Multiple Transmembrane Segments Research Articles

Oligomerization of the γ-Aminobutyric Acid Transporter-1 Is Driven by an Interplay of Polar and Hydrophobic Interactions in Transmembrane Helix II

The available evidence indicates that members of the neurotransmitter:sodium symporter family form constitutive oligomers. Their second transmembrane helix (TM2) contains a leucine heptad repeat proposed to be involved in oligomerization. In artificial transmembrane segments, interhelical interactions are stabilized by polar residues. We searched for these hydrogen bond donors in TM2 by mutating the five polar residues in TM2 of the gamma-aminobutyric acid transporter-1 (GAT1). We tested the ability of the resulting mutants to oligomerize by fluorescence microscopy, Foerster resonance energy transfer, and beta-lactamase fragment complementation. Of all generated mutants, only Y86A- (but not Y86F-), E101A-, E101Q-, and E101D-GAT1 were judged by these criteria to be deficient in oligomerization and were retained intracellularly. The observations are consistent with a model where the leucine heptad repeat in TM2 drives a homophilic association that is stabilized by Tyr(86) and Glu(101); Tyr(86) participates in hydrophobic stacking. Glu(101) is in the a-position of the leucine heptad repeat (where positions 1-7 are denoted a-g, and each leucine is in the central d-position). Thus, Glu(101) is in the position predicted for the hydrogen bond donor (i.e. sandwiched between Leu(97) and Leu(104), which are one helical turn above and below Glu(101)). These key residues, namely Tyr(86) and Glu(101), are conserved in related transporters from archaeae to humans; they are therefore likely to support oligomeric assembly in transporter orthologs and possibly other proteins with multiple transmembrane segments.

Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters.

Capsular polysaccharide (CPS) is a major virulence factor in Streptococcus pneumoniae. CPS gene clusters of S. pneumoniae types 4, 6B, 8, and 18C were sequenced and compared with those of CPS types 1, 2, 14, 19F, 19A, 23F, and 33F. All have the same four genes at the 5' end, encoding proteins thought to be involved in regulation and export. Sequences of these genes can be divided into two classes, and evidence of recombination between them was observed. Next is the gene encoding the transferase for the first step in the synthesis of CPS. The predicted amino acid sequences of these first sugar transferases have multiple transmembrane segments, a feature lacking in other transferases. Sugar pathway genes are located at the 3' end of the gene cluster. Comparison of the four dTDP-L-rhamnose pathway genes (rml genes) of CPS types 1, 2, 6B, 18C, 19F, 19A, and 23F shows that they have the same gene order and are highly conserved. There is a gradient in the nature of the variation of rml genes, the average pairwise difference for those close to the central region being higher than that for those close to the end of the gene cluster and, again, recombination sites can be observed in these genes. This is similar to the situation we observed for rml genes of O-antigen gene clusters of Salmonella enterica. Our data indicate that the conserved first four genes at the 5' ends and the relatively conserved rml genes at the 3' ends of the CPS gene clusters were sites for recombination events involved in forming new forms of CPS. We have also identified wzx and wzy genes for all sequenced CPS gene clusters by use of motifs.

Interaction of the α subunit of Na,K-ATPase with cofilin

The α1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the α subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed that the segment of cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na,K-ATPase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive 86Rb+ uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo.

Interaction of the alpha subunit of Na,K-ATPase with cofilin.

The alpha1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the alpha subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed that the segment of cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na,K-ATPase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive (86)Rb(+) uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo.

Identification of Residues within the Drug-binding Domain of the Human Multidrug Resistance P-glycoprotein by Cysteine-scanning Mutagenesis and Reaction with Dibromobimane

P-glycoprotein (P-gp) can transport a wide variety of cytotoxic compounds that have diverse structures. Therefore, the drug-binding domain of the human multidrug resistance P-gp likely consists of residues from multiple transmembrane (TM) segments. In this study, we completed cysteine-scanning mutagenesis of all the predicted TM segments of P-gp (TMs 1-5 and 7-10) and tested for inhibition by a thiol-reactive substrate (dibromobimane) to identify residues within the drug-binding domain. The activities of 189 mutants were analyzed. Verapamil-stimulated ATPase activities of seven mutants (Y118C and V125C (TM2), S222C (TM4), I306C (TM5), S766C (TM9), and I868C and G872C (TM10)) were inhibited by more than 50% by dibromobimane. The activities of mutants S222C (TM4), I306C (TM5), I868C (TM10), and G872C (TM10), but not that of mutants Y118C (TM2), V125C (TM2), and S776C (TM9), were protected from inhibition by dibromobimane by pretreatment with verapamil, vinblastine, or colchicine. These results and those from previous studies (Loo, T. W. and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948; Loo, T. W. and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388-35392) indicate that the drug-binding domain of P-gp consists of residues in TMs 4, 5, 6, 10, 11, and 12.

Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes

Here, we present a comprehensive analysis of solute transport systems encoded within the completely sequenced genomes of 18 prokaryotic organisms. These organisms include four Gram-positive bacteria, seven Gram-negative bacteria, two spirochetes, one cyanobacterium and four archaea. Membrane proteins are analyzed in terms of putative membrane topology, and the recognized transport systems are classified into 76 families, including four families of channel proteins, four families of primary carriers, 54 families of secondary carriers, six families of group translocators, and eight unclassified families. These families are analyzed in terms of the paralogous and orthologous relationships of their protein members, the substrate specificities of their constituent transporters and their distributions in each of the 18 organisms studied. The families vary from large superfamilies with hundreds of represented members, to small families with only one or a few members. The mode of transport generally correlates with the primary mechanism of energy generation, and the numbers of secondary transporters relative to primary transporters are roughly proportional to the total numbers of primary H+ and Na+ pumps in the cell. The phosphotransferase system is less prevalent in the analyzed bacteria than previously thought (only six of 14 bacteria transport sugars via this system) and is completely lacking in archaea and eukaryotes. Escherichia coli is shown to be exceptionally broad in its transport capabilities and therefore, at a membrane transport level, does not appear representative of the bacteria thus far sequenced. Archaea and spirochetes exhibit fewer proteins with multiple transmembrane segments and fewer net transporters than most bacteria. These results provide insight into the relevance of transport to the overall physiology of prokaryotes.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels.

Isolation of MYADM, a novel hematopoietic-associated marker gene expressed in multipotent progenitor cells and up-regulated during myeloid differentiation

A large number of hematopoietic cytokines and their receptors as well as transcription factors have been shown to be involved in maturation of blood cells. However, many of the genes important for the differentiation of multipotent stem cells to specific cellular lineages are still unknown. To identify novel genes involved in lineage selection of myeloid cells, we have applied differential display analysis during commitment toward granulocytes and macrophages of an IL-3-dependent multipotent progenitor cell line, FDCP-mix. One regulated cDNA represented a novel gene with restricted expression pattern within the hematopoietic system and was strongly up-regulated when FDCP-mix cells differentiated in GM-CSF, G-CSF, and M-CSF. The expression appears to be differentiation stage-specific in myeloid cells and is absent in B and T lymphocytes. Thus we found expression in normal mouse bone marrow enriched for stem cells and multipotent progenitors (c-kit+Sca-1+Lin- cells). When these cells were induced to differentiate toward myeloid cells, MYADM was up-regulated. In contrast, during conditions known to favor the development of B cell progenitors, the gene was down-regulated. The gene, termed MYADM for myeloid-associated differentiation marker gene, shows 100% identity to expressed sequence tags from early mouse embryonic development as well as from the mouse lung and from activated mouse macrophages. The predicted 32-kDa MYADM protein contains multiple hydrophobic putative transmembrane segments and has several potential consensus sites for phosphorylation. In view of its expression pattern, MYADM could serve as a new marker gene for hematopoietic differentiation. Although the function is unknown, antisense oligonucleotides were able to inhibit colony formation of c-kit+ Lin- bone marrow cells, suggesting an important role for MYADM in myeloid differentiation.

Transmembrane biogenesis of Kv1.3.

Using a combination of protease protection, glycosylation, and carbonate extraction assays, we have characterized the topogenic determinants encoded by Kv1.3 segments that mediate translocation events during endoplasmic reticulum (ER) biogenesis. Transmembrane segments S1, S2, S3, S5, and S6 initiate translocation, only S1 and S2 strongly (>60%) anchor themselves in the membrane, S5 exhibits signal anchor activity and contains a cryptic cleavage site, and S3 and S6 fail to integrate into the membrane. Elongation of each single-transmembrane construct to include multiple transmembrane segments alters integration and translocation efficiencies, indicating that multiple topogenic determinants cooperate during Kv1. 3 topogenesis and assembly. Several surprising findings emerged from these studies. First, in the presence of T1, the N-terminal recognition domain, S1 was unable to initiate either translocation or membrane integration. As a result, S2 likely functions as the initial signal sequence to establish Kv1.3 N-terminus topology. Second, S4 independently integrates into the membrane. Third, S6 plus the C-terminus of Kv1.3 is a secretory protein but can be converted to a membrane-integrated protein with a correctly oriented, cytosolic C-terminus by linking S6 to S5 and the pore loop. These results have implications for the role of the N-terminus in Kv biogenesis and on the mechanisms of dominant negative suppression of Kv1.3 by truncated Kv1.3 fragments [Tu et al. (1996) J. Biol. Chem. 271, 18904-18911].

Sequence Analysis of Less Hydrophobic Transmembrane Segments in Polytopic Membrane Proteins

To understand the function of membrane proteins, the knowledge of their structure, especially the knowledge of the position of transmembrane segments (TMSs) and of their membrane topology (i.e., orientation of TMSs across the membrane), is crucial. Since most TMSs of α-type polytopic (i.e., having multiple TMSs) membrane proteins are composed of hydrophobic amino acids, they are usually detected as peaks in the hydropathy plot [1]. However, there are a certain amount of TMSs that are relatively hydrophobic and so it is hard to distinguish them with relatively hydrophobic loop segments. Although how such less hydrophobic segments span the membrane has been a mystery, Ota et al. found an important clue on its mechanism [4]; according their result, each TMS has its own topogenic tendency like membrane proteins with a single TMS. Their topogenic tendency is determined by the distribution of neighboring positively-charged residues like the positive-inside rule [3]. Namely, if a TMS has more positive charges on its nearby N-terminal side, it tends to have the Nin/Cout orientation; otherwise, a TMS tends to be in the Nout/Cin orientation. Thus, if two neighboring TMSs have the same topogenic tendency, a loop segment between them may become a TMS to resolve this contradiction. To confirm this idea, we systematically analyzed the sequence features of both less-hydrophobic TMSs and relatively-hydrophobic loops. Our result not only supports Ota et al.’s conclusion but also implies a more general model of membrane topogenesis.

Identification of Residues in the Drug-binding Domain of Human P-glycoprotein: ANALYSIS OF TRANSMEMBRANE SEGMENT 11 BY CYSTEINE-SCANNING MUTAGENESIS AND INHIBITION BY DIBROMOBIMANE

The drug-binding domain of the human multidrug resistance P-glycoprotein (P-gp) probably consists of residues from multiple transmembrane (TM) segments. In this study, we tested whether the amino acids in TM11 participate in binding drug substrates. Each residue in TM11 was initially altered by site-directed mutagenesis and assayed for drug-stimulated ATPase activity in the presence of verapamil, vinblastine, or colchicine. Mutants G939V, F942A, T945A, Q946A, A947L, Y953A, A954L, and G955V had altered drug-stimulated ATPase activities. Direct evidence for binding of drug substrate was then determined by cysteine-scanning mutagenesis of the residues in TM11 and inhibition of drug-stimulated ATPase activity by dibromobimane, a thiol-reactive substrate. Dibromobimane inhibited the drug-stimulated ATPase activities of two mutants, F942C and T945C, by more than 75%. These results suggest that residues Phe(942) and Thr(945) in TM11, together with residues previously identified in TM6 (Leu(339) and Ala(342)) and TM12 (Leu(975), Val(982), and Ala(985)) (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 31945-31948) form part of the drug-binding domain of P-gp.

Human Acyl-CoA:Cholesterol Acyltransferase-1 in the Endoplasmic Reticulum Contains Seven Transmembrane Domains

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and is involved in atherosclerosis. ACAT-1 protein is located mainly in the ER. The hydropathy plot suggests that ACAT-1 protein contains multiple transmembrane segments. We inserted either the hemagglutinin tag or the HisT7 tag at various hydrophilic regions within the human ACAT-1 protein and used immunofluorescence microscopy to determine the topography of the tagged proteins expressed in mutant Chinese hamster ovary cells lacking endogenous ACAT. All of the tagged proteins are located mainly in the ER and retain full or partial enzyme activities. None of the tagged proteins produces detectable intracellular degradation intermediates. Treating cells with digitonin at 5 micrograms/ml permeabilizes the plasma membranes while leaving the ER membranes sealed; in contrast, treating cells with 0.25% Triton X-100 or with cold methanol permeabilizes both the plasma membranes and the ER membranes. After appropriate permeabilization, double immunostaining using antibodies against the N-terminal region and against the inserted tag were used to visualize various regions of the tagged protein. The results show that human ACAT-1 in the ER contains seven transmembrane domains.

Divergent evolution of membrane protein topology: the Escherichia coli RnfA and RnfE homologues.

Although the molecular evolution of protein tertiary structure and enzymatic activity has been studied for decades, little attention has been paid to the evolution of membrane protein topology. Here, we show that two closely related polytopic inner membrane proteins from Escherichia coli have evolved opposite orientations in the membrane, which apparently has been achieved by the selective redistribution of positively charged amino acids between the polar segments flanking the transmembrane stretches. This example of divergent evolution of membrane protein topology suggests that a complete inversion of membrane topology is possible with relatively few mutational changes even for proteins with multiple transmembrane segments.

Transporters for cationic amino acids in animal cells: discovery, structure, and function.

The structure and function of the four cationic amino acid transporters identified in animal cells are discussed. The systems differ in specificity, cation dependence, and physiological role. One of them, system y+, is selective for cationic amino acids, whereas the others (B[0,+], b[0,+], and y+ L) also accept neutral amino acids. In recent years, cDNA clones related to these activities have been isolated. Thus two families of proteins have been identified: 1) CAT or cationic amino acid transporters and 2) BAT or broad-scope transport proteins. In the CAT family, three genes encode for four different isoforms [CAT-1, CAT-2A, CAT-2(B) and CAT-3]; these are approximately 70-kDa proteins with multiple transmembrane segments (12-14), and despite their structural similarity, they differ in tissue distribution, kinetics, and regulatory properties. System y+ is the expression of the activity of CAT transporters. The BAT family includes two isoforms (rBAT and 4F2hc); these are 59- to 78-kDa proteins with one to four membrane-spanning segments, and it has been proposed that these proteins act as transport regulators. The expression of rBAT and 4F2hc induces system b[0,+] and system y+ L activity in Xenopus laevis oocytes, respectively. The roles of these transporters in nutrition, endocrinology, nitric oxide biology, and immunology, as well as in the genetic diseases cystinuria and lysinuric protein intolerance, are reviewed. Experimental strategies, which can be used in the kinetic characterization of coexpressed transporters, are also discussed.

Role of ribosomes in reinitiation of membrane insertion of internal transmembrane segments in a polytopic membrane protein.

The topogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the topogenesis and membrane assembly of membrane proteins with multiple TM segments (polytopic) is still incomplete. Recently, several studies on P-glycoprotein (Pgp) suggested that the topogenesis of polytopic membrane proteins is likely more complicated than anticipated. While studying the mechanism by which Pgp topogenesis is determined, we unexpectedly found that ribosomes or proteins associated with ribosomes are involved in regulating the membrane insertion and folding of Pgp during its translation. We discovered that when Pgp was translated by wheat germ ribosomes in vitro, TM3 could not reinitiate the insertion of the protein into microsomal membranes following the membrane insertion of TM1 and TM2. In contrast, TM3 could reinitiate membrane insertion when the protein was translated by rabbit reticulocyte ribosomes. These findings suggest that ribosomes or proteins associated with ribosomes play an important role in membrane insertion and folding of TM segments of Pgp and that rabbit reticulocyte and wheat germ ribosomes may use different mechanisms to control the membrane insertion of the same nascent peptide. We propose that ribosomes or proteins associated with ribosomes help reinitiate insertion of internal TM segments into the membrane by dissociation and reassociation with the protein-conducting channel in ER membranes.

Sequence requirements for membrane assembly of polytopic membrane proteins: molecular dissection of the membrane insertion process and topogenesis of the human MDR3 P-glycoprotein.

The biogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the biogenesis and membrane assembly of membrane proteins with multiple TM segments is still incomplete because of the complexity and diversity of polytopic membrane proteins. In an attempt to investigate further the biogenesis of polytopic membrane proteins, I used the human MDR3 P-glycoprotein (Pgp) as a model polytopic membrane protein and expressed it in a coupled cell-free translation/translocation system. I showed that the topogenesis of the C-terminal half MDR3 Pgp molecule is different from that of the N-terminal half. This observation is similar to that of the human MDR1 Pgp. The membrane insertion properties of the TM1 and TM2 in the N-terminal half molecule are different. The proper membrane anchorage of both TM1 and TM2 of the MDR3 Pgp is affected by their C-terminal amino acid sequences, whereas only the membrane insertion of the TM1 is dependent on the N-terminal amino acid sequences. The efficient membrane insertion of TM3 and TM5 of MDR3 Pgp, on the other hand, requires the presence of the putative TM4 and TM6, respectively. The TM8 in the C-terminal half does not contain an efficient stop-transfer activity. These observations suggest that the membrane insertion of putative TM segments in the human MDR3 Pgp does not simply follow the prevailing sequential event of the membrane insertion by signal-anchor and stop-transfer sequences. These results, together with my previous findings, suggest that different isoforms of Pgp can be used in comparison as a model system to understand the molecular mechanism of topogenesis of polytopic membrane proteins.

Hamster UDP-N-Acetylglucosamine:Dolichol-P N-Acetylglucosamine-1-P Transferase Has Multiple Transmembrane Spans and a Critical Cytosolic Loop

UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT) is an endoplasmic reticulum (ER) enzyme responsible for synthesis of GlcNAc-P-P-dolichol, the committed step of dolichol-P-P-oligosaccharide synthesis. The sequence of hamster GPT predicted multiple transmembrane segments (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250-14255). GPT has also been predicted to act on the cytosolic face of the ER membrane, based on topological studies of its substrates and products. In this report we test these predictions by: (i) immunofluorescence microscopy with antibodies specific for native GPT sequences or epitope tags inserted into GPT, after selective permeabilization of the plasma membrane with digitonin; (ii) insertion of Factor Xa cleavage sites; (iii) in vitro translation of GPT; and (iv) site-directed mutagenesis. The loops between the 1st and 2nd and between the 9th and 10th predicted transmembrane spans of GPT were found to be cytosolic. In contrast, the loop between the 6th and 7th transmembrane spans, as well as the carboxyl terminus, were lumenal. Thus, hamster GPT must cross the ER membrane at least three times, consistent with previous computer-assisted predictions. There was no apparent N-glycosylation or signal sequence cleavage detected by in vitro translation. The cytosolic loop between the 9th and 10th transmembrane spans is the largest hydrophilic segment in GPT and, as judged by site-directed mutagenesis, has a number of conserved residues essential for activity. Hence, these results directly support the hypothesis that dolichol-P-P-oligosaccharide assembly is initiated in the cytosol and that a downstream intermediate must translocate to the lumenal face of the ER membrane.

Biogenesis of polytopic membrane proteins: membrane segments of P-glycoprotein sequentially translocate to span the ER membrane.

The initial steps in the biogenesis of membrane proteins parallel those of secretory proteins. However, membrane proteins contain a signal to stop translocation across the membrane. For polytopic membrane proteins, those with multiple transmembrane segments, little is known of the temporal sequence or relationship between synthesis of the nascent proteins, translocation, folding, and integration of the membrane segments into the bilayer. Here we demonstrate that latent membrane segments translocate sequentially as they emerge from the ribosome and do not accumulate on the cytosolic side to form loops, or larger structures, prior to translocation across the membrane.

Molecular imaging of Na +,K +-ATPase in purified kidney membranes

Ion channels and pumps in cell membranes consist of multiple transmembrane segments that are thought to be critical for transport of ions. Channel structures constituted by these transmembrane segments are characteristic of ion channels, whereas such structures have not been identified in ion pumps until now. By applying atomic force microscopy on Na +,K +-ATPase molecules in canine kidney membranes under tapping mode, we identified a hollow in the protein with a characteristic internal diameter of 6–20A˚and an external diameter of 20–55A˚depending upon treatment conditions. This hollow may be interpreted as a channel-like conformation of Na +,K +-ATPase. In the regions where the proteins were absent, lipid head structures with 2A˚width and 6A˚length were imaged in an orthorhombic lattice.

Transmembrane organization of the Na,K-ATPase determined by epitope addition.

The Na,K-ATPase is a membrane-associated enzyme that establishes the internal Na+/K+ environment of most animal cells. The catalytic (alpha) subunit of the Na,K-ATPase contains multiple transmembrane segments, but the number and location of these domains has not been clearly established. We have used epitope addition to determine the transmembrane topology of the alpha subunit. An immunoreactive peptide was inserted into various regions of the cDNA encoding the rat alpha 1 subunit, and the constructs were expressed in transfected mammalian cells. The intra- or extracellular location of the epitope tags was determined by immunofluorescence analysis. Our results indicate that the amino and carboxyl termini of the alpha subunit are situated intracellularly, and the polypeptide is likely to possess eight membrane-spanning segments. The systematic application of epitope tagging may be useful for analyzing the topology of membrane proteins of unknown structure.