Putative Membrane-spanning Domains Research Articles

Cloning of a buffalo (Bubalus bubalis) prostaglandin F(2alpha) receptor: changes in its expression and concentration in the buffalo cow corpus luteum.

Acting primarily through its specific G protein-coupled receptor termed FPr, prostaglandin (PG) F(2alpha) induces regression of the corpus luteum (CL) at the end of a non-fertile oestrous cycle. This study was aimed at cloning a full-length cDNA for FPr and determining its expression and protein concentrations during different stages of CL development in the water buffalo. Serum progesterone and StAR expression were determined to establish temporal relationships between indices of steroidogenesis and changes in FPr expression at different stages of CL development. In contrast to the dairy cow, the stage IV CL (day 20 of the oestrous cycle) did not appear to be functionally regressed in the buffalo. Molecular cloning of a cDNA encoding the buffalo FPr yielded a full length 2193 bp FPr cDNA containing a single open reading frame encoding a 362 amino acid protein with seven putative membrane-spanning domains. The deduced buffalo FPr amino acid sequence possesses a high degree of identity with the other mammalian homologues. Steady state concentration of buffalo FPr transcript increased (P > 0.05) from stage I to stage II/III, and declined at 18 h post PGF(2alpha) injection. The FPr concentration expressed as fmol/microg of plasma membrane protein showed an increase (P > 0.05) from stage I (1.98 +/- 0.10), through stage II/III (2.42 +/- 0.48) to stage IV (2.77 +/- 0.18). High affinity FPr was observed in stage I (K(d) 4.86 nmol) and stage II/III (K(d) 6.28 nmol) while low affinity FPr (K(d) 19.44 nmol) was observed in stage IV. In conclusion, we have cloned a full length FPr cDNA from buffalo cow CL and observed that FPr mRNA expression, receptor number and affinity did not vary significantly (P > 0.05) within the luteal phase of the oestrous cycle.

Kinetic properties of a micronutrient transporter from Pisum sativum indicate a primary function in Fe uptake from the soil.

Fe uptake in dicotyledonous plants is mediated by a root plasma membrane-bound ferric reductase that reduces extracellular Fe(III)-chelates, releasing Fe(2+) ions, which are then absorbed via a metal ion transporter. We previously showed that Fe deficiency induces an increased capacity to absorb Fe and other micronutrient and heavy metals such as Zn(2+) and Cd(2+) into pea ( Pisum sativum L.) roots [Cohen et al. (1998) Plant Physiol 116:1063-1072). To investigate the molecular basis for this phenomenon, an Fe-regulated transporter that is a homologue of the Arabidopsis IRT1 micronutrient transporter was isolated from pea seedlings. This cDNA clone, designated RIT1 for root iron transporter, encodes a 348 amino acid polypeptide with eight putative membrane-spanning domains that is induced under Fe deficiency and can functionally complement yeast mutants defective in high- and low-affinity Fe transport. Chelate buffer techniques were used to control Fe(2+) in the uptake solution at nanomolar activities representative of those found in the rhizosphere, and radiotracer methodologies were employed to show that RIT1 is a very high-affinity (59)Fe(2+) uptake system ( K(m) =54-93 nM). Additionally, radiotracer ((65)Zn, (109)Cd) flux techniques were used to show that RIT can also mediate a lower affinity Zn and Cd influx ( K(m) of 4 and 100 microM, for Zn(2+) and Cd(2+), respectively). These findings suggest that, in typical agricultural soils, RIT1 functions primarily as a high-affinity Fe(2+) transporter that mediates root Fe acquisition. This is consistent with recent findings with Arabidopsis IRT1 knockout mutants that strongly suggest that this transporter plays a key role in root Fe uptake and nutrition. However, the ability of RIT1 to facilitate Zn and Cd uptake when these metals are present at elevated concentrations suggests that RIT1 may be one pathway for the entry of toxic metals into the food chain. Furthermore, the finding that plant Fe deficiency status may promote heavy metal uptake via increased expression of this transporter could have implications both for human nutrition and also for phytoremediation, the use of terrestrial plants to sequester toxic metals from contaminated soil.

Characterization of truncated mutants of human microsomal short-chain dehydrogenase/reductase RoDH-4

Human NAD +-dependent microsomal short-chain dehydrogenase/reductase RoDH-4 oxidizes all- trans-retinol, 13- cis-retinol and 3α-hydroxysteroids to corresponding retinaldehydes and 3-ketones. RoDH-4 behaves as an integral membrane protein, but its topology in the membrane is not known. Analysis of RoDH-4 polypeptide using algorithms for secondary structure predictions suggests that RoDH-4 contains four potential membrane-spanning domains: the N-terminal, the C-terminal, and the two central hydrophobic segments. To determine the role of each segment in association of RoDH-4 with the membrane, we prepared several expression constructs coding for truncated RoDH-4 polypeptides that lacked the putative membrane-spanning domains and expressed them in insect Sf9 cells using the Baculovirus system. Association of truncated RoDH-4 constructs with the microsomal membranes was analyzed by alkaline extraction and floatation in sucrose gradient. Catalytic activity of truncated RoDH-4 constructs was assayed using the 3α-hydroxysteroid androsterone as substrate. Truncated RoDH-4 that lacked the first thirteen amino acids of the N-terminal segment was partially active and exhibited the apparent K m value for androsterone similar to that of the wild-type enzyme. Removal of 23 N-terminal hydrophobic amino acids resulted in significant loss of activity and a 14-fold increase in the apparent K m value. Removal of the C-terminal 27 amino acid segment resulted in a ∼600-fold increase in the apparent K m value. Each truncated mutant behaved as an integral membrane protein. Furthermore, protein that lacked all four hydrophobic segments remained associated with the membrane. Thus, the N-terminal and the C-terminal ends are both important for RoDH-4 activity and the removal of the putative transmembrane segments does not convert RoDH-4 into a soluble protein, suggesting additional sites of membrane interaction.

ABCC13, an unusual truncated ABC transporter, is highly expressed in fetal human liver

In the present study, we have cloned the cDNA of ABCC13, a novel ABC transporter, from the cDNA library of adult human placenta. The ABCC13 gene spans ∼70 kb on human chromosome 21q11.2 and consists of 14 exons. The open reading frame of the ABCC13 cDNA encodes a peptide consisting of 325 amino acid residues. The amino acid sequence corresponding to putative membrane-spanning domains was remarkably similar to ABCC1, ABCC2, ABCC3, and ABCC6. The ABCC13 gene was expressed in the fetal liver at the highest level among the organs studied. While ABCC13 was expressed in the bone marrow, its expression in peripheral blood leukocytes of adult humans was much lower and no detectable levels were observed in differentiated hematopoietic cells. The expression of ABCC13 in K562 cells decreased during cell differentiation induced by TPA. These results suggest that the expression of human ABCC13 is related with hematopoiesis.

Identification and characterization of a novel mitochondrial tricarboxylate carrier

Here we report the identification and functional characterization of a novel mitochondrial tricarboxylate carrier protein, designated BBG-TCC, in rat brain. The cDNA encodes the predicted protein of 342-amino acid residues with five putative membrane-spanning domains. The protein has apparent similarity with a mitochondrial tricarboxylate carrier TCC, but is distinct from the other mitochondria anion transporters. BBG-TCC shows a citrate transport activity. It is specifically expressed in the brain and localizes in the mitochondria of Bergmann glial cells. In contrast, the expression of TCC is rather ubiquitous and strong in neuronal cells in the brain. This new family of proteins may contribute to biosynthesis and bioenergetics in the brain.

Isolation, characterization and differential gene expression of multispecific organic anion transporter 2 in mice.

We isolated cDNA encoding a multispecific organic anion transporter 2 (OAT2) from the mouse kidney cDNA library. Isolated mouse OAT2 (mOAT2) consisted of 1623 base pairs that encoded a 540-amino acid residue protein with 12 putative membrane-spanning domains, and the amino acid sequence was 87% identical to that of rat OAT2 (rOAT2). The gene coding for mOAT2, Slc22a7, is found on chromosome 17C. Northern blot analysis revealed that the mOAT2 mRNA is abundantly expressed in the male mouse kidney, whereas it was predominantly expressed in both the liver and kidney of female mice. When expressed in Xenopus laevis oocytes, mOAT2 mediated the high affinity transport of glutarate (K(m) = 15.8 +/- 3.2 microM) and prostaglandin E2 (K(m) = 5.2 +/- 0.5 nM) in a sodium-independent manner. mOAT2-expressing oocytes also mediated the uptake of alpha-ketoglutarate, glutarate, prostaglandin E2, p-aminohippuric acid, methotrexate, ochratoxin A, valproate, and allopurinol. However, we did not observe mOAT2-mediated uptake of salicylate. A wide range of structurally unrelated organic anions inhibited mOAT2-mediated glutarate uptake especially erythromycin, a potent inhibitor. These results indicate that isolated mOAT2 is a multispecific organic anion transporter having some differences in substrate specificity compared with rOAT2. In addition, we found that there exists a sex- and species-related differential gene expression of the OAT2 isoform.

Lysosome-associated protein transmembrane 4 alpha (LAPTM4 alpha) requires two tandemly arranged tyrosine-based signals for sorting to lysosomes.

Lysosome-associated protein transmembrane 4 alpha (LAPTM4 alpha) and homologues comprise a family of conserved proteins, which are found in mammals, insects and nematodes. LAPTM4 alpha functions to regulate the intracellular compartmentalization of amphipathic solutes and possibly the sensitivity of cells toward anthracyclines, antibiotics, ionophores, nucleobases and organic cations. This is similar to the multidrug-resistance phenotype exhibited by cells synthesizing high levels of P-glycoprotein. Accordingly, it is possible that LAPTM4 alpha may be a suitable target for development of novel chemotherapeutic agents. LAPTM4 alpha contains four putative membrane-spanning domains and a 55 amino acid C-terminal region that faces the cytoplasm. Localization of LAPTM4 alpha to endosomes and lysosomes appears to be tightly controlled as transient high-level expression of LAPTM4 alpha in cultured cells resulted in no detectable protein on the cell surface. Mutagenic analysis of the C-terminus of LAPTM4 alpha indicated that two tandomly arranged tyrosine-containing motifs in the cytoplasmic domain are required for efficient localization of LAPTM4 alpha to vesicles containing the lysosomal marker lysosomal glycoprotein 120. Although a number of membrane proteins that localize to endosomes/lysosomes contain more than one independently functioning sorting signal, to our knowledge, LAPTM4 alpha is the first example of a membrane protein that requires two tandemly arranged tyrosine-based sorting signals for efficient localization in these compartments.

The Human T-Type Amino Acid Transporter-1: Characterization, Gene Organization, and Chromosomal Location

System T is a Na+-independent transport system that selectively transports aromatic amino acids. Here, we determined the structure of the human T-type amino-acid transporter-1 (TAT1) cDNA and gene (SLC16A10). The human TAT1 cDNA encoded a 515-amino-acid protein with 12 putative membrane-spanning domains. Human SLC16A10 was localized on human chromosome 6, mapped to 6q21–q22. SLC16A10 contains six exons spanning 136 kb. In contrast to rat TAT1, which is mainly present in the intestine, human TAT1 was strongly expressed in human kidney as well as in human intestine. Expression of human TAT1 in Xenopus laevis oocytes demonstrated the Na+-independent transport of tryptophan, tyrosine, phenylalanine, and L-dopa, indicating that human TAT1 is a transporter subserving system T. Because human TAT1 is proposed to be crucial to the efficient absorption of aromatic amino acids from intestine and kidney, its defect could be involved in the disruption of aromatic amino-acid transport, such as in blue diaper syndrome.

SLC11A1 (formerly NRAMP1) and disease resistance.

Slc11a1 (formerly Nramp1) has many pleiotropic effects on macrophage (m) activation, including regulation of the CXC chemokine KC, interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), major histocompatibility complex (MHC) class II molecules, tumour necrosis factor α (TNFα), nitric oxide (NO) release, l-arginine flux, oxidative burst and tumoricidal as well as antimicrobial activity (reviewed by Blackwell and Searle, 1999; Blackwell et al., 2000). A naturally occurring Gly→Asp mutation at amino acid 169 of Slc11a1 makes mice as susceptible to Leishmania donovani, Salmonella typhimurium and Mycobacterium bovis as gene-disrupted mice (Vidal et al., 1995). Hence, the mutation is a functional null. This mutation also confers susceptibility to a range of other pathogens in mice, including Mycobacterium lepraemurium (Brown et al., 1982; Skamene et al., 1984), Mycobacterium intracellulare (Goto et al., 1989), Toxoplasma gondii (Blackwell et al., 1994), Candida albicans (Puliti et al., 1995) and Leishmania infantum (Leclercq et al., 1996). In man, SLC11A1 is linked or associated with multiple infectious (Shaw et al., 1997; Abel et al., 1998; Bellamy et al., 1998; Marquet et al., 1999; Cervino et al., 2000; Gao et al., 2000; Greenwood et al., 2000; Ryu et al., 2000; Mohamed et al., 2001) and autoimmune (Shaw et al., 1996; Hofmeister et al., 1997; Esposito et al., 1998; Maliarik et al., 2000; Sanjeevi et al., 2000; Singal et al., 2000; Yang et al., 2000) diseases. The infectious diseases include viral (HIV), bacterial (tuberculosis, leprosy, meningococcal meningitis) and protozoan (visceral leishmaniasis) pathogens. The autoimmune diseases include rheumatoid arthritis, juvenile rheumatoid arthritis, diabetes, sarcoidosis and Crohn's disease. Mutation in the closely related Slc11a2 (Nramp2) gene causes microcytic anaemia in mice (Fleming et al., 1997), but disease association in man has not been reported. Slc11a1 and Slc11a2 are polytopic integral membrane proteins with 10–12 putative membrane-spanning domains (Vidal et al., 1993; Gunshin et al., 1997). In both, the natural functional null mutation occurs in transmembrane domain 4 (Vidal et al., 1993; Fleming et al., 1997). Both Slc11a1 and Slc11a2 have protein kinase C (PKC) binding sites (Vidal et al., 1993; Barton et al., 1994; Gruenheid et al., 1995), but only Slc11a1 has a Pro–Ser-rich N-terminus (Barton et al., 1994). Here, we review current knowledge on the evolution, function and roles of Slc11a1/SLC11A1 in disease.

Mutations within the Putative Membrane-Spanning Domain of the Simian Immunodeficiency Virus Transmembrane Glycoprotein Define the Minimal Requirements for Fusion, Incorporation, and Infectivity

The membrane-spanning domain (MSD) of a number of retroviral transmembrane (TM) glycoproteins, including those from the human and simian immunodeficiency viruses (HIV and SIV), have been predicted to contain a charged arginine residue. The wild-type SIV TM glycoprotein is 354 amino acids long. The entire putative cytoplasmic domain of SIV (amino acids 193 to 354) is dispensable for virus replication in vitro, and such truncation-containing viruses are capable of reaching wild-type titers after a short delay. We show here that further truncation of eight additional amino acids to TM185 results in a protein that lacks fusogenicity but is, nevertheless, efficiently incorporated into budding virions. By analyzing a series of nonsense mutations between amino acids 193 and 185 in Env expression vectors and in the SIVmac239 proviral clone, a region of the SIV TM that contains the minimum requirement for glycoprotein-mediated cell-to-cell fusion and that for virus replication was identified. Virus entry and infectivity were evident in truncations to a minimum of 189 amino acids, whereas cell-cell fusion was observed for a protein of only 187 amino acids. Glycoprotein was efficiently incorporated into budding virions in truncations up to 185 amino acids, indicating that such proteins are membrane anchored and are transported to the cell surface. However, truncation of the TM to 180 amino acids resulted in a protein that displays a transport defect and may be retained in the endoplasmic reticulum. Based on our analyses of these mutants, an alternative model for the MSD of SIV is proposed. Our model suggests that membrane-imbedded charged residues can be neutralized by side-chain interactions with lipid polar head groups. As a consequence, the membrane-spanning region can be reduced by more than a helical turn. This new model accounts for the ability of truncations within the predicted MSD to remain membrane anchored and maintain biological activity.

Structural determinants of Ca2+ transport in the Arabidopsis H+/Ca2+ antiporter CAX1.

Ca(2+) levels in plants, fungi, and bacteria are controlled in part by H(+)/Ca(2+) exchangers; however, the relationship between primary sequence and biological activity of these transporters has not been reported. The Arabidopsis H(+)/cation exchangers, CAX1 and CAX2, were identified by their ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. CAX1 has a much higher capacity for Ca(2+) transport than CAX2. An Arabidopsis thaliana homolog of CAX1, CAX3, is 77% identical (93% similar) and, when expressed in yeast, localized to the vacuole but did not suppress yeast mutants defective in vacuolar Ca(2+) transport. Chimeric constructs and site-directed mutagenesis showed that CAX3 could suppress yeast vacuolar Ca(2+) transport mutants if a nine-amino acid region of CAX1 was inserted into CAX3 (CAX3-9). Biochemical analysis in yeast showed CAX3-9 had 36% of the H(+)/Ca(2+) exchange activity as compared with CAX1; however, CAX3-9 and CAX1 appear to differ in their transport of other ions. Exchanging the nine-amino acid region of CAX1 into CAX2 doubled yeast vacuolar Ca(2+) transport but did not appear to alter the transport of other ions. This nine-amino acid region is highly variable among the plant CAX-like transporters. These findings suggest that this region is involved in CAX-mediated Ca(2+) specificity.

ANT1, an aromatic and neutral amino acid transporter in Arabidopsis.

A new amino acid transporter was identified from the Arabidopsis expressed sequence tag cDNAs by expressing the cDNA in a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast showed that it transports aromatic and neutral amino acids, as well as arginine. This transporter (ANT1, aromatic and neutral transporter) also transports indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. The cDNA is 1.6 kb in length with an open reading frame that codes for a protein with 432 amino acids and a calculated molecular mass of 50 kD. Hydropathy analysis showed ANT1 is an integral membrane protein with 11 putative membrane-spanning domains. Southern analysis and a BLAST search of the Arabidopsis genome database suggests that ANT1 is part of a small gene family containing at least five members. Phylogenetic comparisons with other known amino acid transporters in plants suggests that ANT1 represents a new class of amino acid transporter. RNA gel-blot analysis showed that this transporter is expressed in all organs with highest abundance in flowers and cauline leaves.

Processing of the replicase of murine coronavirus: papain-like proteinase 2 (PLP2) acts to generate p150 and p44.

For Nidoviruses, proteolytic processing of a large polyprotein translated from the 5′-end of the genomic RNA is required for the maturation and assembly of the viral replicase complex. The scheme used to process the arterivirus equine arteritis virus (EAV) replicase polyprotein has been experimentally determined (reviewed in Snijder and Meulenberg, 1998). The EAV replicase polyprotein is processed by 3 distinct proteinases to generate 12 mature protein products. Polyprotein intermediates have been identified as well as major and minor processing pathways. However, the role of the intermediates and the alternate pathways remains to be investigated. For the Coronavirus mouse hepatitis virus (MHV), several laboratories are investigating the pathways used to process the replicase polyprotein. Two major processing models have been postulated and are shown in Figure 1. Studies from our laboratory showed that p150 is an intermediate to the 3C-like Proteinase (3CLpro) product p27 and that p150 likely extends to include the putative membrane-spanning protein domain 1, MPI (Schiller et al 1998). In contrast, other investigators have not detected the p150 precursor and postulate that a p240 product is adjacent to p27 (Denison et al 1992; 1995 and Lu et al 1998). In this study, we developed a specific antiserum to the MPI domain (anti-D11) and determined that the MPI domain is indeed part of the p150 intermediate. Furthermore, we show that MHV papain-like Proteinase 2 (PLP2) is responsible for cleaving the polyprotein at the putative p150 cleavage site. These results show that PLP2 is an active enzyme.

Cloning, characterization and chromosomal assignment of NBC4, a new member of the sodium bicarbonate cotransporter family

We report the cloning, characterization and chromosomal assignment of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC4, from human heart. NBC4 maps to chromosome 2p13 and is a new candidate gene for Alstrom syndrome. NBC4 encodes a 1074-residue polypeptide with 12 putative membrane-spanning domains. Unlike other members of the NBC family, NBC4 has a unique glycine-rich region (amino acids 438–485). In addition, NBC4 lacks the lysine-rich C-terminus of NBC1 with which it is most homologous. The first of two putative stilbene binding motifs (K(M/L)(X)K) is lacking in NBC4 (amino acids 655–658). The ∼6 kb NBC4 transcript is moderately expressed in heart, with the highest expression in liver, testes and spleen.

The multispecific organic anion transporter (OAT) family.

Organic anion transporters play important roles in the elimination of a variety of endogenous substances, xenobiotics and their metabolites from the body. During the last decade, molecular cloning has identified several families of multispecific organic anion transporters mediating the renal and hepatic elimination of organic anions and, most recently, the OAT (organic anion transporter) family, the founding member of which (OAT1) is the basolateral p-aminohippurate (PAH) transporter in the renal proximal tubule. So far, four isoforms have been identified. OATs are membrane proteins with 12 putative membrane-spanning domains and function as sodium-independent exchangers or facilitators. OATs show weak structural similarity to organic cation transporters (OCTs) and OCTN/carnitine transporters. OATs are multispecific organic anion transporters, the substrates of which include both endogenous (e.g. cyclic nucleotides, prostaglandins, urate, dicarboxylates) and exogenous anions (various anionic drugs and environmental substances). All members of the OAT family are expressed in the kidney, while some are also expressed in the liver, brain and placenta. The OAT family represents the renal secretory pathway for organic anions and is also involved in the distribution of organic anions in the body.

An evolutionarily conserved positively charged amino acid in the putative membrane-spanning domain of the foamy virus envelope protein controls fusion activity.

Foamy viruses (FVs) are highly fusogenic, and their replication induces massive syncytium formation in infected cell cultures which is believed to be mediated by expression of the envelope (Env) protein. The FV Env is essential for virus particle egress. The unusually long putative membrane-spanning domain (MSD) of the transmembrane subunit carries dispersed charged amino acids and has an important function for particle envelopment. To better understand the capsid-envelope interaction and Env-mediated cell fusion, we generated a variety of FV MSD mutations. C-terminal deletions revealed the cytoplasmic domain to be dispensable but the full-length MSD to be required for fusogenic activity. The N-terminal 15 amino acids of the MSD were found to be sufficient for membrane anchorage and promotion of FV particle release. Expression of wild-type Env protein rarely induced syncytia due to intracellular retention. Coexpression with FV Gag-Pol resulted in particle export and a dramatic increase in fusion activity. A nonconservative mutation of K(959) in the middle of the putative MSD resulted in increased fusogenic activity of Env in the absence of Gag-Pol due to enhanced cell surface expression as well as structural changes in the mutant proteins. Coexpression with Gag-Pol resulted in a further increase in the fusion activity of mutant FV Env proteins. Our results suggest that an interaction between the viral capsid and Env is required for FV-induced giant-cell formation and that the positive charge in the MSD is an important determinant controlling intracellular transport and fusogenic activity of the FV Env protein.

Molecular Cloning and Characterization of Multispecific Organic Anion Transporter 4 Expressed in the Placenta

A cDNA encoding a novel multispecific organic anion transporter, OAT4, was isolated from a human kidney cDNA library. The OAT4 cDNA consisted of 2210 base pairs that encoded a 550-amino acid residue protein with 12 putative membrane-spanning domains. The amino acid sequence of OAT4 showed 38 to 44% identity to those of other members of the OAT family. Northern blot analysis revealed that OAT4 mRNA is abundantly expressed in the placenta as well as in the kidney. When expressed in Xenopus oocytes, OAT4 mediated the high affinity transport of estrone sulfate (K(m) = 1.01 microM) and dehydroepiandrosterone sulfate (K(m) = 0.63 microM) in a sodium-independent manner. OAT4 also mediated the transport of ochratoxin A. OAT4-mediated transport of estrone sulfate was inhibited by several sulfate conjugates, such as p-nitrophenyl sulfate, alpha-naphthyl sulfate, beta-estradiol sulfate, and 4-methylumbelliferyl sulfate. By contrast, glucuronide conjugates showed little or no inhibitory effect on the OAT4-mediated transport of estrone sulfate. OAT4 interacted with chemically heterogeneous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, sulfobromophthalein, penicillin G, and bile salts, whereas tetraethylammonium, an organic cation, did not. OAT4 is the first member of the multispecific organic anion transporter family, which is expressed abundantly in the placenta. OAT4 might be responsible for the elimination and detoxification of harmful anionic substances from the fetus.

Cloning and Functional Identification of a Neuronal Glutamine Transporter

Glutamine is the preferred precursor for the neurotransmitter pool of glutamate, the major excitatory transmitter in the mammalian central nervous system. We have isolated a complementary DNA clone (designated GlnT) encoding a plasma membrane glutamine transporter from glutamatergic neurons in culture, and its properties have been examined using the T7 vaccinia system in fibroblasts. When GlnT is transfected into CV-1 cells, L-glutamine is the preferred substrate. Transport is Na(+)-dependent and inhibited by alpha-methylaminoisobutyric acid, a specific inhibitor of neutral amino acid transport system A. Kinetic analysis of glutamine uptake by GlnT is saturable, with a Michaelis constant (K(m)) of 489 +/- 88 microM at pH 7.4. Glutamine uptake mediated by GlnT is pH-sensitive with a 5-fold greater efficiency of uptake at pH 8.2 than at pH 6.6. Only the maximal velocity of transport increases without a significant change in K(m). The distribution of GlnT mRNA and protein in the central nervous system is widespread and is expressed on neurons that use glutamate as their neurotransmitter. In cultured cerebellar granule cells, GlnT is expressed only on neurons and is absent from astrocytes. GlnT expression increases concomitantly with the morphologic and functional differentiation of these cells in vitro, consistent with its role of supplying glutamatergic neurons with their neurotransmitter precursor. GlnT is the first member of the system A family of neutral amino acid transporters with 11 putative membrane-spanning domains and is a potential target to modulate presynaptic glutamatergic function.

Localization of Somatostatin Receptors at the Light and Electron Microscopial Level by Using Antibodies Raised Against Fusion Proteins

Somatostatin mediates its multiple biological effects via specific plasma membrane receptors belonging to the family of G-protein coupled receptors with seven putative membrane-spanning domains. Five somatostatin receptor subtypes (sst 1-sst 5) have been cloned in human, mouse, and rat. We have raised specific antibodies against the five human somatostatin receptors by using the fusion protein technique. DNA sequences encoding C-terminal parts of the somatostatin receptors were inserted into a pGEX-2T plasmid vector. E. coli bacteria were transformed with the recombinant plasmid and fusion proteins were expressed and purified using the glutathione S-transferase Gene Fusion System. The fusion proteins were emulsified with Freund's complete adjuvant and polyclonal antibodies were raised in rabbits. The antisera were tested for specificity in Western blot analysis of membrane preparations from cell lines expressing the receptors and in membrane preparations of brain tissues. The receptors were visualized at the light microscopical level in paraformaldehyde fixed tissue sections by use of biotin labelled secondary antibodies as well as by amplification with biotinylated tyramide. The final step in the immunohistochemical visualization of the receptors was done by both peroxidase labelled streptavidin/biotin and different fluorophores. At the electron microscopical level, some of the receptors could be visualized in tissues fixed with a combination of paraformaldehyde and low concentrations of glutaraldehyde. In the hamster brain, sst 2 receptors labelling was observed in both neuronal processes and perikarya. The staining was present in neo-, and allocortical areas of the forebrain, the hypothalamus, brain stem, and spinal cord. In the rat and human, sst 1 receptor was shown to be an auto receptor on somatostatinergic neurons located in the hypothalamus. In the retina both sst 1 and sst 2 receptors were present. sstl receptors were confined to amacrine cells, few ganglionic cells, and Müller cell-end feet. sst 2 receptors were more widespread than the sst 1 receptors. sst 2-immunoreactivity was present in dopaminergic amacrine cells, the Müller cell-end feet, and in the inner segments of the cone photoreceptors. Thus, the availability of subtype specific antibodies against the five somatostatin receptors makes it possible to identify the receptors involved in the multiple somatostatinergic system in the body.

Hexose transporters of tomato: molecular cloning, expression analysis and functional characterization.

A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H+/hexose symporter. The Km of the symporter for glucose is 45 microM.