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)
Holappa
K
K
Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland. kholappa@cc.oulu.fi
Suokas
M
M
Soininen
P
P
Kellokumpu
S
S
D000818:Animals; D027321:Anion Transport Proteins; D017920:Antiporters; D015152:Blotting, Northern; D002460:Cell Line; D006224:Cricetinae; D003902:Detergents; D005347:Fibroblasts / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D017830:Octoxynol; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D020033:Protein Isoforms / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D064606:SLC4A Proteins
D027321:Anion Transport Proteins; D017920:Antiporters; D003902:Detergents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D064606:SLC4A Proteins; D049452:Green Fluorescent Proteins; D017830:Octoxynol