Functional activation of plasma membrane anion exchangers occurs in a pre-Golgi compartment

Folding and oligomerization of most plasma membrane glycoproteins, including those involved in ion transport, occur in the ER and are frequently required for their exit from this organelle. It is currently unknown, however, where or when in the biosynthetic pathway these proteins become functionally active. AE1 and AE2 are tissue-specific, plasma membrane anion transport proteins. Transient expression of AE2 in a eukaryotic cell line leads to an increase in stilbene inhibitable whole cell 35SO4(2-)-efflux consistent with its function as a plasma membrane anion exchanger. No such increased transport activity was observed in AE1 transfectants, despite the fact that the two proteins were synthesized in roughly equal portions. In contrast, both AE1 and AE2 expression resulted in significant increase in Cl-/SO4(2-)-exchange in crude microsomes demonstrating that both AE1 and AE2 cDNAs encode functional proteins. Immunofluorescence staining and pulse-chase labeling experiments revealed that while 60% of AE2 is processed to the cell surface of transfectants, AE1 is restricted to an intracellular compartment and never acquires mature oligosaccharides. Crude microsomes from transfected cells were fractionated into plasma membrane and ER-derived vesicles by con A affinity chromatography. All of the AE1 and approximately half of the cellular AE2 was eluted with the ER vesicles, confirming their intracellular localization. Anion transport measurements on these fractions confirmed that the ER- restricted anion exchangers were functional. We conclude that AE1 and AE2 acquire the ability to mediate anion exchange at an early stage of their biosynthesis, before their exit from the ER.     

Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain

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.     

Expression and characterization of the anion transporter homologue YNL275w in Saccharomyces cerevisiae

A search of the yeast Saccharomyces cerevisiae genome has revealed an open reading frame, YNL275w, which encodes a 576-amino acid protein that shows sequence similarity to the family of mammalian Cl-/HCO3- anion exchangers and Na+/HCO3- cotransporters. This yeast protein also has a very similar hydropathy profile to the mammalian HCO3- transporters, indicating a similar membrane topology and structure. A V5 epitope and His6-tagged version of Ynl275wp was expressed in yeast and was localized to the plasma membrane by subcellular fractionation and immunofluorescence labeling. The protein was purified by nickel affinity chromatography and was found not to be N-glycosylated. The protein's mobility on SDS-PAGE gels was not altered by treatment with N-glycanase F, alpha-mannosidase, or by mutation of each of the five consensus N-glycosylation sites. The protein did not bind to concanavalin A by lectin blotting or lectin affinity chromatography. The expressed protein bound specifically to a stilbene disulfonate inhibitor resin (SITS-Affi-Gel), and this binding could be competed by certain anions (HCO3-, Cl-, NO3-, and I-) but not by others (SO4(2-) and PO4(3-)). These results suggest that the yeast gene YNL275w encodes a nonglycosylated anion transport protein, localized to the plasma membrane.     

Purification and characterization of the recombinant human prostaglandin H synthase-2 expressed in Pichia pastoris

Prostaglandin H synthase-1 and -2 (PGHS-1 and PGHS-2, EC 1.14.99.1) are membrane associated glycoproteins that catalyze the first two steps in prostaglandin synthesis. As the enzymes play an important regulatory role in several physiological and pathophysiological processes, recombinant PGHS isoforms are widely used in biomedical research. In the present study, we expressed human PGHS-2 (hPGHS-2) with and without a six histidine sequence tag (His(6) tag) near the amino- or carboxy-terminus of the protein in the Pichia pastoris (P. pastoris) expression system using native or yeast signal sequences. The recombinant His(6) tagged hPGHS-2 was purified using Ni-affinity and anion exchange chromatography, whereas the purification of the C-terminally His(6) tagged hPGHS-2 was more efficient. K(m), k(cat) and IC(50) values were determined to characterize the protein. The data obtained indicate that both the N- and C-terminally His(6) tagged hPGHS-2 are functional and the catalytic properties of the recombinant protein and the enzyme produced in other expression systems are comparable. As the yeast culture is easy to handle, the P. pastoris system could serve as an alternative to the most commonly used baculovirus-insect cell expression system for the production of the recombinant PGHS-2.     

Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris

A DNA fragment containing the mature human interleukin (IL)-6 sequence was cloned into pPICZαA, generating a fusion protein with the alpha factor from baker's yeast and integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level rhIL-6 production were identified, secreting as much as 280 mg L(-1) rhIL-6 after 4 days of induction by methanol. The rhIL-6 was purified by PEG-8000 precipitation, followed by DEAE anion exchange and Sephadex G-75 gel filtration, yielding over 95% pure rhIL-6 at about 170 mg L(-1) . Mass spectrometry analysis showed that the rhIL-6 has a molecular weight of 20,908.85 Da, which is close to the mass calculated from the sequence of the protein. Functional analysis of the purified rhIL-6 using the lymphocyte proliferation assay by an MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazoliumbromide] method demonstrated a specific activity that is at least fivefold higher than the commercial rhIL-6 produced in Escherichia coli. In summary, the experimental procedure we have reported here allows us to obtain a large amount of rhIL-6 from P. pastoris suitable for subsequent biophysical studies.     

In situ localization of anion exchanger-2 in the human kidney

Na+-independent anion exchangers (AE) are a family of membrane carriers that mediate the electroneutral exchange of Cl- for HCO3- ions across plasma membranes. They are involved in intracellular pH and cell volume regulation as well as in transepithelial acid-base transport. While anion exchanger-1 (AE1) has been localized previously in the human kidney, thus far there has been no definite report on anion exchanger-2 (AE2) in this human tissue. Accordingly, immunohistochemistry was carried out on surgical specimens of the human kidney (fixed in formalin and embedded in paraffin), using a specific AE2 monoclonal antibody. Strong immunostaining was observed at the basolateral membrane of cells of thick ascending limbs and distal convoluted tubules, colocalizing with the basal membranous labyrinth of cellular interdigitations, typical of these segments. In fact, AE2 staining was attenuated at the macula densa, where basal infoldings are scarce. Additionally, in situ hybridization experiments on formalin-fixed tissue demonstrated the presence of AE2 mRNA in the same segments of the distal nephron. On the other hand, control immunohistochemistry with a monoclonal antibody against AE1 gave the expected immunoreactivity at the basal pole of the type A intercalated cells of connecting tubules and cortical collecting ducts, and in erythrocytes. Our results indicate that, depending on the nephron segment and corresponding cell types, AE1 and AE2 proteins are differentially involved in the Na+-independent exchange of Cl- for HCO3- at the basolateral membrane of polarized kidney epithelial cells.     

Anion exchangers in the red cell and beyond.

The anion exchanger genes (AE1-3) encode a family of transport proteins responsible for the electroneutral exchange of bicarbonate and chloride across membranes. These transporters are important in processes such as pH regulation and bicarbonate metabolism. This article reviews recent progress in this field based on presentations made at a satellite workshop on anion exchangers held in conjunction with the 8th Fisher Winternational Symposium on Cellular and Molecular Biology entitled "Membrane proteins in health and disease." The transmembrane topology of AE1 has been refined using various combinations of protein chemistry and site-directed mutagenesis. The use of specific inhibitors and novel expression systems continues to reveal fundamental features of the anion exchanger mechanism and its regulation. The importance of anion exchangers in blood and kidney diseases is underscored by the identification and characterization of a plethora of novel mutations in the AE1 gene. Investigations of anion exchanger structure and function have moved beyond studies of the red cell protein into the larger arenas of cellular and molecular biology.     

Determination of structural models of the complex between the cytoplasmic domain of erythrocyte band 3 and ankyrin-R repeats 13-24

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.     

Identification of the binding interface involved in linkage of cytoskeletal protein 4.1 to the erythrocyte anion exchanger.

Linkages of the cytoskeleton to integral membrane proteins of the plasma membrane have been shown to be important for diverse cellular functions. The erythrocyte membrane provides the best studied example of how the spectrin-actin based membrane cytoskeleton is linked via two proteins, ankyrin and protein 4.1, to the anion exchanger (anion exchanger 1, AE1). Although these and other types of cytoskeleton-membrane connections have been well documented by in vitro binding studies it has not been possible to establish any of such interactions by defining the binding interface at the amino acid level. In the present study we have performed binding studies between protein 4.1 and AE1 using peptides and corresponding idiotypic and anti-idiotypic antibodies to show that arginine-rich clusters of the cytoplasmic domain of AE1 (IRRRY/LRRRY) serve as a major binding site for a motif with opposite charge and identical hydrophobicity present on the membrane-binding domain of protein 4.1 (LEEDY). Both motifs appear to be highly conserved during evolution and may also be involved in other types of cytoskeleton-membrane association, i.e. in binding of protein 4.1 to the glycophorins.     

Topological and segmental phylogenetic analyses of the anion exchanger (band 3) family of transporters

Eleven sequenced anion exchanger (AE; band 3) proteins, including five AE1, four AE2 and two AE3 proteins, comprise the anion exchanger family (AEF) of homologous proteins. Eliminating the rat and rabbit proteins that are nearly Identical to the corresponding mouse proteins, seven dissimilar members of this family were selected for study, divided into N-terminal, central and C-terminal segments (designated segments 0, 1 and 2, respectively) and analysed separately for sequence similarity and phylogenetic relatedness. Segments 0 are variable in length and sequence, are essentially lacking in some of the members of the AEF, and are not demonstrably homologous in other members of the family. All segments 1 and 2 are homologous, but they exhibit widely differing degrees of sequence divergence. Segments 2 are highly conserved in all AEF proteins. Segments 1 of the AE2 and AE3 proteins are as conserved as are segments 2, but segments 1 of the AE1 proteins have diverged from each other and from the AE2 and AE3 segments 1 much more than have segments 2 of these same proteins.

The distributions of various types of amino acid residues in the putative transmembrane helical spanners of the seven dissimilar members of the AEF, based on a modification of the 14-spanner model of Wang et al. (1994) was determined, and this distribution was compared with those of other transmembrane transport proteins of known structure (bacterial rhodopsins, outer membrane porins of Gram-negative bacteria and bacterial photosynthetic reaction centres). Anion exchangers exhibit a predominance of aromatic residues (F, W, Y) at the ends of the putative spanners and of aliphatic residues (L, I, V, M) in the centres of these spanners. This feature was also a characteristic of bacteriorhodopsins (of λ- structure) and of bacterial porins (of β-structure) but not of photosynthetic reaction centres (of a-structure). Almost all membrane-embedded charged residues in the AEF proteins occur in odd-numbered spanners, a unique characteristic of anion exchangers.     

A comparative study of the human T-cell leukemia virus type 2 integrase expressed in and purified from Escherichia coli and Pichia pastoris

The human T-cell leukemia virus type-2 (HTLV-2) integrase (IN) catalyzes the insertion of the viral genome into the host chromosome. HTLV-2 IN was expressed as an N-terminal hexa-histidine tagged protein in the methylotrophic yeast Pichia pastoris and as a C-terminal hexa-histidine fusion in Escherichia coli. Maximal IN expression was observed at 48h post-induction for the yeast system and 2h post-induction for E. coli. Effective purification strategies were developed using non-ionic and zwitterionic detergents for initial protein extraction, followed by a one-step nickel-chelating chromatography purification. IN from both sources was routinely greater than 90% pure with yields exceeding 1.5mg of purified IN per liter of culture for P. pastoris. The relative pI was defined for both INs, pH 5.0-5.4, by 2D-gel electrophoresis. Specific activities for IN purified from E. coli and P. pastoris were calculated from in vitro 3(') processing assays and were comparable. In vitro IN assays were also performed to optimize reaction buffer pH and metal concentrations for both 3(') processing and strand transfer assays. Strand transfer was optimal from pH 6.2-6.8, more than 1.5 pH units below the optimal 3(') processing pH of 8.3. IN from both sources showed no enhancement in activity with MnCl(2) concentrations greater than 5mM. The specific activity of P. pastoris purified IN was 0.35 product (pmol)/h/microg IN, and E. coli produced IN was 0.48 product (pmol)/h/microg IN.     

Targeting of the AE2 anion exchanger to the Golgi apparatus is cell type-dependent and correlates with the expression of Ank(195), a Golgi membrane skeletal protein

Sodium-independent anion exchangers (AE1-4) show remarkable variability in their tissue-specific expression and subcellular localization. Currently, isoform-specific targeting mechanisms are considered to be responsible for this variable localization. Here, we report that targeting can also be cell type-specific. We show that the full-length AE2 protein and its green fluorescent protein- or DsRed-tagged variants localize predominantly either to the Golgi apparatus in COS-7 cells, or to the plasma membrane in HeLa cells. This alternative targeting did not seem to result from either translational or post-translational differences, but rather from differential expression of at least one of the Golgi membrane skeletal proteins, ankyrin(195) (Ank(195)), between the two cell types. Comparative studies with several different cell lines revealed that the Golgi localization of the AE2 protein correlated strictly with the expression of Ank(195) in the cells. The two Golgi-associated proteins also co-localized well and similarly resisted detergent extraction in the cold, whereas the plasma membrane-localized AE2 in Ank(195)-deficient cells was mostly detergent-soluble. Collectively, our results suggest that Ank(195) expression is a key determinant for the variable and cell type-dependent localization of the AE2 protein in the Golgi apparatus in mammalian cells.     

Identification of the full-length AE2 (AE2a) isoform as the Golgi-associated anion exchanger in fibroblasts.

Na(+)-independent Cl(-)/HCO(3)(-) exchangers (AE1, AE2, AE3) are generally known as ubiquitous, multispanning plasma membrane proteins that regulate intracellular pH and transepithelial acid-base balance in animal tissues. However, previous immunological evidence has suggested that anion exchanger (AE) proteins may also be present in intracellular membranes, including membranes of the Golgi complex and mitochondria. Here we provide several lines of evidence to show that an AE protein is indeed a resident of the Golgi membranes and that this protein corresponds to the full-length AE2a isoform in fibroblasts. First, both the N- and C-terminal antibodies to AE2 (but not to AE1) detected an AE protein in the Golgi membranes. Golgi localization of this AE2 antigen was evident also in cycloheximide-treated cells, indicating that it is a true Golgi-resident protein. Second, our Northern blotting and RT-PCR analyses demonstrated the presence of only the full-length AE2a mRNA in cells that show prominent Golgi staining with antibodies to AE2. Third, antisense oligonucleotides directed against the translational initiation site of the AE2a mRNA markedly inhibited the expression of the endogenous AE2 protein in the Golgi. Finally, transient expression of the GFP-tagged full-length AE2a protein resulted in predominant accumulation of the fusion protein in the Golgi membranes in COS-7 and CHO-K1 cells. Golgi localization of the AE2a probably involves its oligomerization and/or association with the recently identified Golgi membrane skeleton, because a substantial portion of both the endogenous AE2a and the GFP-tagged fusion protein resisted detergent extraction in cold. (J Histochem Cytochem 49:259-269, 2001)     

Purification, partial sequencing and characterization of an insect membrane dipeptidyl aminopeptidase that degrades the insect neuropeptide proctolin.

Two proctolin-binding proteins solubilized from 1600 cockroach hindgut membranes were purified 1000-fold using five chromatography steps. Twenty-five micrograms of protein were recovered from the final size-exclusion chromatography as a single peak eluting at 74 kDa, whereas two major bands at 80 and 76 kDa were identified after silver staining of electrophoresis gels. The fragments, sequenced by tandem mass spectrometry and the Edman method, revealed a high homology with rat liver dipeptidyl aminopeptidase (DPP) III and a significant homology between the cockroach-purified proteins. From analysis of the Drosophila genome sequence database, it was possible to identify a putative DPP sharing high homology with the sequences obtained from the cockroach purified proteins and with the rat DPP III. Anti-(rat liver DPP III) Ig reacted specifically with both cockroach-purified proteins in Western blot analysis. The purified proteins removed the N-terminal dipeptide from the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) with a Km value of 3.8 +/- 1.1 microM. The specific DPP III inhibitor tynorphin prevented the degradation of proctolin by the purified insect DPP (IC50 = 0.68 microM). These results provide strong evidence that the cockroach-purified proteins represent an insect membrane DPP, presumably present in Drosophila, and that it is closely related to vertebrate DPP III.     

Identification, cloning, and expression of the Scytalidium acidophilum XYL1 gene encoding for an acidophilic xylanase

We cloned XYL1, a Scytalidium acidophilum gene encoding for an acidophilic family 11 xylanase. The XYL1p protein was expressed in Pichia pastoris using the pPICZalphaA expression plasmid. The secreted protein was purified by TAXI affinity column chromatography. The purified XYL1p showed an optimum activity at pH 3.2 and 56 degrees C. The Michaelis-Menten constants were determined.     

Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics

We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.