Transmembrane-spanning Segments Research Articles

Abstract 528: Characterization Of Sitosterolemia-causing Mutations In The Extracellular Domain Of Abcg8

Background: ABCG5 and ABCG8 form a heterodimer that promotes biliary secretion and opposes intestinal absorption of cholesterol and phytosterols. Whole-genome and exome sequencing has revealed over 2000 variants of ABCG5 and ABCG8, many of which are predicted to be pathogenic or likely pathogenic. Missense mutations in clinically confirmed cases of Sitosterolemia are of particular interest for their potential to reveal structure-function relationships in ABCG5 ABCG8 and other ABC transporter family members. Mutations in ABCG5 ABCG8 with clinically confirmed Sitosterolemia generally cluster within the ATP binding cassette and transmembrane spanning segments that form the substrate-binding domain. However, four disease-causing mutations occur in the short extracellular loop in ABCG8, suggesting that these mutations may reveal novel elements of the ABCG5 ABCG8 function. Methods: Mutants of ABCG8 were generated by site-directed mutagenesis. Plasmids encoding normal ABCG5 and normal or mutant ABCG8s were co-transfected into human Huh7 hepatocytes, lysates prepared, and analyzed by SDS-PAGE and immunoblot analysis. Maturation and trafficking of the ABCG5 ABCG8 heterodimer beyond the endoplasmic reticulum was assessed by the appearance of the higher molecular weight, mature form of each glycoprotein. Results: Mutants G574R, G575D, and L596R failed to support trafficking of ABCG5 ABCG8 and were designated as Class II mutants causative in Sitosterolemia. Only G574E supported maturation of the ABCG5 ABCG8 transporter in cultured human hepatocytes and remains unclassified. Conclusion: Formation and trafficking of the ABCG5 ABCG8 heterodimer is dependent on key residues within the third extracellular loop in ABCG8. Maturation of ABCG5 ABCG8 is tolerant of substitutions in glycine 574, including the addition of a larger and negatively charged residue. However, neither substitution forms a functional ABCG5 ABCG8 transporter.

Glutamate Transporter Homolog-based Model Predicts That Anion-π Interaction Is the Mechanism for the Voltage-dependent Response of Prestin

Prestin is the motor protein of cochlear outer hair cells. Its unique capability to perform direct, rapid, and reciprocal electromechanical conversion depends on membrane potential and interaction with intracellular anions. How prestin senses the voltage change and interacts with anions are still unknown. Our three-dimensional model of prestin using molecular dynamics simulations predicts that prestin contains eight transmembrane-spanning segments and two helical re-entry loops and that tyrosyl residues are the structural specialization of the molecule for the unique function of prestin. Using site-directed mutagenesis and electrophysiological techniques, we confirmed that residues Tyr(367), Tyr(486), Tyr(501), and Tyr(508) contribute to anion binding, interacting with intracellular anions through novel anion-π interactions. Such weak interactions, sensitive to voltage and mechanical stimulation, confer prestin with a unique capability to perform electromechanical and mechanoelectric conversions with exquisite sensitivity. This novel mechanism is completely different from all known mechanisms seen in ion channels, transporters, and motor proteins.

Role of a major facilitator superfamily transporter in adaptation capacity of Penicillium funiculosum under extreme acidic stress

Fungal species present in extreme low pH environments are expected to have adapted for tolerance to high H+ concentrations. However, their adaptability mechanism is unclear. In this study, we isolated an acid-tolerant strain of Penicillium funiculosum, which can grow actively at pH 1.0 and thrived in pH 0.6. A major facilitator superfamily transporter (PfMFS) was isolated from an acid-sensitive random insertional mutant (M4) of the fungus. It encodes a putative protein of 551 residues and contains 14 transmembrane-spanning segments. A targeted mutant (M7) carrying an inactivated copy of PfMFS showed an obvious reduction of growth compared with the wild type (WT) and complementation of M7 with PfMFS restored the wild-type level of growth at pH 1.0. Further data showed that the wild-type showed higher intracellular pH than M7 in response to pH 1. Subcellular localization showed that PfMFS was a cell membrane protein. Homology modeling showed structural similarity with an MFS transporter EmrD from Escherichiacoli. These results demonstrate that the PfMFS transporter is involved in the acid resistance and intracellular pH homeostasis of P. funiculosum.

Identification of life-stage and tissue-specific splice variants of an inward rectifying potassium (Kir) channel in the yellow fever mosquito Aedes aegypti

Inward-rectifier potassium (Kir) channels play key roles in nerve, muscle, and epithelial cells in mammals, but their physiological roles in insects remain to be determined. The yellow fever mosquito (Aedes aegypti) possesses five different genes encoding Kir channel subunits: Kir1, Kir2A, Kir2B, Kir2B′, and Kir3. We have recently cloned and characterized the Kir1, Kir2B, and Kir3 cDNAs in the renal (Malpighian) tubules of adult female Ae. aegypti. Here we characterize the expression of the Kir2A gene in Ae. aegypti, which was not abundantly expressed in Malpighian tubules. We find that the 1) Kir2A gene is expressed primarily in the midgut and hindgut of adult female mosquitoes, and 2) Kir2A mRNAs are alternatively spliced into three distinct variants (Kir2A-a, -b, and -c). The deduced Kir2A proteins from these splice forms share a completely conserved transmembrane domain (a pore-forming domain flanked by two transmembrane-spanning segments), but possess novel NH2-terminal and/or COOH-terminal domains. Semi-quantitative RT-PCR analyses indicate that the splice variants exhibit both developmental- and tissue-specific expression. Lastly, we provide insights into the conservation of alternative splicing among the Kir2A genes of dipterans, which may add molecular diversity that compensates for the relatively limited number of Kir channel genes in insects compared to mammals.

NMR structures of polytopic integral membrane proteins

Membrane proteins represent up to 30% of the proteins in all organisms, they are involved in many biological processes and are the molecular targets for around 50% of validated drugs. Despite this, membrane proteins represent less than 1% of all high-resolution protein structures due to various challenges associated with applying the main biophysical techniques used for protein structure determination. Recent years have seen an explosion in the number of high-resolution structures of membrane proteins determined by NMR spectroscopy, especially for those with multiple transmembrane-spanning segments. This is a review of the structures of polytopic integral membrane proteins determined by NMR spectroscopy up to the end of the year 2010, which includes both β-barrel and α-helical proteins from a number of different organisms and with a range in types of function. It also considers the challenges associated with performing structural studies by NMR spectroscopy on membrane proteins and how some of these have been overcome, along with its exciting potential for contributing new knowledge about the molecular mechanisms of membrane proteins, their roles in human disease, and for assisting drug design.

Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.

Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)

Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2.

Genetic evidence for an essential oscillation of transmembrane-spanning segment 5 in the Escherichia coli ammonium channel AmtB.

Ammonium channels, called Amt or Mep, concentrate NH(4)(+) against a gradient. Each monomer of the trimer has a pore through which substrate passes and a C-terminal cytoplasmic extension. The importance of the C-terminal extension to AmtB activity remains unclear. We have described lesions in conserved C-terminal residues that inactivate AmtB and here characterize 38 intragenic suppressors upstream of the C terminus ( approximately 1/3 of total suppressors). Three that occurred repeatedly, including the previously characterized W148L at the pore entry, restored growth at low NH(3) to nearly wild-type levels and hence restored high activity. V116L completely restored function to two of the mutant proteins and, when separated from other lesions, did not damage wild-type AmtB. A179E notably altered folding of AmtB, compensated for all inactivating C-terminal lesions, and damaged wild-type AmtB. V116L and A179E lie at the cytoplasmic end of transmembrane-spanning segments (TM) 3 and 5, respectively, and the proximal part of the C-terminal tail makes intimate contacts with the loops following them before crossing to the adjacent monomer. Collectively, the properties of intragenic suppressor strains lead us to postulate that the C-terminal tail facilitates an oscillation of TM 5 that is required for coordinated pore function and high AmtB activity. Movement of TM 5 appears to control the opening of both the periplasmic entry and the cytoplasmic exit to the pore.

Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Escherichia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.

Interaction of transmembrane-spanning segments of the α2-adrenergic receptor with model membranes

Adrenergic receptors are integral membrane proteins involved in cellular signalling that belong to the G protein-coupled receptors. Synthetic peptides resembling the putative transmembrane (TM) segments TM4, TM6 and TM7, of the human alpha2-adrenergic receptor subtype C10 (P08913) and defined lipid vesicles were used to assess protein-lipid interactions that might be relevant to receptor structure/function. P6 peptide contains the hydrophobic core of TM6 plus the N-terminal hydrophilic motif REKR, while peptides P4 and P7 contained just the hydrophobic stretches of TM4 and TM7, respectively. All the peptides increase their helical tendency at moderate concentrations of TFE (30-50%) and in presence of 1,2-dielaidoyl-sn-glycero-3-phosphatidylethanolamine (DEPE) lipids. However, only P6 displays up to 19% of alpha-helix in the presence of just the DEPE lipids, evidences a transmembrane orientation and stabilizes the Lalpha lipid phase. Conversely, P4 and P7 peptides form only stable beta-sheet structures in DEPE and favour the non-lamellar, inverted hexagonal (H(II)) phase of DEPE by lowering its phase transition temperature. This study highlights the potential of using synthetic peptides derived from the amino acid sequence in the native proteins as templates to understand the behaviour of the transmembrane segments and underline the importance of interfacial anchoring interactions to meet hydrophobic matching requirements and define membrane organization.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Insertion and Topology of Normal and Mutant Bestrophin-1 in the Endoplasmic Reticulum Membrane

The vitelliform macular dystrophy type 2 (VMD2) gene mutated in Best macular dystrophy encodes a 585-amino acid putative transmembrane protein termed bestrophin-1. The vast majority of known disease-associated alterations are of the missense type, which cluster near predicted transmembrane domains (TMDs). To investigate bestrophin-1 membrane topology and to assess consequences of point mutations on membrane integration, we have analyzed the insertion of putative TMDs into the endoplasmic reticulum (ER) membrane. Out of six potential TMDs, our data suggest a topological model of bestrophin-1 with four transmembrane-spanning segments and one large cytoplasmatic loop between putative TMD2 and TMD5. Consequently, a relatively hydrophobic segment containing putative TMD3 (aa 130-149) and TMD4 (aa 179-201) is located within the cytoplasm. Furthermore, we show that three out of 18 disease-associated alterations investigated (I73N, Y85H, F281del) reveal measurable effects on membrane insertion suggesting that defective membrane integration of bestrophin-1 may represent a potential disease mechanism for a small subset of Best macular dystrophy-related mutations.

An overview of recent developments in GPCR modelling: methods and validation

The superfamily of G-protein-coupled receptors (GPCRs) are single polypeptide chains possessing seven hydrophobic transmembrane-spanning segments that couple with an effector molecule through a trimeric G protein complex. A knowledge of their three-dimensional structure could be of great help in the task of understanding their function and in the rational design of specific ligands. However, as GPCRs are membrane-bound proteins, high-resolution structural characterisation is still an extremely difficult task. For this reason, great importance has been placed on molecular modelling studies, and in the last few years especially on homology modelling (HM) techniques. The HM procedure starts from the recently resolved crystal structure of the bovine rhodopsin, but other experimental data such as site-directed mutagenesis and substituted-cysteine accessibility method studies are extremely necessary. In this review the most common HM computational steps are reported and discussed together with the most recent alternative approaches and main validation methods. Future possible targets of the HM are presented and expected improvements in computational methods are considered.

A voltage-gated proton-selective channel lacking the pore domain.

Voltage changes across the cell membrane control the gating of many cation-selective ion channels. Conserved from bacteria to humans, the voltage-gated-ligand superfamily of ion channels are encoded as polypeptide chains of six transmembrane-spanning segments (S1-S6). S1-S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged lever that moves in response to voltage changes. The VSD 'ligand' transmits force via a linker to the S5-S6 pore domain 'receptor', thereby opening or closing the channel. The ascidian VSD protein Ci-VSP gates a phosphatase activity rather than a channel pore, indicating that VSDs function independently of ion channels. Here we describe a mammalian VSD protein (H(V)1) that lacks a discernible pore domain but is sufficient for expression of a voltage-sensitive proton-selective ion channel activity. H(v)1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. Mutagenesis of H(v)1 identified three arginine residues in S4 that regulate channel gating and two histidine residues that are required for extracellular inhibition of H(v)1 by Zn2+. H(v)1 is expressed in immune tissues and manifests the characteristic properties of native proton conductances (G(vH+)). In phagocytic leukocytes, G(vH+) are required to support the oxidative burst that underlies microbial killing by the innate immune system. The data presented here identify H(v)1 as a long-sought voltage-gated H+ channel and establish H(v)1 as the founding member of a family of mammalian VSD proteins.

Evolutionary conservation and diversification of Rh family genes and proteins.

Rhesus (Rh) proteins were first identified in human erythroid cells and recently in other tissues. Like ammonia transporter (Amt) proteins, their only homologues, Rh proteins have the 12 transmembrane-spanning segments characteristic of transporters. Many think Rh and Amt proteins transport the same substrate, NH(3)/NH(4)(+), whereas others think that Rh proteins transport CO(2) and Amt proteins NH(3). In the latter view, Rh and Amt are different biological gas channels. To reconstruct the phylogeny of the Rh family and study its coexistence with and relationship to Amt in depth, we analyzed 111 Rh genes and 260 Amt genes. Although Rh and Amt are found together in organisms as diverse as unicellular eukaryotes and sea squirts, Rh genes apparently arose later, because they are rare in prokaryotes. However, Rh genes are prominent in vertebrates, in which Amt genes disappear. In organisms with both types of genes, Rh had apparently diverged away from Amt rapidly and then evolved slowly over a long period. Functionally divergent amino acid sites are clustered in transmembrane segments and around the gas-conducting lumen recently identified in Escherichia coli AmtB, in agreement with Rh proteins having new substrate specificity. Despite gene duplications and mutations, the Rh paralogous groups all have apparently been subject to strong purifying selection indicating functional conservation. Genes encoding the classical Rh proteins in mammalian red cells show higher nucleotide substitution rates at nonsynonymous codon positions than other Rh genes, a finding that suggests a possible role for these proteins in red cell morphogenetic evolution.

CDNA Cloning and Expression of the Cardiac Na+/Ca2+ Exchanger from Mozambique Tilapia (Oreochromis mossambicus) Reveal a Teleost Membrane Transporter with Mammalian Temperature Dependence

The complete cDNA sequence of the tilapia cardiac Na(+)/Ca2+ exchanger (NCX-TL1.0) was determined. The 3.1-kb transcript encodes a protein 957 amino acids in length, with a predicted signal peptide cleaved at residue 31 and two potential N-glycosylation sites in the extracellular N terminus. Hydropathy analysis and sequence comparison predicted a mature protein with nine transmembrane-spanning segments, consistent with the structural topologies of other known mammalian and teleost NCX isoforms. Overall sequence comparison shows high identity to both trout NCX-TR1.0 ( approximately 81%) and mammalian NCX1.1 ( approximately 73%), and phylogenetic analyses confirmed its identity as a member of the NCX1 gene family, expressing exons A, C, D, and F in the alternative splice site. Sequence identity is even higher in the alpha-repeats, the exchanger inhibitory peptide (XIP) site, and Ca(2+)-binding domains, which is reflected in the functional and regulatory properties of tilapia NCX-TL1.0. When NCX-TL1.0 was expressed in Xenopus oocytes and the currents were measured in giant excised patches, they displayed both positive regulation by Ca2+ and Na(+)-dependent inactivation in a manner similar to trout NCX-TR1.0. However, tilapia NCX-TL1.0 exhibited a relatively high sensitivity to temperature compared with trout NCX-TR1.0. Whereas trout NCX-TR1.0 currents displayed activation energies of approximately 7 kJ/mol, tilapia NCX-TL1.0 currents showed mammal-like temperature dependence, with peak and steady-state current activation energies of 53 +/- 9 and 67 +/- 21 kJ/mol, respectively. Using comparative sequence analysis, we highlighted 10 residue positions in the N-terminal domain of the NCX that, in combination, may confer exchanger temperature dependence through subtle changes in protein flexibility. Tilapia NCX-TL1.0 represents the first non-mammalian NCX to exhibit a mammalian temperature dependence phenotype and will prove to be a useful model in defining the interplay between molecular flexibility and stability in NCX function.

Isolation of white gene orthologue of the sawfly, Athalia rosae (Hymenoptera) and its functional analysis using RNA interference

We isolated and characterized the white gene orthologue of the sawfly, Athalia rosae (Hymenoptera). The A. rosae white ( Ar white) cDNA cloned was 2058-bp long encoding 685 amino acids in a single open reading frame (ORF). Comparison of the cDNA sequence with the genomic DNA sequence revealed that the ORF was derived from 11 exons. Ar white was a single copy gene as evidenced by genomic Southern blotting and its cytological localization on the metaphase chromosomes. The deduced amino acid sequence aligned well with known insect white orthologous gene products sharing conserved regions such as the ATP-binding motif and the six transmembrane-spanning segments. Expression of Ar white was detected at embryonic and pupal stages by Northern blotting. In situ hybridization detected the embryonic expression in a pair of the lateral tips of protocephalic placodes from where optic organs are formed. Ar white function was examined using double-stranded RNA (dsRNA)-mediated interference. The synthesized dsRNA targeting Ar white transcripts caused a decrease in the level of the original mRNAs, and resulted in the white phenocopy in the embryonic eye pigmentation when microinjected into eggs from wild-type females. The effects occurred in a dose-dependent manner.

Mutational analysis of the large periplasmic loop 7–8 of the putrescine transporter PotE in Escherichia coli

The PotE protein is a putrescine–ornithine antiporter found in many gram-negative bacteria. It is a member of the APA family of transporters and has 12 predicted α-helical transmembrane spanning segments (TMS). While the substrate binding site has previously been mapped to a region near the surface of the cytoplasmic lipid layer, no structural feature within the periplasmic domains of PotE have been shown to be important for function. We examined the role of the only large outer loop, situated between transmembrane spanning segment 7 and 8, in putrescine uptake. Deletion of the highly conserved amino acids in the region closest to transmembrane spanning segment 7 produced a protein with little activity. Glycine-scanning mutagenesis of this region showed that Val 249 and Leu 254 were required for optimal transporter function. The V249G mutant transported putrescine at a lower maximal rate compared to wild-type (WT) but with the same substrate binding affinity. In contrast, the L254G mutant had a higher substrate affinity. A series of Val 249 mutants indicated that the hydrophobicity of this residue, which is located at or near the membrane surface, is important for PotE function. Secondary structure predictions of the large outer loop indicated the presence of a hydrophobic α-helix in the centre with a hydrophobic region at each end suggesting that the loop was not entirely exposed to the aqueous periplasmic space. The study shows that loop 7–8 is important for PotE function, possibly by forming a re-entrant loop in the channel of the transporter.

Topology of the retinal cone NCKX2 Na/Ca-K exchanger.

The Na/Ca-K exchanger (NCKX) is a polytopic membrane protein that plays a critical role in Ca(2+) homeostasis in retinal rod and cone photoreceptors. The NCKX1 isoform is found in rods, while the NCKX2 isoform is found in cones, in retinal ganglion cells, and in various parts of the brain. The topology of the Na/Ca-K exchanger is thought to consist of two large hydrophilic loops and two sets of transmembrane spanning segments (TMs). The first large hydrophilic loop is located extracellularly at the N-terminus; the other is cytoplasmic and separates the two sets of TMs. The TMs consist of either five and five membrane spanning helices or five and six membrane spanning helices, depending upon the predictive algorithm used. Little specific information is yet available on the orientation of the various membrane spanning helices and the localization of the short loops connecting these helices. In this study, we have determined which of the connecting loops are exposed to the extracellular milieu using two different methods: accessibility of substituted cysteine residues and insertion of N-glycosylation sites. The two methods resulted in a consistent NCKX topology in which the two sets of TMs each contain five membrane spanning helices. Our new model places what was previously membrane spanning helix six in the cytoplasm, which places the C-terminus on the extracellular surface. Surprisingly, this NCKX topology model is different from the current NCX topology model with respect to the C-terminal three membrane helices.

An ATP-binding Cassette Transporter Is a Major Glycoprotein of Sea Urchin Sperm Membranes

Sperm are terminally differentiated cells that undergo several membrane-altering events before fusion with eggs. One event, the sea urchin sperm acrosome reaction (AR), is blocked by the lectin wheat germ agglutinin (WGA). In an effort to identify proteins involved in the AR induction, the peptide sequence was obtained from a 220-kDa WGA-binding protein. Degenerate PCR and library screening resulted in the full-length deduced amino acid sequence of an ATP-binding cassette transporter, suABCA. The protein of 1,764 residues has two transmembrane regions, two nucleotide-binding domains, and is most closely related to the human ABC subfamily A member 3 transporter (ABCA3). Sequence analysis suggests a large extracellular loop between transmembrane spanning segments 7 and 8, with five N-linked glycosylation sites. An antibody made to the loop region binds to non-permeabilized cells, supporting that this region is extracellular. suABCA is found in sperm membrane vesicles, it can be solubilized with nonionic detergents, and it shifts from 220 to 200 kDa upon protein:N-glycanase F digestion. suABCA localizes to the entire surface of sperm in a punctate pattern, but is not detected in lipid rafts. Based on its relationship to subfamily A, suABCA is most likely involved in phospholipid or cholesterol transport. This is the first investigation of an ABC transporter in animal sperm.