Chicken erythroid AE1 anion exchangers associate with the cytoskeleton during recycling to the Golgi.
ABSTRACT: Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.
Project description:The AE1 gene encodes band 3 Cl-/HCO3- exchangers that are expressed both in the erythrocyte and in the acid-secreting, type A intercalated cells of the kidney. Kidney AE1 contributes to urinary acidification by providing the major exit route for HCO3- across the basolateral membrane. Several AE1 mutations cosegregate with dominantly transmitted nonsyndromic renal tubular acidosis (dRTA). However, the modest degree of in vitro hypofunction exhibited by these dRTA-associated mutations fails to explain the disease phenotype in light of the normal urinary acidification associated with the complete loss-of-function exhibited by AE1 mutations linked to dominant spherocytosis. We report here novel AE1 mutations linked to a recessive syndrome of dRTA and hemolytic anemia in which red cell anion transport is normal. Both affected individuals were triply homozygous for two benign mutations M31T and K56E and for the loss-of-function mutation, G701D. AE1 G701D loss-of-function was accompanied by impaired trafficking to the Xenopus oocyte surface. Coexpression with AE1 G701D of the erythroid AE1 chaperonin, glycophorin A, rescued both AE1-mediated Cl- transport and AE1 surface expression in oocytes. The genetic and functional data both suggest that the homozygous AE1 G701D mutation causes recessively transmitted dRTA in this kindred with apparently normal erythroid anion transport.
Project description:Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO3- and Cl- exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl-/HCO3- exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO3- and outward Cl- gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO3- for stomatin-deficient patients. Similarly, kinetics of Cl- efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.
Project description:Autosomal dominant distal renal tubular acidosis (dRTA) has been associated with several mutations in the anion exchanger AE1 gene. The effect of an 11-amino-acid C-terminal dRTA truncation mutation (901 stop) on the expression of kidney AE1 (kAE1) and erythroid AE1 was examined in transiently transfected HEK-293 cells. Unlike the wild-type proteins, kAE1 901 stop and AE1 901 stop mutants exhibited impaired trafficking from the endoplasmic reticulum to the plasma membrane as determined by immunolocalization, cell-surface biotinylation, oligosaccharide processing and pulse-chase experiments. The 901 stop mutants were able to bind to an inhibitor affinity resin, suggesting that these mutant membrane proteins were not grossly misfolded. Co-expression of wild-type and mutant kAE1 or AE1 resulted in intracellular retention of the wild-type proteins in a pre-medial Golgi compartment. This dominant negative effect was due to hetero-oligomer formation of the mutant and wild-type proteins. Intracellular retention of kAE1 in the alpha-intercalated cells of the kidney would account for the impaired acid secretion into the urine characteristic of dRTA.
Project description:AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.
Project description:The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na(+) and K(+) conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracil-proton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3-5 and TM8 that should function as an anion exchanger and a cation leak.
Project description:The Anion Cl-/HCO3- Exchangers AE1, AE2, and AE3 are membrane pH regulatory ion transporters ubiquitously expressed in vertebrate tissues. Besides relieving intracellular alkaline and CO2 loads, the AEs have an important function during development and cell death and play a central role in such cellular properties as cell shape, metabolism, and contractility. The activity of AE(s) are regulated by neurohormones. However, little is known as to the intracellular signal transduction pathways that underlie this modulation. We show here that, in cardiomyocytes that express both AE1 and AE3, the purinergic agonist, ATP, triggers activation of anion exchange. The AE activation is observed in cells in which AE3 expression was blocked but not in cells microinjected with neutralizing anti-AE1 antibodies. ATP induces tyrosine phosphorylation of AE1, activation of the tyrosine kinase Fyn, and association of both Fyn and FAK with AE1. Inhibition of Src family kinases in vivo by genistein, herbimycin A, or ST638 prevents purinergic activation of AE1. Microinjection of either anti-Cst.1 antibody or recombinant CSK, both of which prevent activation of Src family kinase, significantly decreases ATP-induced activation of AE. Microinjection of an anti-FAK antibody as well as expression in cardiomyocytes of Phe397 FAK dominant negative mutant, also prevents purinergic activation of AE. Therefore, tyrosine kinases play a key role in acute regulation of intracellular pH and thus in cell function including excitation-contraction coupling of the myocardium.
Project description:Anion exchanger 1 (AE1) is responsible for the exchange of bicarbonate and chloride across the erythrocyte plasma membrane. Human AE1 consists of a cytoplasmic and a membrane domain joined by a 33-residue flexible linker. Crystal structures of the individual domains have been determined, but the intact AE1 structure remains elusive. In this study, we use molecular dynamics simulations and modeling to build intact AE1 structures in a complex lipid bilayer that resembles the native erythrocyte plasma membrane. AE1 models were evaluated using available experimental data to provide an atomistic view of the interaction and dynamics of the cytoplasmic domain, the membrane domain, and the connecting linker in a complete model of AE1 in a lipid bilayer. Anionic lipids were found to interact strongly with AE1 at specific amino acid residues that are linked to diseases and blood group antigens. Cholesterol was found in the dimeric interface of AE1, suggesting that it may regulate subunit interactions and anion transport.
Project description:All affected patients in four families with autosomal dominant familial renal tubular acidosis (dRTA) were heterozygous for mutations in their red cell HCO3-/Cl- exchanger, band 3 (AE1, SLC4A1) genes, and these mutations were not found in any of the nine normal family members studied. The mutation Arg589--> His was present in two families, while Arg589--> Cys and Ser613--> Phe changes were found in the other families. Linkage studies confirmed the co-segregation of the disease with a genetic marker close to AE1. The affected individuals with the Arg589 mutations had reduced red cell sulfate transport and altered glycosylation of the red cell band 3 N-glycan chain. The red cells of individuals with the Ser613--> Phe mutation had markedly increased red cell sulfate transport but almost normal red cell iodide transport. The erythroid and kidney isoforms of the mutant band 3 proteins were expressed in Xenopus oocytes and all showed significant chloride transport activity. We conclude that dominantly inherited dRTA is associated with mutations in band 3; but both the disease and its autosomal dominant inheritance are not related simply to the anion transport activity of the mutant proteins.
Project description:We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na(+) efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na(+) efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl(-) transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl(-) transport activity, but R730 substitution with I, E, or H inactivated Cl(-) transport. AE1 R730C expression substantially increased endogenous oocyte Na(+)-K(+)-ATPase-mediated (86)Rb(+) influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pH(o)) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pH(o)-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pH(o) insensitive. MTSES-treated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl(-) influx by acid pH(o). Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb(+) and Li(+) influxes. Selective rescue of acid pH(o)-stimulated sulfate uptake and conferral of acid pH(o)-stimulated Cl(-) influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and regulation of anion transport by AE1.
Project description:Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the ?-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1.