Identification of the Full-length AE2 (AE2a) Isoform as the Golgi-associated Anion Exchanger in Fibroblasts

Abstract

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)

Ionic determinants of pH of acidic compartments under hypertonic conditions in trout hepatocytes

“…pH of acid compartments combine with translocated H + , preventing the formation of a large chemical gradient. Apart from this study, and while anion exchangers have already been detected in acidic compartments (Holappa et al, 2001;Holappa and Kellokumpu, 2003), evidence for a contribution of HCO 3 -to organelle pH regulation is lacking (Paroutis et al, 2004). Actually, in the present study, removal of extracellular HCO 3 -appeared to have a slight effect on steady-state pH L while no effect on the hypertonicity-induced acidification or the subsequent restoration of isotonicity was seen.…”

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra‐Golgi pH

“…This interaction is essential for the maintenance of the characteristic flattened shape of the cell and its mechanical stability [Marchesi, 1985;Nishi and Forgac, 2002]. Hence, in the Golgi complex, the potential ankyrin-interacting Golgi membrane protein anion exchanger 2 (AE2) [Holappa et al, 2001;Holappa and Kellokumpu, 2003] could provide a functional membrane anchorage site for the Golgi-associated spectrin/ ankyrin/actin cytoskeleton [De Matteis et al, 2000;Valderrama et al, 2000]. The structural role of the AE2 protein in the Golgi complex would be linked to its function (the reversible electroneutral exchange of Cl À for HCO 3 À ), thus contributing to the regulation of intracellular pH, organelle volume, and chloride concentration.…”

Localization and function of the anion exchanger Ae2 in developing teeth and orofacial bone in rodents

“…Endogenous peroxidase was blocked by incubation in 3% H 2 O 2 in water for 3 min at room temperature. Next, non-specific binding sites were blocked by incubating sections with normal goat serum (30%) followed by overnight incubation at 4 °C with affinity purified primary polyclonal rabbit antibodies raised against a synthetic peptide, identical to the 12 C-terminal amino acids of the Ae2a protein as described by Holappa et al (2001). These antibodies can recognise all five Ae2 isotypes (Ae2a, Ae2b1, Ae2b2 and Ae2c1 and Aec2) as well as erythrocyte Ae1 (9 of the 12 C- terminal amino acid residues are identical to Ae2).…”