF2, and SMF3 are yeast homologues of the Nramp proteins with 51-54% identity in amino acid sequence to each other and 33-36% identity to DCT1. SMF1 was originally thought to be localized in the yeast mitochondrial membrane (19) and was named SMF, which stands for suppressor of mitochondria import function. However, more recent studies using an antibody demonstrated that SMF1 is located in the yeast plasma membrane, where it is thought to mediate uptake of Mn 2ϩ and Zn 2ϩ into the cytoplasm (20). There was indirect evidence that other divalent metal ions such as Cd 2ϩ , Co 2ϩ , and Cu 2ϩ are also substrates of SMF1 (21). In analogy to HFE in mammalian cells, the product of the yeast BSD2 (bypass superoxide dismutase deficiency gene 2), localized in the endoplasmic reticulum, regulates metal ion absorption by exerting a negative control on SMF1 activity (21,22). Despite these findings, a functional characterization of SMF1 has not yet been reported.In the present study, we expressed SMF1, SMF2, and SMF3 in Xenopus oocytes and used both a radiotracer approach and the two-microelectrode voltage-clamp technique to investigate the function of these proteins. We show that SMF1 mediates H ϩ -dependent Fe 2ϩ transport and uncoupled Na ϩ currents. SMF2 also mediates significant H ϩ -coupled Fe 2ϩ transport and uncoupled Na ϩ currents, which are much smaller than those mediated by SMF1. SMF3 exhibited no detectable activities when expressed in oocytes. Because Na ϩ inhibited metal ion uptake in oocytes expressing SMF1, we investigated the effect of Na ϩ on yeast growth. EXPERIMENTAL PROCEDURESOocyte Preparation-Yeast SMF1, SMF2, and SMF3 cDNAs were subclo...

A rat cDNA clone encoding the novel membrane protem of the nemotransmitter transporters family was cloned and sequenced. The cDNA was identified as a transcript of the gene NTT4 of which a partial genomic clone was previously sequenced. Alignment of the amino acid sequence of NTT4 with other members of the neurotransmitter transporter family revealed a marked deviation from the conserved structure of all other members of the family. The largest extracellular loop with a potential glycosylation site was identified between membrane segments 7 and 8. The protem retains the common glycosylated loop between transmembrane helices 3 and 4 in all members of the family. The transcript of NTT4 was found exclusively in the central nervous system and is more abundant in the cerebellum and the cerebral cortex.