An N-terminal GFP tag does not alter the functional expression to the plasma membrane of red cell and kidney anion exchanger (AE1) in mammalian cells.
Two isoforms of the band 3 anion exchanger are expressed in mammalian cells, a 911 residue protein (B3) in red cells, and a truncated protein (KB3) in the alpha-intercalated cells of the kidney. Mutants of both isoforms are known to be associated with human disease, and mistargeting of the mutated proteins has been suggested as the mechanism of pathogenesis in several cases but has been difficult to prove. The present study demonstrates the feasibility of using confocal microscopy for investigating the targeting of homozygous and heterozygous B3 and KB3 mutants. K562 erythroleukemia cells offer several advantages for the stable expression of B3, but have not previously been used for its visualization. A wide range of cell attachment factors, growth conditions, fixation reagents and primary antibodies were investigated to enable imaging of B3 and endogenous GPA by immunofluorescence confocal microscopy in stable K562/B3 clones. B3 co-localized with GPA at the cell surface and also in an intracellular compartment. Functional cell surface expression of KB3 in stable K562 clones was also obtained. Importantly, both B3 and KB3 could be expressed as stable fusion proteins tagged with green fluorescent protein (GFP) in K562 cells, and it was demonstrated that N-terminal GFP-tagging does not affect the targeting or chloride transport properties of B3 or KB3. The use of GFP as well as double-labelling methods developed for immunostaining will be invaluable for investigating the interactions of band 3 with itself and other proteins during its trafficking in erythroid and kidney cells. This will help elucidate how band 3 mutations can cause human diseases such as hereditary spherocytosis and distal renal tubular acidosis.
Beckmann
Roland
R
Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK. rb311@cam.ac.uk
Toye
Ashley M
AM
Smythe
Jonathan S
JS
Anstee
David J
DJ
Tanner
Michael J A
MJ
D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000906:Antibodies / Q000378:metabolism; D001692:Biological Transport; D002712:Chlorides / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation; D006021:Glycophorins / Q000032:analysis / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents / Q000737:chemistry; D020014:K562 Cells / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods
D001457:Anion Exchange Protein 1, Erythrocyte; D000906:Antibodies; D002712:Chlorides; D006021:Glycophorins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins