A Transport Metabolon

Abstract

The cytoplasmic carboxyl-terminal domain of AE1, the plasma membrane chloride/bicarbonate exchanger of erythrocytes, contains a binding site for carbonic anhydrase II (CAII). To examine the physiological role of the AE1/CAII interaction, anion exchange activity of transfected HEK293 cells was monitored by following the changes in intracellular pH associated with AE1-mediated bicarbonate transport. AE1-mediated chloride/bicarbonate exchange was reduced 50 -60% by inhibition of endogenous carbonic anhydrase with acetazolamide, which indicates that CAII activity is required for full anion transport activity. AE1 mutants, unable to bind CAII, had significantly lower transport activity than wild-type AE1 (10% of wild-type activity), suggesting that a direct interaction was required. To determine the effect of displacement of endogenous wild-type CAII from its binding site on AE1, AE1-transfected HEK293 cells were co-transfected with cDNA for a functionally inactive CAII mutant, V143Y. AE1 activity was maximally inhibited 61 ؎ 4% in the presence of V143Y CAII. A similar effect of V143Y CAII was found for AE2 and AE3cardiac anion exchanger isoforms. We conclude that the binding of CAII to the AE1 carboxyl-terminus potentiates anion transport activity and allows for maximal transport. The interaction of CAII with AE1 forms a transport metabolon, a membrane protein complex involved in regulation of bicarbonate metabolism and transport.Carbon dioxide, the metabolic end product of oxidative respiration, must be effectively cleared from the human body. CO 2 diffuses out of cells into the blood stream and into erythrocytes, where it is hydrated by cytosolic carbonic anhydrase (CA). 1 The resulting membrane-impermeant HCO 3 Ϫ is exported into the plasma by the plasma membrane Cl Ϫ /HCO 3 Ϫ anion exchanger (AE1), thus increasing the blood capacity for carrying CO 2 . Upon returning to the lungs the process is reversed; HCO 3 Ϫ is transported into the erythrocyte in exchange for Cl Ϫ by AE1 and dehydrated by CA, and the resulting CO 2 diffuses across the erythrocyte and alveolar membranes to be expired from the body. The 5 ϫ 10 4 s Ϫ1 turnover rate of AE1 (1) and the high content of AE1 in the membrane (2) facilitate completion of bicarbonate transport within 50 ms during passage of an erythrocyte through a capillary (3).AE1 is a 911-amino acid polytopic glycoprotein that facilitates the one for one electroneutral exchange of Cl Ϫ for HCO 3

Role of tubular secretion and carbonic anhydrase in vertebrate renal sulfate excretion

“…In addition to CAII acceleration of transport activity, the COOH-terminal motif of AE1 (LDADD) increases CAII activity (81). Numerous other ion transporters, including Pendrin (SLC26A4), a member of the mammalian SLC26 gene family, possess potential binding sites for CAII (85). Figure 4 shows that the SO 4 2Ϫ transporters SLC26A1, SLC26A2, SLC26A7, and SLC26A11, which are expressed in the mammalian kidney, also possess potential CAII binding sites at their COOH terminals.…”

“…For anion exchange, this association may effectively create a functional asymmetry, which favors the transport of a particular substrate (HCO 3 Ϫ or OH Ϫ ) in one direction. The association of mammalian CAII and AE1 occurs between a histidine-rich region in the NH 2 terminal of CAII and a hydrophobic residue followed by 2-3 acidic residues in the COOH terminal of AE1 (85,90,91). In addition to CAII acceleration of transport activity, the COOH-terminal motif of AE1 (LDADD) increases CAII activity (81).…”

“…The complex that is formed from the association of CA with a transport protein has been termed a "transport metabolon" (85). A metabolon is defined as a series of physically associated enzymes involved in a coupled metabolic pathway that allows metabolites to move efficiently between active sites by limiting the loss of intermediates to diffusion (84,85). Even for enzymes that approach "kinetic perfection," such as CA, the rate-limiting step in enzymatic catalysis is diffusion (19).…”

“…An allosteric effect can be ruled out because coexpression of AE1 (85) and NBC-1 (1) with an inactive CAII mutant (V143Y), which retains binding affinity, inhibits transport activity. The complex that is formed from the association of CA with a transport protein has been termed a "transport metabolon" (85). A metabolon is defined as a series of physically associated enzymes involved in a coupled metabolic pathway that allows metabolites to move efficiently between active sites by limiting the loss of intermediates to diffusion (84,85).…”

A thiol-mediated active membrane transport of selenium by erythroid anion exchanger 1 protein

“…The MSD mediates the anion exchange, while the C-CPD contains acidic motifs that constitute a core binding site for cytoplasmic carbonic anhydrase II 9 and is required for the Cl -/HCO 3 -exchange process. 23,24 The extreme sequence of the N-CPD binds multiple glycolytic enzymes 25 The AE1 contains five cysteine residues: Cys201 and Cys317 in the N-CPD, Cys479 and inhibition of the selenium export from red cells. 40 In the present study, we investigated the selenium transport mechanism across red cell membranes from Hb to the plasma and proposed a possible mechanism for a thiol-mediated membrane transport of selenium by the erythroid AE1 protein.…”