The cellobiose permease of Escherichia coli consists of three proteins and is homologous to the lactose permease of Staphylococcus aureus

The cellobiose (cel) operon of Escherichia coli was recently sequenced and shown to consist of five genes, celABCDF (Parker and Hall, 1990). We have shown that the CelA, CelB and CelC proteins possess amino acid sequences which are homologous to different domains of the lactose permease of Staphylococcus aureus. CelB corresponds to the integral membrane portion of the permease (IIcel) while CelC (IIIcel) and CelA (IVcel) correspond to the two cytoplasmic domains which appear to comprise the first and second phosphorylation sites in the permease, respectively. The cellobiose permease is the only one of several homologous sequenced permeases of the phosphoenolpyruvate:sugar phosphotransferase system which has its three known functional domains residing on distinct polypeptide chains.     

Molecular and functional characterization of the first nucleobase transporter gene from African trypanosomes

African trypanosomes are unable to synthesize purines and depend upon purine nucleoside and nucleobase transporters to salvage these compounds from their hosts. To understand the crucial role of purine salvage in the survival of these parasites, a central objective is to identify and characterize all of the purine permeases that mediate uptake of these essential nutrients. We have cloned and functionally expressed in a purine nucleobase transport deficient strain of Saccharomyces cerevisiae a novel nucleobase transporter gene, TbNT8.1, from Trypanosoma brucei. The permease encoded by this gene mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with Kms in the low micromolar range. The TbNT8.1 protein is a member of the equilibrative nucleoside transporter (ENT) family of permeases that occur in organisms as diverse as protozoa and mammals. TbNT8.1 is distinct from other ENT permeases that have been identified in trypanosomes in utilizing multiple purine nucleobases, rather than purine nucleosides, as substrates and is hence the first bona fide nucleobase permease identified in these parasites. Furthermore, unlike the mRNAs for other purine transporters, TbNT8.1 mRNA is significantly more abundant in insect stage procyclic forms than in mammalian stage bloodstream forms, and the TbNT8.1 permease thus may represent a major route for purine nucleobase uptake in procyclic trypanosomes.     

Functional analysis of <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> carboxylic acids permeases: heterologous expression of <Emphasis Type="Italic">KlJEN1</Emphasis> and <Emphasis Type="Italic">KlJEN2</Emphasis> genes

The present work describes a detailed physiological and molecular characterization of the mechanisms of transport of carboxylic acids in Kluyveromyces lactis. This yeast species presents two homologue genes to JEN1 of Saccharomyces cerevisiae: KlJEN1 encodes a monocarboxylate permease and KlJEN2 encodes a dicarboxylic acid permease. In the strain K. lactis GG1888, expression of these genes does not require an inducer and activity for both transport systems was observed in glucose-grown cells. To confirm their key role for carboxylic acids transport in K. lactis, null mutants were analyzed. Heterologous expression in S. cerevisiae has been performed and chimeric fusions with GFP showed their proper localization in the plasma membrane. S. cerevisiae jen1Δ cells transformed with KlJEN1 recovered the capacity to use lactic acid, as well as to transport labeled lactic acid by a mediated mechanism. When KlJEN2 was heterologously expressed, S. cerevisiae transformants gained the ability to transport labeled succinic and malic acids by a mediated mechanism, exhibiting, however, a poor growth in malic acid containing media. The results confirmed the role of KlJen1p and KlJen2p as mono and dicarboxylic acids permeases, respectively, not subjected to glucose repression, being fully functional in S. cerevisiae. O. Queirós and L. Pereira contributed equally to this work.     

Structure of the Mycobacterium tuberculosis antigen 88, a protein related to the Escherichia coli PstA periplasmic phosphate permease subunit.

We report the cloning and sequencing of the gene coding for antigen 88 from Mycobacterium tuberculosis by using monoclonal antibodies to screen an expression library in lambda gt11. The gene encodes a 403-amino-acid-residue protein with a calculated molecular mass of 43,790 Da which contains seven putative transmembrane alpha-helical domains and presents a significant homology to the PstA protein of Escherichia coli. In its N-terminal region, it contains a 61-amino-acid region highly homologous to the fifth transmembrane helix of E. coli PstC. PstA and PstC are the two hydrophobic subunits of an E. coli periplasmic phosphate permease. Since the phosphate-binding subunit of this putative permease in M. tuberculosis has previously been characterized, i.e., the 38-kDa mycobacterial protein (also called protein antigen b, Ag 5, and Ag 78) homologous to PstS of E. coli, it seems likely that functional permeases analogous to the periplasmic permeases of gram-negative bacteria also exist in mycobacteria.     

GNP1, the high-affinity glutamine permease of S. cerevisiae

  Glutamine uptake in S. cerevisiae is mediated by at least three transporters: high- and low-affinity glutamine permeases and the general amino-acid permease. We have isolated the gene encoding the high-affinity glutamine permease and named it GNP1. The amino-acid sequence of GNP1, and its hydropathy profile of 12 transmembrane domains, closely resemble those of known amino-acid permeases. The Km of GNP1 for glutamine uptake was determined to be 0.59 mM. Cells lacking GNP1 exhibit reduced levels of glutamine transport, and are resistant to a toxic analog of glutamine, L-glutamic acid γ-monohydroxamate. Unlike other amino-acid permeases, whose expression is nitrogen-source limited, GNP1 is expressed on both rich and poor nitrogen sources. Received: 27 February / 30 March 1996     

The lac permease of Escherichia coli: site-directed mutagenesis studies on the mechanism of beta-galactoside/H+ symport

In this communication, we summarize site-directed mutagenesis studies of the lac permease from Escherichia coli, a prototypic H(+)-coupled active transport protein. We classify mutant permeases by phenotype, and suggest possible roles for some individual residues in the mechanism of H+/lactose symport. Although high-resolution structural information is not presently available, kinetic analysis of the partial reactions catalysed by the mutant permeases, as well as biophysical studies, suggest an evolving model for the mechanism of H+/lactose symport.     

Structural identification of brush border membrane transport systems — towards an understanding of regulatory mechanisms

Summary Until recently proximal tubular membrane transport systems were defined only on the basis of their function. However, in the past 4–5 years the use of molecular biology techniques has permitted structural identification of several proximal tubular transport systems. For example, sodium/D-glucose, sodium/phosphate, and sodium/sulfate cotransport systems have been identified. Also (a component of) an amino acid transport system accepting cystine and dibasic amino acids has been identified. These transport pathways may be targets for genetic alterations in proximal tubular solute reabsorption and/or be under physiological control.     

A new family of high-affinity ABC manganese and zinc permeases.

Phylogenetic analysis of 47 extracellular putative metal binding receptors (MBRs) belonging to the newly defined cluster suggests the existence of two subclusters. The question of substrate specificity of the corresponding ATP binding cassette (ABC) permeases is discussed, based on data collected from 19 of them concerning their regulation, metal requirement of permease mutants, metal uptake and metal binding. The proposal that the two subclusters correspond to paralogous metal permeases dedicated primarily to manganese and to zinc transport is made. The question of a direct role of MBRs as adhesins of Gram-positive bacteria is then discussed and the importance of metal permeases for cellular processes and host-bacteria interactions is reviewed.     

Inhibition of specific amino acid uptake in Candida albicans by lysosomal extracts from rabbit alveolar macrophages.

Lysosomal-rich fractions, obtained from normal rabbit alveolar macrophages, were extracted and tested for their effects on Candida albicans. The uptake and incorporation of various compounds (amino acids, uridine, 2-deoxy-D-glucose, and Rb+) by C. albicans were measured in the presence and absence of extract. These studies demonstrated that the extract had a specific effect on the uptake of certain amino acids by C. albicans. Of the amino acids tested, the uptake of methionine valine, lysine, phenylalanine, and leucine was drastically reduced in the presence of extract, whereas proline and glutamic acid uptake was unaffected. Those amino acids whose uptake was inhibited have been shown to be transported in other yeasts by a general amino acid permease. The existence of a general amino acid permease in C. albicans is compatible with our data. Additionally, the extract had no effect on the uptake of uridine, 2-deoxy-D-glucose, and Rb+. Leakage of 86Rb by C. albicans was detected in the presence of the extract. Viability of Candida, as measured by colony-forming ability, decreased after a 16-h incubation of C. albicans with the extract.     

Activity of the yeast Tat2p tryptophan permease is sensitive to the anti-tumor agent 4-phenylbutyrate

4-Phenylbutyrate (PB) induces differentiation and is being intensively studied as a treatment for brain, prostate, breast, and hematopoietic cancer. While many different primary targets for PB have been proposed, the mechanism by which it causes cellular differentiation remains unknown. To identify the primary cellular target, we investigated its effects on Saccharomyces cerevisiae and showed that it inhibits tryptophan transport. We show here that PB and sorbic acid induce an ubiquitin-dependent turnover of the tryptophan permease Tat2p. However, the inhibition of transport is not a consequence of the loss of Tat2p, since it also occurs when turnover is prevented by deleting the Tat2p ubiquitination sites. When we tested the effects of PB and other growth inhibitory agents on the growth of amino acid auxotrophs, we found that several auxotrophs are hypersensitive to a number of chemically unrelated agents, including PB and some, but not all, weak acids; and this sensitivity is due to the inhibition of amino acid transport. For the inhibitory weak acids, inhibition is not confined to aromatic amino acid auxotrophs, nor is it a general weak acid stress response, since the degree of inhibition is independent of weak acid hydrophobicity and pK_a. Our results show that diverse agents affect the activity of the Tat2p permease rather than its stability and suggest the hypothesis that the anti-neoplastic action of PB is due to a decrease in the activity of surface receptors or other membrane proteins needed to maintain the transformed state.     

Interrelationships between protein phosphorylation and oligomerization in transport and chemotaxis via the Escherichia coli mannitol phosphotransferase system.

The membrane-bound enzymes II of the bacterial carbohydrate phosphotransferase system (PTS) are multifunctional: they are required for the transport, phosphorylation and chemotactic sensing of their substrates. An oligomer (minimally a dimer) of at least one of these PTS permeases, the Escherichia coli mannitol permease, appears to be necessary for this protein to optimally carry out these functions. Much indirect evidence is consistent with this hypothesis, and recent experiments show that transport and phosphorylation of, and chemotaxis to, mannitol in E. coli involves an intersubunit phosphotransfer reaction, which can only occur in a protein oligomer. Membrane topological studies of the mannitol permease also argue in favour of an oligomeric structure in the membrane which may be necessary to form the hydrophilic channel through which mannitol must traverse the phospholipid bilayer. The possibility that the oligomerization state of the mannitol permease is a target for regulation of its activity in vivo is proposed, but has not yet been explored experimentally.     

Activation of amino acid transport during steady-state growth of Escherichia coli

Accumulation of amino acids in exponentially increasing cultures of Escherichia coli was linear, supporting the interpretation that the biphasic response observed when cultures grew without these acids reflects a transient perturbation in accumulation. Rates of accumulation of glutamine, histidine and glycine were compared in steady-state and non-steady-state cultures. Their uptake rates were markedly enhanced in steady-state cultures at low exogenous concentrations, 10 microM or less. The results support the activation of amino acid transport systems by low concentrations of the particular amino acid present during growth. This activation was decreased when exogenous concentrations of the amino acid were markedly increased or when cells were washed free of the amino acid. Upon readdition of the amino acid after washing, recovery of enhanced transport required several generations, supporting a process of recovery other than enzymatic induction. The observation of amino acid enhancement of transport for eight other amino acids examined in steady-state culture suggests that this enhancement is a common process.     

Molecular cloning and characterization of a novel human gene homologous to the murine ecotropic retroviral receptor.

A novel human cDNA homologous to the murine ecotropic retroviral receptor was cloned from a cDNA library derived from a human T-cell line. The human cDNA is highly homologous to the murine counterpart (87.6% amino acid identity), and its sequence predicts a protein with 629 amino acids (approximately 68 kDa), which is 7 amino acids more than the murine counterpart (622 amino acids). The predicted protein is highly hydrophobic and contains 14 potential transmembrane-spanning domains. No other gene and protein with significant homology to the cloned human gene and the predicted protein were identified by a computer-based search of sequence data banks other than the murine T-cell early activation gene (52.5% amino acid identity) and the murine ecotropic retroviral receptor gene. The human gene is ubiquitously expressed in human tissues and conserved among mammalian species. The genomic gene was also isolated from a cosmid library derived from human lymphocytes, and its organization was elucidated. The gene mapped to human chromosome 13.     

Puromycin effect on amino acid transport: differential rates of carrier protein turnover.

In fetal rat calvaria, puromycin selectively inhibited the uptake of certain groups of amino acids. Puromycin treatment decreased the uptake of glycine, L-proline, and alpha-aminoisobutyric acid but was without effect on the active uptake of all other amino acids tested. In studies of alpha-aminoisobutyric acid uptake, puromycin decreased the maximal transport velocity by 70% but had no effect on the affinity of the transport system for the amino acid. With puromycin treatment, the fall-off in rates of alpha-aminoisobutyric acid uptake was first order with a half-life of 68 min. Insulin treatment increased this half-life to 118 min. These findings suggest that protein components of specific transport systems are degraded at varying rates after puromycin blockade of protein synthesis. Hormones that stimulate amino acid transport (e.g., insulin) may decrease the rate of degradation of these protein components.     

The SLC36 family: proton-coupled transporters for the absorption of selected amino acids from extracellular and intracellular proteolysis

Whilst Na(+) has replaced H(+) as a major transport driving force at the plasma membrane of animal cells, the evolutionarily older H(+)-driven systems persist on endomembranes and at the plasma membrane of specialized cells. The first member of the SLC36 family, present in both intracellular and plasma membranes, was identified independently as a lysosomal amino acid transporter (LYAAT1) responsible for the export of lysosomal proteolysis products into the cytosol and as a proton/amino acid transporter (PAT1) responsible for the absorption of amino acids in the gut. In addition to LYAAT1/PAT1, the family comprises another characterized member, PAT2, and two orphan transporters. Both PAT1 and PAT2 mediate 1:1 symport of protons and small neutral amino acids such as glycine, alanine, and proline. Their mRNAs are broadly and differentially expressed in mammalian tissues. The PAT1 protein localizes to lysosomes in brain neurons, but is also found in the apical membrane of intestinal epithelial cells with a role in the absorption of amino acids from luminal protein digestion. In both cases, protons supplied by the lysosomal H(+)-ATPase or by the acidic microclimate of the brush border membrane drive transport of the amino acids into the cytosol. The subcellular localization and physiological role of PAT2 have still to be determined. SLC36 transporters are related distantly to other proton-coupled amino acid transporters, such as the vesicular neurotransmitter transporter VIAAT/VGAT (SLC32) and system N transporters (SLC38 family).     

System L: heteromeric exchangers of large,neutral amino acids involved in directional transport

The plasma membrane transport system L is in many cells the only (efficient) pathway for the import of large branched and aromatic neutral amino acids. The corresponding transporters are hetero(di)mers composed of a catalytic subunit (LAT1 or LAT2=light chain=glycoprotein-associated amino acid transporter) associated covalently with the glycoprotein 4F2hc/CD98 (heavy chain). The tissue distribution of LAT1 suggests that it is involved mainly in transporting amino acids into growing cells and across some endothelial/epithelial secretory barriers, whereas the localization of LAT2 indicates that it is mainly involved in the basolateral efflux step of transepithelial (re)absorptive amino acid transport. However, system L transporters are obligatory amino acid exchangers with 1:1 stoichiometry, with similar (but not identical) intra- and extracellular substrate selectivities and with highly asymmetrical apparent affinities (low affinity inside). Therefore, net directional transport of large, neutral amino acids by system L depends on the parallel expression of a unidirectional transporter with overlapping selectivity (for instance systems A or N) that provides/recycles amino acids that drive system L exchange function. By mediating the regulated flux of these exchange substrates, unidirectional transporters control the activity of system L.     

Amino acid transport into cultured McCoy cells infected with Chlamydia trachomatis

Amino acid transport into McCoy cells infected with strains representative of the two major biovars of Chlamydia trachomatis has been studied to determine if uptake is increased during infection. Preliminary work suggested that the transport systems L, A/ASC (for neutral amino acid transport), N (for transport of Asn, Gln, and His) and y+ (for cationic amino acids) were present in McCoy cells. With lymphogranuloma venereum biovar strain 434, little difference in the influx of representative amino acids Trp, His, and Lys or the analogue 2-aminoisobutyric acid (AIB) was observed during infection. With trachoma biovar strain DK20, a small increase in the initial entry rate and equilibrium concentration of each amino acid was found. McCoy cells appear to have great capacity for concentrating amino acids, which might obviate the need for transport induction by chlamydiae under conditions favoring the growth of infectious organisms.