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Purification of functional human Cl-/HCO3 - exchanger, AE1, over-expressed in Saccharomyces cerevisiae
Abstract
There is no high-resolution structure for the membrane domain of the human erythrocyte anion exchanger, AE1 (Band 3). In this report, we have developed an expression and purification strategy for AE1 to be used in crystallization trials. Saccharomyces cerevisiae strain BJ5457 was transformed with an expression vector encoding the AE1 membrane domain (AE1MD, amino acids 388-911), fused C-terminally to an epitope tag, corresponding to the nine C-terminal amino acids of rhodopsin. The fusion protein, AE1MD-Rho, was expressed at a concentration of 0.3 mg/l of culture. Confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation revealed that AE1MD-Rho did not process to the plasma membrane of S. cerevisiae, but was retained in an intracellular membrane fraction. Treatment with the endoglycosidase, PNGase F, showed that AE1MD-Rho is not N-glycosylated. AE1MD-Rho solubilized from yeast membranes, with Fos-choline detergent, was purified to 93% homogeneity in a single-step, using a 1D4 antibody affinity resin, in amounts up to 2.5 mg from 18 l of culture. The ability of purified AE1MD-Rho to transport sulfate was examined in reconstituted vesicles. The rate of sulfate efflux mediated by vesicles reconstituted with AE1MD-Rho was indistinguishable from vesicles with purified erythrocyte-source AE1. Using this purification strategy, sufficient amounts of functional, homogeneous AE1MD-Rho can be purified to enable crystallization trials.
... shown that numerous autosomal mutations in the respective kAE1-encoding gene SLC4A1 are associated with the misfolding, retention in the secretory pathway and/or premature degradation of the transporter, eventually cumulating in the development of a pathophysiological syndrome named distal renal tubular acidosis (dRTA) (Bonar & Casey, 2010). Some proteins involved in the trafficking of kAE1 have already been identified, however, the exact molecular mechanisms causing the development of dRTA remain unclear (Groves et al., 1999;. ...
... However, a partial transport to the plasma membrane as well as in vivo activity could only be shown for the 54 truncated derivative AE1 361-911 ; all other shortened variants were accumulated in various intracellular membranes (Sekler et al., 1995;Groves et al., 1996). In addition, attempts to express full-length versions of human AE1 in yeast failed because AE1 was not only unable to reach the yeast cell surface but was also not functional (Bonar & Casey, 2010). However, a recent report demonstrated that a full-length variant of yeast codon-optimized kAE1 was successfully expressed in S. cerevisiae, partially reached the cell surface, and showed anion transport activity (Sarder et al., 2020;Li et al., 2021). ...
... Yeast is a popular model for studying the biogenesis, expression, trafficking, and functionality of human proteins, including membrane proteins. However, a prerequisite to successfully examining these types of proteins in yeast is the correct trafficking to the yeast plasma membrane (Bonar & Casey, 2010). Concerning the heterologous expression of the renal membrane protein kAE1 in yeast, proper plasma membrane localization could only be achieved for the N-terminally truncated version of the protein kAE1 B3mem (Sekler et al., 1995). ...
... Aside from the high conservation of major cellular processes between yeast and humans, yeast cells further possess critical advantages compared with human cell culture including affordability and rapid cultivation, availability of a broad range of genetic manipulation tools (e.g., plasmid and knock-out strain collections) and the potential for performing high-throughput screening assays (Kolb et al., 2011;Mackie & Brodsky, 2018). Nevertheless, heterologous expression of human proteins often failed and/or ended up with proteins lacking their native function, localization and/or folding state (Bonar & Casey, 2010;Groves et al., 1999;Kolb et al., 2011;Romanos et al., 1992). A current example represents human kidney anion exchanger 1 (kAE1) which was found to be trapped in intracellular membrane structures after its heterologous expression in S. cerevisiae (Bonar & Casey, 2010;Groves et al., 1999;Sarder et al., 2020). ...
... Nevertheless, heterologous expression of human proteins often failed and/or ended up with proteins lacking their native function, localization and/or folding state (Bonar & Casey, 2010;Groves et al., 1999;Kolb et al., 2011;Romanos et al., 1992). A current example represents human kidney anion exchanger 1 (kAE1) which was found to be trapped in intracellular membrane structures after its heterologous expression in S. cerevisiae (Bonar & Casey, 2010;Groves et al., 1999;Sarder et al., 2020). ...
... As illustrated in Figure 1d,e, cells expressing the EV showed the highest growth rate whereas the expression of the full-length kAE1 variants kAE1 WT and kAE1 HA significantly slowed cell growth. Interestingly, expression of N-terminally truncated kAE1 (kAE1 B3Mem ) (Bonar & Casey, 2010;Groves et al., 1996) and an inactive kAE1 E681Q variant (Jennings & Smith, 1992) lacking its bicarbonate exchange function did not improve cell growth. ...
... Aside from the high conservation of major cellular processes between yeast and humans, yeast cells further possess critical advantages compared with human cell culture including affordability and rapid cultivation, availability of a broad range of genetic manipulation tools (e.g., plasmid and knock-out strain collections) and the potential for performing high-throughput screening assays (Kolb et al., 2011;Mackie & Brodsky, 2018). Nevertheless, heterologous expression of human proteins often failed and/or ended up with proteins lacking their native function, localization and/or folding state (Bonar & Casey, 2010;Groves et al., 1999;Kolb et al., 2011;Romanos et al., 1992). A current example represents human kidney anion exchanger 1 (kAE1) which was found to be trapped in intracellular membrane structures after its heterologous expression in S. cerevisiae (Bonar & Casey, 2010;Groves et al., 1999;Sarder et al., 2020). ...
... Nevertheless, heterologous expression of human proteins often failed and/or ended up with proteins lacking their native function, localization and/or folding state (Bonar & Casey, 2010;Groves et al., 1999;Kolb et al., 2011;Romanos et al., 1992). A current example represents human kidney anion exchanger 1 (kAE1) which was found to be trapped in intracellular membrane structures after its heterologous expression in S. cerevisiae (Bonar & Casey, 2010;Groves et al., 1999;Sarder et al., 2020). ...
... As illustrated in Figure 1d,e, cells expressing the EV showed the highest growth rate whereas the expression of the full-length kAE1 variants kAE1 WT and kAE1 HA significantly slowed cell growth. Interestingly, expression of N-terminally truncated kAE1 (kAE1 B3Mem ) (Bonar & Casey, 2010;Groves et al., 1996) and an inactive kAE1 E681Q variant (Jennings & Smith, 1992) lacking its bicarbonate exchange function did not improve cell growth. ...
Human kidney anion exchanger 1 (kAE1) facilitates simultaneous efflux of bicarbonate and absorption of chloride at the basolateral membrane of α‐intercalated cells. In these cells, kAE1 contributes to systemic acid‐base balance along with the proton pump v‐H+‐ATPase and the cytosolic carbonic anhydrase II. Recent electron microscopy analyses in yeast demonstrate that heterologous expression of several kAE1 variants causes a massive accumulation of the anion transporter in intracellular membrane structures. Here, we examined the origin of these kAE1 aggregations in more detail. Using various biochemical techniques as well as advanced light and electron microscopy, we showed that accumulation of kAE1 mainly occurs in ER membranes which eventually leads to strong UPR activation and severe growth defect in kAE1 expressing yeast cells. Furthermore, our data indicates that UPR activation is dose‐dependent and uncoupled from the bicarbonate transport activity. By using truncated kAE1 variants, we identified the C‐terminal region of kAE1 as crucial factor for the increased ER stress level. Finally, a redistribution of ER‐localized kAE1 to the cell periphery was achieved by boosting the ER folding capacity. Our findings not only demonstrate a promising strategy for preventing intracellular kAE1 accumulation and improving kAE1 plasma membrane targeting, but also highlight the versatility of yeast as model to investigate kAE1‐related research questions including the analysis of structural features, protein degradation and trafficking. Furthermore, our approach might be a promising strategy for future analyses to further optimize the cell surface targeting of other disease‐related PM proteins, not only in yeast but also in mammalian cells.
... Numerous attempts were previously made to express full-length human kAE1 (hkAE1) in yeast, but those efforts either failed or resulted in an inactive protein that did not get transported to the cell surface (Bonar & Casey, 2010;Groves et al., 1999). In order to obtain a successful expression of full-length kAE1 in S. cerevisiae, two main approaches were applied in this study. ...
... Secondly, the human cDNA sequence of kAE1 was optimized for the synonymous yeast codon (Bonar & Casey, 2010;Groves et al., 1996;Sekler et al., 1995). The idea behind introducing a Kar2 SS was the attachment of a yeast signal peptide to the human protein, which has already been shown to facilitated ER translocation. ...
... Former studies in yeast reported that kAE1 is highly prone to be trapped in different subcellular membrane structures (Bonar & Casey, 2010). The observation of spinning disk microscopy data suggested that a majority of the expressed kAE1 was intracellularly accumulated in protein-rich membrane-like structure (Figure 3.11). ...
... Codon optimization leads to heterologous expression of human kAE1 in yeast. Previous studies already demonstrated the heterologous expression of various truncated versions of red cell anion exchanger 1 (AE1; 361 to 911 amino acids [aa], 183 to 911 aa, and 388 to 911 aa) in the bakers's yeast species S. cerevisiae (27)(28)(29). So far, the results have revealed that only AE1 361-911 was partially transported to the PM and showed anion transport activity in vivo, whereas other truncated variants seemed to be trapped in intracellular membranes (28,30). ...
... In these experiments, there was no detectable size shift for any of the tested variants, indicating that full-length kAE1 versions are not glycosylated when expressed in yeast (data not shown). These findings are consistent with early reports demonstrating that AE1 is likewise not N-glycosylated when heterologously expressed in yeast (29). ...
... However, it is known that human membrane proteins, when expressed in yeast, show less maturation than the corresponding fungal or plant homologs and often fail to reach their correct intracellular localization and function (16). Nevertheless, previous studies already demonstrated that yeast is suitable for high-level expression of different AE1 variants (27,29,30). However, efforts to express full-length AE1/kAE1 variants In the presence of kAE1, intracellular Cl Ϫ seems to be excreted (solid arrow) as a consequence of the action of kAE1, leading to a decrease in [Cl Ϫ ] in and an increase in [Cl Ϫ ] ex. ...
Distal renal tubular acidosis (dRTA) is a common kidney dysfunction characterized by impaired acid secretion via urine. Previous studies revealed that α-intercalated cells of dRTA patients express mutated forms of human kidney anion exchanger 1 (kAE1) which result in inefficient plasma membrane targeting or diminished expression levels of kAE1. However, the precise dRTA-causing processes are inadequately understood, and alternative model systems are helpful tools to address kAE1-related questions in a fast and inexpensive way. In contrast to a previous study, we successfully expressed full-length kAE1 in Saccharomyces cerevisiae . Using advanced microscopy techniques as well as different biochemical and functionality assays, plasma membrane localization and biological activity were confirmed for the heterologously expressed anion transporter. These findings represent first important steps to use the potential of yeast as a model organism for studying trafficking, activity, and degradation of kAE1 and its mutant variants in the future.
... Deletion of histidinol dehydrogenase [63] S. cerevisiae EBY.S7 MATα hxt1-17∆gal2∆agt1∆stl1∆leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8c SUC2 hxt∆fgy1 Deletion of hexose transporters [64] S. cerevisiae EBY.F4-1 MATα hxt1-17∆gal2∆agt1∆stl1∆leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8c SUC2 hxt∆fgy1 fgy41 Deletion of hexose transporters [64] S. cerevisiae EBY.VW4000 MATa leu2-3,112 ura3-52 trp1-289 his3-1 MAL2-8c SUC2 ∆hxt1-17 ∆gal2 ∆stl1::loxP ∆agt1::loxP ∆mph2::loxP ∆mph3::loxP Deletion of hexose transporters [65] S. cerevisiae SDY.022 MATa leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8C SUC2 ∆hxt1-17 ∆gal2 ∆agt1 ∆stl1 fgy1-1 erg4::kanMX Deletion of hexose transporters [66] S. cerevisiae BJ5457 MATα ura3-52 trp1 lys2-801 leu2-∆1 his3-∆200 pep4:HIS3 prb1-delta1.6R can1 GAL Protease deficient [67] S. cerevisiae RE700A MATa hxt1::HIS3::hxt4 hxt5::LEU2 hxt2::HIS3hxt3::LEU2::hxt6 hxt7::HIS3 Deletion of hexose transporters [68] S. cerevisiae BY4742 MATα his3∆1 leu2∆0 lys2∆0 ura3∆ Minimize homologous recombination [59] S. cerevisiae BY4742 GEV MATa, (PGAL10+gal1)∆::loxP, leu2∆0::PACT1-GEV-NatMX, gal4∆::LEU2, HAP1+ Minimize homologous recombination [69] P. pastoris SMD1168H pep4 Protease A deficiency [59] S. cerevisiae FAB158 MATa his3-∆200 leu2-∆1 lys2-801 trp1-∆1 ade2-101 ura3-52 tat2 ∆::HIS3 Deletion of tryptophan transporter [70] S. cerevisiae TMY203 MATa his3-∆200 leu2-∆1 lys2-801 trp1-∆1 ade2-101 ura3-52 tat1 ∆::kanMX4 tat2∆::LEU2 Deletion of tryptophan transporters [70] S. cerevisiae FAY18A MATa his3-∆200 leu2-∆1 lys2-801 trp1-∆1 ade2-101 ura3-52 HPG1-1, Rsp5 P514T Deletion of Rsp5 ubiquitin ligase [70] S. cerevisiae XPY1263a MATa thi3∆::natMX thi7D::kanMX Deletion of thiamine transporter [71] S. cerevisiae BY4741mp MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; mir1∆; pic2∆ Deletion of phosphate and copper transporter [72] S. cerevisiae BY4741 pic2∆ MATa, leu2,met15, ura3, his3, PIC2::KANMX Deletion of copper transporter [73] S. cerevisiae WB-12 MATα ade2-1 trp1-1 ura3-1 can1-100 aac1::LEU2 aac2::HIS3 Deletion of adenine nucleotide carriers [74] ...
... In some cases, the expressed transporter does not reach the correct destination completely or at all. An example is the SLC4A1 transporter for which two different isoforms are described: the full-length erythrocyte variant (eAE1) and the kidney variant (kAE1) lacking the first 65 N-terminal amino acids present in the erythrocyte isoform [67]. Concerning the kidney variant kAE1, trafficking studies revealed that only a part of the transporter molecules efficiently enters the secretory pathway reaching the plasma membrane, the rest being accumulated in other membranes. ...
For more than 20 years, yeast has been a widely used system for the expression of human membrane transporters. Among them, more than 400 are members of the largest transporter family, the SLC superfamily. SLCs play critical roles in maintaining cellular homeostasis by transporting nutrients, ions, and waste products. Based on their involvement in drug absorption and in several human diseases, they are considered emerging therapeutic targets. Despite their critical role in human health, a large part of SLCs’ is ‘orphans’ for substrate specificity or function. Moreover, very few data are available concerning their 3D structure. On the basis of the human health benefits of filling these knowledge gaps, an understanding of protein expression in systems that allow functional production of these proteins is essential. Among the 500 known yeast species, S. cerevisiae and P. pastoris represent those most employed for this purpose. This review aims to provide a comprehensive state-of-the-art on the attempts of human SLC expression performed by exploiting yeast. The collected data will hopefully be useful for guiding new attempts in SLCs expression with the aim to reveal new fundamental data that could lead to potential effects on human health.
... Interestingly, the Band 3 cytoplasmic domain is not strongly associated with its membrane domain [Grinstein et al., 1978;Bonar and Casey, 2010], providing a strong point of structural difference between the two SLC4 proteins [Shnitsar et al., 2013]. If cytoplasmic domain interactions with the cytoplasmic face of the membrane domain occur in SLC4A11, but not in Band 3, we would expect greater evolutionary pressure to conserve the cytoplasmic face of SLC4A11 than Band 3. The greater amino acid sequence conservation on the cytoplasmic face of SLC4A11 than Band 3, in particular at the cytoplasmic ends of TM5, 13, and 14 (Fig. 6), is consistent with a role of this region in protein-protein interaction, potentially the interaction of the membrane domain with the cytoplasmic domain. ...
We studied the structural effects of point mutations of a membrane protein that cause genetic disease. SLC4A11 is a membrane transport protein (OH−/H+/NH3/H2O) of basolateral corneal endothelium, whose mutations cause some cases of Congenital Hereditary Endothelial Dystrophy and Fuchs Endothelial Corneal Dystrophy. We created a three-dimensional homology model of SLC4A11 membrane domain, using Band 3 (SLC4A1) crystal structure as template. The homology model was assessed in silico and by analysis of mutants designed on the basis of the model. Catalytic pathway mutants p.Glu675Gln, p.His724Arg and p.His724Ala impaired SLC4A11 transport. p.Ala720Leu, in a region of extended structure of the proposed translocation pore, failed to mature to the cell surface. p.Gly509Lys, located in an open region at the core domain/gate domain interface, had wild-type level of transport function. The molecular phenotype of 37 corneal dystrophy-causing point mutants was rationalized, based on their location in the homology model. Four map to the substrate translocation pathway, 25 to regions of close transmembrane helix packing, three to the dimeric interface, and five lie in extramembraneous loops. The model provides a view of the spectrum of effects of disease mutations on membrane protein structure and provides a tool to analyze pathogenicity of additional newly-discovered SLC4A11 mutants. This article is protected by copyright. All rights reserved
... AE1 and mNect . AE1 , were quantified by densitometry in comparison to a known amount of AE1 - Ct standard ( a purified GST fusion protein containing the last 40 amino acids ( residues 872 – 911 ) of AE1 ) ( Bonar & Casey , 2010 ) , using the anti - AE1 ( IVF12 ) antibody . The third standard is a sample in which cells were harvested , counted , and a known amount used as a cell number standard on subsequent blots . ...
Microdomains, regions of discontinuous cytosolic solute concentration enhanced by rapid solute transport and slow diffusion rates, have many cellular roles. pH-regulatory membrane transporters, like the Cl−/HCO3− exchanger AE1, could develop H+ microdomains since AE1 has a rapid transport rate and cytosolic H+ diffusion is slow. We examined whether the pH environment surrounding AE1 differs from other cellular locations. As AE1 drives Cl−/HCO3− exchange, differences in pH, near and remote from AE1, were monitored by confocal microscopy using two pH-sensitive fluorescent proteins: deGFP4 (GFP) and mNectarine (mNect). Plasma membrane (PM) pH (defined as ∼1 μm region around the cell periphery) was monitored by GFP fused to AE1 (GFP.AE1), and mNect fused to an inactive mutant of the Na+-coupled nucleoside co-transporter, hCNT3 (mNect.hCNT3). GFP.AE1 to mNect.hCNT3 distance was varied by co-expression of different amounts of the two proteins in HEK293 cells. As the GFP.AE1–mNect.hCNT3 distance increased, mNect.hCNT3 detected the Cl−/HCO3− exchange-associated cytosolic pH change with a time delay and reduced rate of pH change compared to GFP.AE1. We found that a H+ microdomain 0.3 μm in diameter forms around GFP.AE1 during physiological HCO3− transport. Carbonic anhydrase isoform II inhibition prevented H+ microdomain formation. We also measured the rate of H+ movement from PM GFP.AE1 to endoplasmic reticulum (ER), using mNect fused to the cytosolic face of ER-resident calnexin (CNX.mNect). The rate of H+ diffusion through cytosol was 60-fold faster than along the cytosolic surface of the plasma membrane. The pH environment surrounding pH regulatory transport proteins may differ as a result of H+ microdomain formation, which will affect nearby pH-sensitive processes.
... The coding sequence for these amino acids was amplified from pJRC9 (33) using the forward primer 5 0 -CGC GGA TCC GTC CTG CTG CCG CTC ATC TTC-3 0 and the reverse primer 5 0 -CGC GGA TCC TCA CAC AGG CAT GGC CAC TTC GT-3 0 . The PCR product was inserted into pGEX-6p-1 (GE Healthcare Life Sciences), a GST expression vector, by BamHI digestion and subsequent ligation to generate expression construct pHJC1 (36). ...
AE1, which exists in the erythrocyte plasma membrane as a noncovalent dimer, facilitates transmembrane Cl⁻/HCO₃⁻ exchange. Here a concatamer of AE1 (two AE1 monomers fused via a two-residue linker to form an intramolecular dimer) was designed to facilitate fluorescence resonance energy transfer (FRET) studies. The concatameric protein (AE1·AE1) was expressed at the plasma membrane at levels similar to that of wild-type AE1 and had Cl⁻/HCO₃⁻ exchange activity indistinguishable from that of wild-type AE1. Nondenaturing gel electrophoresis revealed that AE1·AE1 does not associate into higher-order oligomers when expressed in HEK293 cells and Xenopus laevis oocytes. The cysteine-less concatamer (AE1·AE1-C⁻) enabled introduction of unique cysteine residues into the whole intramolecular dimer. AE1(Q434C)·AE1(Q434C)-C⁻, with a single cysteine residue in each AE1 subunit, was labeled with the donor Alexa Fluor 488 C(5)-maleimide (AF) and the acceptor tetramethylrhodamine methanethiosulfonate (TMR-MTS). Energy transfer efficiency revealed that the distance between these residues in the AE1 dimer is 49 ± 5 Å. The 72% FRET efficiency observed between AE1(Q434C)·AE1-C⁻ labeled with AF and the lipid bilayer labeled with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate indicates that Q434 is less than 33 Å from the lipid bilayer. We thus provide two distance constraints for the position of Q434, which is located in extracellular loop 1, connecting the first two transmembrane segments of AE1.
Microbes play an important role in biotransformation and biosynthesis of biofuels, natural products, and polymers. Therefore, microbial manufacturing has been widely used in medicine, industry, and agriculture. However, common strategies including enzyme engineering, pathway optimization, and host engineering are generally inadequate to obtain an efficient microbial production system. Transporter engineering provides an alternative strategy to promote the transmembrane transfer of substrates, intermediates, and final products in microbial cells and thus enhances production by alleviating feedback inhibition and cytotoxicity caused by final products. According to the current studies in transport engineering, native transporters usually have low expression and poor transportation ability, resulting in inefficient transport processes and microbial production. In this review, we comprehensively summarized current approaches for transporter mining, characterization, and verification. Practical approaches to enhance the transport system in engineered cells, such as balancing transporter overexpression and cell growth, and evolution of native transporters were discussed. Furthermore, the applications of transporter engineering in microbial manufacturing, including enhancement of substrate utilization, concentration of metabolic flux to the target pathway, and acceleration of efflux and recovery of products, demonstrate its outstanding advantages and promising prospects.
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Large cytoplasmic domains (CD) are a common feature amongst integral membrane proteins. In virtually all cases these CD have a function (e.g. binding cytoskeleton or regulatory factors) separate from the membrane domain (MD). Strong associations between cytosolic and membrane domains are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, whose mutations cause genetic corneal blindness. SLC4A11 has a 41 kDa CD and 57 kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that CD-SLC4A11 has similar structure to the CD of Cl(-)/HCO3 (-) exchange protein, AE1 (SLC4A1). Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that CD-AE1 has a structure similar to CD-SLC4A11. CD-AE1/MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensible role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD and MD-SLC4A11, these domains specifically associate when co-expressed in HEK293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the cytoplasmic domain has strong associations with the integral membrane domain, which contribute to membrane transport function.
Three genetic corneal dystrophies congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy) arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channeling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in X. laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: i) similar to some aquaporins in rate, ii) uncoupled from solute-flux, iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors), iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. Slc4a11(-/-) mice manifest corneal edema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues.
Many cellular processes are controlled via either stable or transient protein-protein interactions (PPIs). Protein complexes are 'molecular machines' in which multiple interactive partners carry out various cellular functions. Given that almost a third of the proteome consists of membrane proteins and that more than 50% of currently available drugs are targeted toward them, investigation of membrane protein complexes has taken center stage over the past years. Thus, gaining an in-depth understanding of PPI networks will give us more insight into the functional relationship as well as downstream effectors of protein complexes, hence opening strategies for new drug target definitions.
Studying membrane proteins in yeast has recently been applied to many different classes of proteins with diverse functions and structures including membrane transporters. Techniques such as the split-ubiquitin membrane yeast two-hybrid or variants of the protein-fragment complementation assay have been successfully applied to both large-scale genome-wide screens and as smaller-scale PPI studies in a reliable and robust fashion.
Yeast-based methods to study membrane PPI in vivo offer a powerful tool for the investigation of protein complexes from various organisms, including mammals. The investigation of global protein maps will serve as a foundation for mechanistic and quantitative studies of poorly characterized gene products and disease-associated proteins. Identification of PPIs is also of great interest for drug discovery as many human diseases result from abnormal PPIs.
The red blood cell membrane (RBCM) is a primary model for animal cell plasma membranes. One of its major organizing centers is the cytoplasmic domain of band 3 (cdb3), which links multiple proteins to the membrane. Included among its peripheral protein ligands are ankyrin (the major bridge to the spectrin-actin skeleton), protein 4.1, protein 4.2, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, deoxyhemoglobin, p72syk protein tyrosine kinase, and hemichromes. The crystal structure of cdb3 is reported at 0.26 nm (2.6 Å) resolution. A tight symmetric dimer is formed by cdb3; it is stabilized by interlocked dimerization arms contributed by both monomers. Each subunit also includes a larger peripheral protein binding domain with an α+ β-fold. The binding sites of several peripheral proteins are localized in the structure, and the nature of the major conformational change that regulates membrane-skeletal interactions is evaluated. An improved structural definition of the protein network at the inner surface of the RBCM is now possible.
Two monoclonal antibodies designated as rhodopsin (rho) 1D4 and rho 4A2 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of a mouse immunized with bleached bovine rod outer segment disk membranes. These antibodies were specific for rhodopsin as determined by radioimmune labeling of bovine rod outer segment disk membrane proteins electrophoretically transferred from sodium dodecyl sulfate gels to CNBr-activated paper. Limited proteolytic digestion of rhodopsin in sealed disk membranes in conjunction with radioimmune assays indicated that the rho 1D4 antibody bound to the carboxyl-terminal segment of rhodopsin on the cytoplasmic side of disk membranes, whereas the rho 4A2 antibody bound to a determinant along the amino-terminal third of the rhodopsin polypeptide chain. Binding of the rho 4A2 antibody was sensitive to solubilization and photobleaching of rhodopsin. The rho 4A2 antibody did not bind to rhodopsin in sealed membrane disks but did bind to detergent-solubilized rhodopsin. Detergent-solubilized bleached rhodopsin was 13 times more antigenic than unbleached rhodopsin. Rhodopsin solubilized in Triton X-100 was more antigenic than rhodopsin solubilized in cholate. These results indicate that the 4A2 antibody serves as a sensitive immunological probe for structural changes of rhodopsin caused by solubilization and photobleaching. Both the rho 1D4 and 4A2 antibodies were also found to cross-react with frog rhodopsin but not Halobacterium halobium bacteriorhodopsin. The rho 4A2 antibody bound to the three forms of frog rhodopsin resolved by sodium dodecyl sulfate gel electrophoresis whereas rho 1D4 bound to only the two higher molecular weight frog rhodopsins. Finally, lectin inhibition studies using 125I-labeled succinyl-Con A and antibody inhibition studies confirmed previous results indicating freshly prepared bovine disks were sealed with the lectin binding sites oriented toward the inside of the disk, whereas frozen-thawed disks were predominantly unsealed with both membrane surfaces exposed. Frog disk membrane vesicles were shown to have the same orientation.
The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months.
This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic understanding to improve membrane protein production. Omics technologies provide new concepts to engineer microbial hosts for membrane protein production.
Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.
The family of mammalian bicarbonate transport proteins are involved in a wide-range of physiological processes. The importance of bicarbonate transport follows from the biochemistry of HCO(3)(-) itself. Bicarbonate is the waste product of mitochondrial respiration. HCO(3)(-) undergoes pH-dependent conversion into CO(2) and in doing so converts from a membrane impermeant anion into a gas that can diffuse across membranes. The CO(2)-HCO(3)(-) equilibrium forms the most important pH buffering system of our bodies. Bicarbonate transport proteins facilitate the movement of membrane-impermeant HCO(3)(-) across membranes to accelerate disposal of waste CO(2), control cellular and whole-body pH, and to regulate fluid movement and acid/base secretion. Defects of bicarbonate transport proteins manifest in diseases of most organ systems. Fourteen gene products facilitate mammalian bicarbonate transport, whose physiology and pathophysiology is discussed in the present review.
The structural and functional roles of the single asparagine (N)-linked oligosaccharide chain of Band 3 (AE1), the anion transport protein of the human erythrocyte membrane, were examined. Purified Band 3 (M(r) = 95,000) in 0.1% octaethylene glycol mono n-dodecyl ether (C12E8) detergent solution was deglycosylated using N-glycosidase F. This treatment sharpened the protein band on sodium dodecyl sulfate gel electrophoresis and decreased its apparent molecular weight by 5,000. The purified membrane domain could be deglycosylated under similar conditions, causing a shift from a broad band centered at 55 kDa to a sharp 46-kDa band. Band 3 was shown to bind tomato lectin, and loss of lectin binding on blots provided a sensitive assay for deglycosylation. Carbohydrate analysis revealed that greater than 80% of the oligosaccharide could be removed from Band 3 by N-glycosidase F digestion. The deglycosylated protein maintained its dimeric structure and level of detergent binding but had a smaller Stokes radius (RS = 72 A) than native Band 3 (RS = 75 A). The Stokes radius of the membrane domain (RS = 60 A) also decreased upon deglycosylation (RS = 58 A). Circular dichroism studies showed that deglycosylation did not change the secondary structure of Band 3 or the membrane domain. The sensitivity of Band 3 or the membrane domain to proteolytic digestion by trypsin or proteinase K was also unaffected by deglycosylation. The deglycosylated protein aggregated more rapidly and was much more readily precipitable by ammonium sulfate. The deglycosylated protein bound the anion transport inhibitor 4-benzamido-4'-amino-stilbene-2,2'-disulfonate with the same affinity (Kd = 1 microM) as the native protein. Transport studies using reconstituted Band 3 and resealed ghosts showed that deglycosylated Band 3 retained its ability to transport anions. We conclude that removal of the oligosaccharide chain from Band 3 and any resultant structural changes had no effect on the transport function of this protein.
The oligomeric state of human Band 3 (Mr = 95,000), the erythrocyte membrane anion exchanger, was examined by size exclusion high performance liquid chromatography in solutions containing the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). Band 3 was heterogeneous with respect to oligomeric composition, the predominant (70%) species being a dimer that bound 0.57 mg of C12E8/mg of protein (Stokes radius = 78 A, s20,w = 6.9 S). Variable amounts of larger oligomers were also present; however, no evidence for equilibration between oligomeric species was observed in detergent solution. Analytical and large zone size exclusion chromatography showed that Band 3 could not be dissociated to monomers, other than by protein denaturation. The membrane domain of Band 3 (Mr = 52,000) was also dimeric, but without evidence for higher oligomeric forms, which implies that the interactions responsible for higher associations involve the cytoplasmic domain. Prelabeling of Band 3 with the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate had no effect upon the oligomeric state of either intact Band 3 or its 52-kDa membrane domain. Band 3 oligomeric state could be reversibly changed in the membrane by altering the pH of the solution. The fraction of Band 3 not associated with the cytoskeleton was almost entirely dimeric. Band 3 purified from erythrocytes separated by density gradient centrifugation revealed that older red cells contained a larger proportion of higher oligomers than did younger cells. We conclude that Band 3, in the membrane and in C12E8 solution, exists as a mixture of dimers and larger oligomers. The higher oligomers interact with the cytoskeleton, increase in amount with cell age, and are held together by interactions of the cytoplasmic domain.
A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.
Purification de la proteine a partir des globules rouges humains, necessitant une elimination des proteines extrinseques, a une solubilisation des proteines intrinseques par un detergent, et a un isolement de la bande 3 par chromatographie d'echanges d'ions et d'affinite. Caracterisation des proprietes fonctionnelles de la proteine purifiee a l'aide d'inhibitions fluorescentes des transports d'anions, et de mesure par dichroisme circulaire et experiences de reticulation par la ophenanthroline
Monoclonal antibodies against the membrane domain of human red blood cell band 3 protein have been prepared and used in topographical studies of the arrangement of the polypeptide in the membrane. One of the antibodies binds to a site near the N terminus of the membrane domain; another binds to a site near the C terminus. The latter has been used to localize a site of intracellular trypsin digestion. The cleavage site, in human band 3, corresponds to Lys-761 in mouse band 3; the site is 168 residues from the C terminus of the protein. This is the first intracellular site in the membrane domain (other than the N terminus) that has been localized in the primary structure. The antibody that binds to the N-terminal portion of the membrane domain has been used to identify a new S-cyanylation cleavage site about 7,000 daltons from the C terminus. Proteolysis/cross-linking experiments with the stilbenedisulfonate derivative H2DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) reveal that one end of the H2DIDS reacts covalently with a lysine residue that is between about 70 and 168 residues from the C terminus of band 3. In addition to placing restrictions on the location of the H2DIDS-binding lysine, these studies provide direct evidence that the C-terminal 28,000-dalton papain fragment crosses the membrane at least three times. With previous data on the remainder of the membrane domain, there is now direct evidence that the band 3 polypeptide crosses the membrane at least eight times.
The structure of branched lactosaminoglycan isolated from Band 3 glycoprotein of adult human erythrocytes was elucidated. The glycopeptides were digested by endo-beta-galactosidase under various conditions and oligosaccharides and core glycopeptides thus obtained and intact glycopeptides were analyzed by methylation, exoglycosidase digestion, and fast atom bombardment mass spectrometry. Based on these experiments, the structure of adult lactosaminoglycan was found to have the following unique features: 1) two (as major components) or three (as minor components) polylactosaminyl side chains composed of (Gal beta 1----4 GlcNAc beta 1----3) repeating units are attached to the core portion, the structure of which is Man alpha 1----6(Man alpha 1----3) [+/- GlcNAc beta 1----4]Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc----Asn, 2) the polylactosaminyl side chain arising from the mannose C-6 side is composed of 10-12 N-acetyllactosaminyl units and three branches; the lactosaminyl side chain arising from the mannose C-3 side is composed of 5-6 lactosaminyl units and contains one or two branches, 3) each branch is short and is composed of the Gal beta 1----4GlcNAc beta 1----6 structure, 4) fucose and sialic acid are preferentially linked to nonreducing terminal regions but not on the branches in the internal portion of the polylactosaminyl chain.
Human erythroid anion exchanger AE1 (Band 3) was expressed in the yeast Saccharomyces cerevisiae under the control of the constitutive promoter and transcriptional terminator of the yeast phosphoglycerate kinase gene. AE1 expression in stable yeast transformants was estimated to be approximately 0.7 mg AE1 per liter. Density gradient sedimentation analysis indicated that the AE1 protein was associated with a membrane fraction distinct from plasma membrane, most likely the endoplasmic reticulum. AE1 protein was solubilized from yeast membranes with lysophosphatidyl choline, and the protein, tagged with six histidines at its amino terminus, was purified to 35% homogeneity by metal chelation affinity chromatography. Size-exclusion chromatography in the presence of octaethylene glycol monododecyl ether indicated that the solubilized yeast-expressed AE1, like endogenous erythroid AE1, eluted at a stokes radius of 77 A, consistent with a dimeric oligomeric state. Binding of partially purified yeast-expressed AE1 to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate resin was competitive with the transportable substrate chloride but not the nontransported anion citrate, suggesting that the structure of the anion binding site is preserved. The specific activity of sulfate transport by partially purified yeast AE1 was determined in proteoliposomes to be similar to that of authentic AE1 purified from erythrocyte membranes. These data show that this expression system has the capacity to produce functional mammalian plasma membrane anion exchangers at levels sufficient for biochemical and biophysical analysis.
AE1 (Band 3), a congruent to 110-kDa integral plasma membrane protein, facilitates the electroneutral movement of Cl- and HCO3- across the erythrocyte membrane and serves as the primary attachment site for the erythrocyte spectrin-actin cytoskeleton. In this investigation, we have characterized the role of native cysteines in the function of AE1. We have constructed a mutant version of human AE1 (AE1C-) in which all five cysteines of AE1 were replaced with serines. Wild-type and AE1C- cDNAs were expressed by transient transfection of human embryonic kidney cells. Two of the mutated cysteines in AE1C- are in a region involved in ankyrin binding, and ankyrin binding has previously been shown to be sensitive to the oxidation state of these cysteines. However, the KD values for ankyrin binding by AE1 and AE1C- were indistinguishable, suggesting that AE1 cysteines are not essential components of the ankyrin-binding site. Using size exclusion chromatography, both AE1 and AE1C- were found to associate as a mixture of dimers and high molecular mass complexes. The rate of anion exchange by AE1C-, as measured in a reconstituted microsome sulfate transport assay, was indistinguishable from that by AE1 and was inhibited by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate. We conclude that the cysteines of AE1 are not required for the anion exchange or cytoskeletal binding roles of the protein.
The electroneutral exchange of chloride and bicarbonate across the human erythrocyte membrane is facilitated by Band 3, a 911 amino acid glycoprotein consisting of a 43 kDa N-terminal cytosolic domain that binds the cytoskeleton, haemoglobin and glycolytic enzymes and a 52 kDa C-terminal membrane domain that mediates anion transport. Electron microscopy and three-dimensional image reconstruction of negatively stained two-dimensional crystals of the dimeric membrane domain revealed a U-shaped structure with dimensions of 60 x 110 A, and a thickness of 80 A. The structure is open on the top and at the sides, with the monomers in close contact at the base. The basal domain is 40 A thick and probably spans the lipid bilayer. The upper part of the dimer consists of two elongated protrusions measuring 25 x 80 A in projection, with a thickness of 40 A. The protrusions form the sides of a canyon, enclosing a wide space that narrows down and converges into a depression at the centre of the dimer on the top of the basal domain. This depression may represent the opening to a transport channel located at the dimer interface. Based on the available protein-chemical data, the two protrusions face the cytosolic side of the membrane and they appear to be dynamic.
We used baculovirus to transiently express a rat anion exchanger (AE2) in Spodoptera frugiperda (Sf9) insect cells. No detectable Cl(-)-HCO3- exchange activity was observed in wild type or sham-infected Sf9 cells, monitored using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, a pH-sensitive fluorescent dye. Functional expression of anion exchange activity in the AE2 recombinant baculovirus-infected cells was observed within the first day after infection and sustained over the next 3 days. The expressed AE2 anion exchange activity was Na+ independent and could be reversibly and irreversibly inhibited by the specific anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). The reversible inhibition was sensitive to the concentration of DIDS, with a half inhibition of 4 microM. These results indicate that the rat AE2 protein produced in the recombinant baculovirus-infected insect cells is inserted into the plasma membrane in a biologically active form that appears suitable for functional studies of AE2.
The membrane domain of human erythrocyte Band 3 protein (M(r) 52,000) was reconstituted with lipids into two-dimensional crystals in the form of sheets or tubes. Crystalline sheets were monolayers with six-fold symmetry (layer group p6, a = b = 170 A, gamma = 60 degrees), whereas the symmetry of the tubular crystals was p2 (a = 104 A, b = 63 A, gamma = 104 degrees). Electron image analysis of negatively stained specimens yielded projection maps of the protein at 20 A resolution. Maps derived from both crystal forms show that the membrane domain is a dimer of two monomers related by two-fold symmetry, with each monomer consisting of three subdomains. In the dimer, two subdomains of each monomer form a roughly rectangular core (40 x 50 A in projection), surrounding a central depression. The third subdomain of the monomer measures approximately 15 x 25 A in projection and appears to be connected to the other two by a flexible link. We propose that the central depression may represent the channel for anion transport while the third subdomain appears not to be directly involved in channel formation.
We expressed the 52-kDa integral membrane domain (B3mem) of the human erythrocyte anion transporter (band 3; AE1) in a protease-deficient strain of the yeast Saccharomyces cerevisiae under the control of the inducible GAL10-CYC1 promoter. Immunoblots of total protein from transformed yeast cells confirmed that the B3mem polypeptide was overexpressed shortly after induction with galactose. Cell surface expression of the functional anion transporter was detected by using a simple transport assay to measure stilbene disulfonate-inhibitable chloride influx into intact yeast cells. The B3mem polypeptide was recycled and degraded by the cells with a half-life of approximately 1-3 hr, which led to a steady-state level of expression in exponentially growing cultures. Our data suggest that 5-10% of total B3mem is functionally active at the cell surface at any one time and that overexpression of this anion transport protein does not interfere with cell growth or survival. This is one of only a few reports of the functional expression of a plasma membrane transport protein in the plasma membrane of yeast cells and to our knowledge is the first report of red cell band 3-mediated anion transport at the plasma membrane of cDNA-transformed cells. The cell surface expression system we describe will provide a simple means for future study of the functional properties of band 3 by using site-directed mutagenesis.
AE1 is the chloride/bicarbonate anion exchanger of the erythrocyte plasma membrane. We have used scanning cysteine mutagenesis and sulfhydryl-specific chemistry to identify pore-lining residues in the Ser643-Ser690 region of the protein. The Ser643-Ser690 region spans transmembrane segment 8 of AE1 and surrounds Glu681, which may reside at the transmembrane permeability barrier. Glu681 also directly interacts with some anions during anion transport. The introduced cysteine mutants were expressed by transient transfection of HEK293 cells. Anion exchange activity was assessed by measurement of changes of intracellular pH, which follow transmembrane bicarbonate movement mediated by AE1. To identify residues that might form part of an aqueous transmembrane pore, we measured anion exchange activity of each introduced cysteine mutant before and after incubation with the sulfhydryl reagents para-chloromercuribenzene sulfonate and 2-(aminoethyl)methanethiosulfonate hydrobromide. Our data identified transmembrane mutants A666C, S667C, L669C, L673C, L677C, and L680C and intracellular mutants I684C and I688C that could be inhibited by sulfhydryl reagents and may therefore form a part of a transmembrane pore. These residues map to one face of a helical wheel plot. The ability to inhibit two intracellular mutants suggests that transmembrane helix 8 extends at least two helical turns beyond the intracellular membrane surface. The identified hydrophobic pore-lining residues (leucine, isoleucine, and alanine) may limit interactions with substrate anions.
The red blood cell membrane (RBCM) is a primary model for animal cell plasma membranes. One of its major organizing centers is the cytoplasmic domain of band 3 (cdb3), which links multiple proteins to the membrane. Included among its peripheral protein ligands are ankyrin (the major bridge to the spectrin-actin skeleton), protein 4. 1, protein 4.2, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, deoxyhemoglobin, p72syk protein tyrosine kinase, and hemichromes. The crystal structure of cdb3 is reported at 0.26 nm (2.6 A) resolution. A tight symmetric dimer is formed by cdb3; it is stabilized by interlocked dimerization arms contributed by both monomers. Each subunit also includes a larger peripheral protein binding domain with an alpha(+) beta-fold. The binding sites of several peripheral proteins are localized in the structure, and the nature of the major conformational change that regulates membrane-skeletal interactions is evaluated. An improved structural definition of the protein network at the inner surface of the RBCM is now possible.
The human erythrocyte anion-exchanger isoform 1 (AE1) is a dimeric membrane protein that exchanges chloride for bicarbonate across the erythrocyte plasma membrane. Crystallographic studies suggest that the transmembrane anion channel lies at the interface between the two monomers, whereas kinetic analysis provides evidence that each monomer contains an anion channel. We have studied the structure-function relationship of residues at the dimeric interface of AE1 by cysteine-directed cross-linking. Single cysteine mutations were introduced in 16 positions of putative loop regions throughout AE1. The ability of these residues to be chemically cross-linked to their partner within the dimeric protein complex was assessed by mobility of the protein on immunoblots. Introduced cysteine residues in extracellular loops (ECs) 1-4 and intracellular loop 1 formed disulphide cross-linked dimers. Treatment with homobifunctional maleimide cross-linkers of different lengths (6, 10 and 16 A; 1 A identical with 0.1 nm) also cross-linked AE1 with introduced cysteines in EC5 and close to the start of transmembrane segment (TM) 1. On the basis of these data, tentative positional constraints of TMs 1-4 and 6 relative to the dimeric interface are proposed. Neither disulphide- nor maleimide-mediated cross-linking perturbed AE1 transport function, suggesting that loop-loop contacts across the dimeric interface are not primarily responsible for allosteric interactions between monomers within the functional dimeric protein complex.
The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.
The plasma membrane H(+)-ATPase, Pma1, is an essential and long-lived integral membrane protein. Previous work has demonstrated that the Pma1-D378N mutant is a substrate for endoplasmic reticulum (ER)-associated degradation and causes a dominant negative effect on cell growth by preventing ER export of wild-type Pma1. We now show that Pma1-D378N is ubiquitylated, and it heterooligomerizes with wild-type Pma1, resulting in ubiquitylation and ER-associated degradation of wild-type Pma1. In temperature-sensitive lcb1-100 cells, defective in sphingoid base synthesis, Pma1 fails to oligomerize. At 30 degrees C, lcb1-100 is a suppressor of pma1-D378N because wild-type Pma1 fails to heterooligomerize with Pma1-D378N; wild-type Pma1 moves to the cell surface, indicating that oligomerization is not required for delivery to the plasma membrane. Even in the absence of Pma1-D378N, wild-type Pma1 is ubiquitylated and it undergoes internalization from the cell surface and vacuolar degradation at 30 degrees C in lcb1-100 cells. At 37 degrees C in lcb1-100 cells, a more severe defect occurs in sphingoid base synthesis, and targeting of newly synthesized Pma1 to the plasma membrane is impaired. These data indicate requirements for sphingolipids at three discrete stages: Pma1 oligomerization at the ER, targeting to the plasma membrane, and stability at the cell surface.
The mechanosensitive channel of small conductance (MscS) responds both to stretching of the cell membrane and to membrane depolarization. The crystal structure at 3.9 angstroms resolution demonstrates that Escherichia coli MscS folds as a membrane-spanning heptamer with a large cytoplasmic region. Each subunit contains three transmembrane helices (TM1, -2, and -3), with the TM3 helices lining the pore, while TM1 and TM2, with membrane-embedded arginines, are likely candidates for the tension and voltage sensors. The transmembrane pore, apparently captured in an open state, connects to a large chamber, formed within the cytoplasmic region, that connects to the cytoplasm through openings that may function as molecular filters. Although MscS is likely to be structurally distinct from other ion channels, similarities in gating mechanisms suggest common structural elements.
Human AE1 performs electroneutral exchange of Cl(-) for HCO(3)(-) across the erythrocyte membrane. We examined the topology of the AE1 C-terminal region using cysteine-scanning mutagenesis and sulfhydryl-specific chemistry. Eighty individual cysteine residues, introduced into an otherwise cysteine-less mutant between Phe(806) and Cys(885), were expressed by transient transfection of HEK293 cells. Topology of the region was determined by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent biotin maleimide, with or without prior labeling with the membrane-impermeant reagents, bromotrimethylammoniumbimane bromide (qBBr) and lucifer yellow iodoacetamide (LYIA). Phe(806)-Leu(835), Ser(852)-Ala(855), and Ile(872)-Cys(885) were labeled by biotin maleimide, suggesting their location in an aqueous environment. In contrast, Phe(836)-Lys(851) and Ser(856)-Arg(871) were not labeled by biotin maleimide and therefore localize to the plane of the bilayer, as transmembrane segments (TM). Labeling by qBBr revealed that Pro(815)-Lys(829) and Ser(852)-Ala(855) are accessible to the extracellular medium. Pro(815)-Lys(829) mutants were also labeled with LYIA. Mutants Ile(872)-Cys(885) were inaccessible to the extracellular medium and thus localized to the intracellular surface of AE1. Functional assays revealed that one face of each of two AE1 TMs was sensitive to mutation. Based on these results, we propose a topology model for the C-terminal region of the membrane domain of human AE1.
Tubular acid-base transport regulates systemic acid-base balance. Transepithelial acid-base transport across nephron segments requires the coordinated control of intracellular pH and cellular volume by transporters of protons and bicarbonate. Bicarbonate transporter polypeptides are encoded by at least two gene families, SLC4 and SLC26. The SLC4 gene family includes at least three Na+-independent chloride-bicarbonate exchanger genes and multiple Na+-bicarbonate cotransporter and Na+-dependent anion exchanger genes. The most extensively studied among them are the Na+-independent anion exchangers, AE1, AE2, and AE3, all of which are expressed in kidney. The AE1 gene encodes eAE1 (band 3), the major intrinsic protein of the erythrocyte, as well as kAE1, the basolateral Cl/HCO3 exchanger of the acid-secreting Type A intercalated cell. Mutations in A-E I are responsible for some forms of heritable distal renal tubular acidosis. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by ammonium ion. These properties are not shared by the closely related AE1 anion exchanger. Less is known about AE3 in kidney. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2 which are important in exhibition of the regulatory properties absent from AE1. These include regions of the tran membrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2μ yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0.4–0.7 μmol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated. By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s−1 were observed. The kinetic parameters KMgATPM, Vmax, and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies.
The endogenous Escherichia coli porin OmpF was crystallized as an accidental by-product of our efforts to express, purify, and crystallize the E. coli integral membrane protein KdpD in the presence of foscholine-12 (FC12). FC12 is widely used in membrane protein studies, but no crystal structure of a protein that was both purified and crystallized with this detergent has been reported in the Protein Data Bank. Crystallization screening for KdpD yielded two different crystals of contaminating protein OmpF. Here, we report two OmpF structures, the first membrane protein crystal structures for which extraction, purification, and crystallization were done exclusively with FC12. The first structure was refined in space group P21 with cell parameters a = 136.7 A, b = 210.5 A, c = 137 A, and beta = 100.5 degrees , and the resolution of 3.8 A. The second structure was solved at the resolution of 4.4 A and was refined in the P321 space group, with unit cell parameters a = 215.5 A, b = 215.5 A, c = 137.5 A, and gamma = 120 degrees . Both crystal forms show novel crystal packing, in which the building block is a tetrahedral arrangement of four trimers. Additionally, we discuss the use of FC12 for membrane protein crystallization and structure determination, as well as the problem of the OmpF contamination for membrane proteins overexpressed in E. coli.
The membrane domain of human erythrocyte anion exchanger 1 (AE1) works as a Cl(-)/HCO(3)(-) antiporter. This exchange is a key step for CO(2)/O(2) circulation in the blood. In spite of their importance, structural information about AE1 and the AE (anion exchanger) family are still very limited. We used electron microscopy to solve the three-dimensional structure of the AE1 membrane domain, fixed in an outward-open conformation by cross-linking, at 7.5-A resolution. A dimer of AE1 membrane domains packed in two-dimensional array showed a projection map similar to that of the prokaryotic homolog of the ClC chloride channel, a Cl(-)/H(+) antiporter. In a three-dimensional map, there are V-shaped densities near the center of the dimer and slightly narrower V-shaped clusters at a greater distance from the center of the dimer. These appear to be inserted into the membrane from opposite sides. The structural motifs, two homologous pairs of helices in internal repeats of the ClC transporter (helices B+C and J+K), are well fitted to those AE1 densities after simple domain movement.
Inward-rectifier potassium (K+) channels conduct K+ ions most efficiently in one direction, into the cell. Kir2 channels control the resting membrane voltage in many electrically
excitable cells, and heritable mutations cause periodic paralysis and cardiac arrhythmia. We present the crystal structure
of Kir2.2 from chicken, which, excluding the unstructured amino and carboxyl termini, is 90% identical to human Kir2.2. Crystals
containing rubidium (Rb+), strontium (Sr2+), and europium (Eu3+) reveal binding sites along the ion conduction pathway that are both conductive and inhibitory. The sites correlate with
extensive electrophysiological data and provide a structural basis for understanding rectification. The channel’s extracellular
surface, with large structured turrets and an unusual selectivity filter entryway, might explain the relative insensitivity
of eukaryotic inward rectifiers to toxins. These same surface features also suggest a possible approach to the development
of inhibitory agents specific to each member of the inward-rectifier K+ channel family.
The C-terminal membrane domain of erythrocyte band 3 functions as an anion exchanger. Here, we report the three-dimensional (3D) structure of the membrane domain in an inhibitor-stabilized, outward-open conformation at 18A resolution. Unstained, frozen-hydrated tubular crystals containing the membrane domain of band 3 purified from human red blood cells (hB3MD) were examined using cryo-electron microscopy and iterative helical real-space reconstruction (IHRSR). The 3D image reconstruction of the tubular crystals showed the molecular packing of hB3MD dimers with dimensions of 60 x 110 A in the membrane plane and a thickness of 70A across the membrane. Immunoelectron microscopy and carboxyl-terminal digestion demonstrated that the intracellular surface of hB3MD was exposed on the outer surface of the tubular crystal. A 3D density map revealed that hB3MD consists of at least two subdomains and that the outward-open form is characterized by a large hollow area on the extracellular surface and continuous density on the intracellular surface.
G-protein-coupled receptors (GPCRs) and other structurally and functionally related membrane proteins represent particularly attractive targets for drug discovery. Integral membrane proteins are often difficult to purify from native contexts, and lack of sufficient quantities hampers subsequent structural and functional proteomic studies. We describe here an optimized enrichment strategy involving a membrane protein-compatible 1D4 affinity tag that is derived from the carboxy-terminal nine amino residues of bovine rhodopsin, and its corresponding tag-specific, high-affinity monoclonal antibody. When two GPCRs as well as two related ATP binding cassette (ABC) transporters are expressed in their functional forms in human cell lines, we have shown that a single detergent and wash condition can be employed for the purification of all said membrane proteins. Subsequent in-gel digestion with trypsin and mass spectrometric peptide analysis resulted in high sequence coverage for the ABC transporters ABCA1-1D4 and ABCA4-1D4. In contrast, digestion by various enzymatic combinations was necessary to obtain the best sequence coverage for affinity-enriched GPCRs CXCR4-1D4 and CCR5-1D4 as compared against other entries in an annotated spectrum library. Furthermore, specific enzyme combinations were necessary to produce suitable peptides for deducing N-glycosylation sites on CXCR4. Our results demonstrate that the 1D4-tag enrichment strategy is a versatile tool for the characterization of integral membrane proteins that can be employed for functional proteomic studies.
Voltage-dependent H(+) (Hv) channels mediate proton conduction into and out of cells under the control of membrane voltage. Hv channels are unusual compared to voltage-dependent K(+), Na(+), and Ca(2+) channels in that Hv channel genes encode a voltage sensor domain (VSD) without a pore domain. The H(+) currents observed when Hv channels are expressed heterologously suggest that the VSD itself provides the pathway for proton conduction. In order to exclude the possibility that the Hv channel VSD assembles with an as yet unknown protein in the cell membrane as a requirement for H(+) conduction, we have purified Hv channels to homogeneity and reconstituted them into synthetic lipid liposomes. The Hv channel VSD by itself supports H(+) flux.
A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-beta-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others.
Sulfate efflux was measured in inside-out vesicles obtained from human red cells. Inhibition was observed in vesicles derived from cells pretreated with DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate) or after addition of dipyridamole to the vesicles, both agents being specific and potent inhibitors of anion transport in cells. Trypsinization of the cytoplasmic side of the membrane in order to release a 40 000 dalton fragment from band 3 (the purported anion transport protein) had no effect on sulfate efflux. Further degradation of band 3 to a 17 000 dalton segment, by trypsinization of inside-out vesicles derived from cells that had been pretreated with chymotrypsin, also showed little reduction in transport activity. Furthermore, such vesicles derived from DIDS pretreated cells were inhibited by over 90%. In DIDS-treated cells, the agent is highly localized in band 3. In trypsinized inside-out vesicles, it is largely found in a 55000 fragment and in trypsinized vesicles derived from cells pretreated with chymotrypsin it is largely located in the 17 000 fragment. The data suggest that both the anion transport and inhibitor binding sites are located in a 17 000 transmembrane segment of band 3.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.
This chapter provides protocols for the application of immunofluorescence procedures to yeast. It should perhaps be stressed that immunofluorescence and other light microscopic techniques play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light microscopy. The potential advantages of light microscopic approaches derive from various facts: (1) they can be applied to lightly processed or (in some cases) living cells, (2) Much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) Some structures (for example, the cytoplasmic microtubules) have simply been easier to see by light microscopy than by electron microscopy.
The gene family of anion exchangers consists of at least four or five members, of which three have been characterized at the cDNA level. AE1-3 encode polypeptides that share significant homology with the erythrocyte anion exchanger, band 3 (AE1). Expression of cDNAs encoding these genes in heterologous systems confirms that this sequence similarity is reflected in the capacity to mediate reversible Cl/HCO3 exchange. While the NH2-terminal domain of band 3 is known to interact with several cytoplasmic proteins in erythrocytes, the function of the analogous domains of AE2 and AE3 remains unknown. The AE1 gene is expressed coordinately with other erythroid genes during erythropoiesis in both avian and mammalian erythroid progenitor cells. In addition, AE1 is expressed at the basolateral plasma membrane of the acid-secreting intercalated cells of the kidney. AE2 is expressed in a number of epithelial and nonepithelial cells; it may be expressed in the Golgi apparatus of some of these cells. AE3 is expressed in excitable tissues, including neurons and muscle. It is likely that these proteins play a role in regulation of intracellular pH and chloride in their respective tissue. Understanding of the physiological roles of these proteins, both for ion transport and for plasma membrane organization, remains a central issue.
The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H2DIDS and flufenamate (approx.K
l-values at 30°C: 0.1 and 0.7 μm, respectively), and “right-side-out” orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H2DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
A short procedure for the isolation of band-3 protein, the protein responsible for anion exchange in erythrocytes, in a reasonable degree of purity was developed. Using this protein preparation and a novel procedure for membrane-protein reconstitution, vesicles displaying the basic features of the anion-exchange system of the erythrocyte were obtained. The reconstitution procedure is based on slow direct removal of Triton X-100 from aqueous lipid/detergent solutions. According to the composition of the reconstitution medium, either small single-walled or large multi-walled vesicles are obtained. The procedure conserves protein properties well, as is revealed by the similarity of the rates of SO4(2-) exchange in erythrocytes and reconstituted vesicles when corrected for the relevant volumes. A number of functional features of the exchange system were studied and compared with those of the native membrane.
The electroneutral exchange of chloride and bicarbonate across the human erythrocyte membrane is facilitated by Band 3, a 911 amino acid glycoprotein. The 43 kDa amino-terminal cytosolic domain binds the cytoskeleton, haemoglobin and glycolytic enzymes. The 52 kDa carboxyl-terminal membrane domain mediates anion transport. The protein is a functional dimer, in which the two subunits probably interact with one another by an allosteric mechanism. It is proposed that the link between the mobile cytoplasmic and the membrane-spanning domains of the protein is flexible, based on recent biochemical, biophysical and structural data. This explains the long-standing puzzle that attachment to the cytoskeletal spectrin and actin does not appear to restrict the rotational movement of the Band 3 protein in the erythrocyte membrane. In the Band 3 isoform from the Southeast Asian Ovalocytes (SAO) this link is altered, resulting a tighter attachment of the cytoskeleton to the plasma membrane and a more rigid red blood cell.
The interaction of band 3, the 95-kDa anion-exchange protein of the human erythrocyte membrane, with a variety of nonionic detergents was studied. Band 3 dimers (Stokes radius = 76 A) prepared in octaethylene glycol monododecyl ether (C12E8) could be exchanged into a variety of detergents by size-exclusion high-performance liquid chromatography (HPLC), with complete removal of C12E8 from band 3 being confirmed using radiolabeled detergent. Critical micellar concentration (cmc) values, determined for all detergents in the buffer used for HPLC analysis, ranged from 0.47 microM to 223 mM. Band 3 was found to aggregate in all detergents below their cmc, and concentrations of detergents 2-200 times the cmc were required to prevent aggregation. For detergents with a low cmc, it was important to ensure that the concentration of detergent micelles minimally equalled the concentration of protein. Hydrodynamic measurements and cross-linking studies showed that band 3 remained dimeric in most detergents above their cmc. Furthermore, circular dichroism and inhibitor binding studies supported the view that band 3 can retain its native structure after detergent exchange. Detergents with short alkyl chains (C8) denature band 3, while detergents with longer alkyl chains (C12) maintained the native structure of band 3. The ability to exchange band 3 into a variety of detergents with the maintenance of native structure is an essential prerequisite for crystallization trials. The results obtained in this study of band 3, a model polytopic (multispanning) membrane protein, may be generally applicable to other membrane proteins.
Tubular acid-base transport regulates systemic acid-base balance. Transepithelial acid-base transport across nephron segments requires the coordinated control of intracellular pH and cellular volume by transporters of protons and bicarbonate. Bicarbonate transporter polypeptides are encoded by at least two gene families, SLC4 and SLC26. The SLC4 gene family includes at least three Na()+)-independent chloride-bicarbonate exchanger genes and multiple Na(+)-bicarbonate cotransporter and Na(+)-dependent anion exchanger genes. The most extensively studied among them are the Na(+)-independent anion exchangers, AE1, AE2, and AE3, all of which are expressed in kidney. The AE1 gene encodes eAE1 (band 3), the major intrinsic protein of the erythrocyte, as well as kAE1, the basolateral Cl/HCO3 exchanger of the acid-secreting Type A intercalated cell. Mutations in AE1 are responsible for some forms of heritable distal renal tubular acidosis. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by ammonium ion. These properties are not shared by the closely related AE1 anion exchanger. Less is known about AE3 in kidney. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2 which are important in exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.
Three-dimensional crystals were obtained for the membrane domain of the human erythrocyte anion exchanger (AE1, Band 3). Protein homogeneity and stability and the delicate balance between the detergent used and the amount of phospholipids copurifying are critical to the formation of three-dimensional crystals of the AE1 membrane domain. While deglycosylation improved the protein homogeneity, its stability was significantly increased by inhibitor binding. Size-exclusion chromatography showed that the protein was monodisperse in detergents with acyl chains of 10-12 carbons over a pH range of 5.5-10.0. This pH range and the detergents that retained the protein's monodispersity were used for crystallization screening. Crystals were obtained with the protein purified in C(12)E(8), dodecylmaltoside, decylthiomaltoside, and cyclohexyl-hexylmaltoside. Five to 13 lipid molecules per protein were required for the protein crystal formation. Those crystals grown in dodecylmaltoside diffracted X-rays to 14 A. With these factors taken into consideration, ways to further improve the crystal quality are suggested.
We report here the synthesis and characterization of two gene constructs designed to facilitate structure/function studies of the human neuronal cannabinoid receptor, CB1. The first gene, which we call shCB1, is a synthetic gene containing unique restriction sites spaced roughly 50-100 bases apart to facilitate rapid mutagenesis and cloning. A nine amino acid epitope tag (from the rhodopsin C-terminus) is also present in the shCB1 C-terminal tail to enable detection and purification using the monoclonal antibody 1D4. We find that that the shCB1 gene can be transiently expressed in COS cells with yield of approximately 10-15 micro g receptor per 15 cm plate and is wild type like in its ability to bind cannabinoid ligands. Our confocal microscopy studies indicate shCB1 targets to the membrane of HEK293 cells and is internalized in response to agonist. To facilitate functional studies, we also made a chimera in which the C-terminus of shCB1 was fused with the N-terminus of a G-protein alpha subunit, Galphai. The shCB1/Galphai chimera shows agonist stimulated GTPgammaS binding, and thus provides a simplified way to measure agonist induced CB1 activation. Taken together, the shCB1 and shCB1/Galphai gene constructs provide useful tools for biochemical and biophysical examinations of CB1 structure, activation and attenuation.