We examined the assembly of the membrane domain of the human red cell anion transporter (band 3; AE1) by co-expression of recombinant N- and C-terminal fragments in Xenopus oocytes and in cell-free translation with canine pancreatic microsomes. Co-immunoprecipitation was performed in non-denaturing detergent solutions using antibodies directed against the N- and C-termini of the membrane domain. Eleven of the twelve fragments were expressed stably in oocytes in the presence or absence of their respective partners. However, the fragment containing from putative span nine to the C-terminus could be detected in oocytes only when co-expressed with its complementary partner containing the first eight spans. Co-expression of pairs of fragments divided in the first, second, third and fourth exofacial loops and in the fourth cytoplasmic loop resulted in a concentration-dependent association, but a pair of fragments divided in the sixth cytoplasmic loop did not co-immunoprecipitate. When two complementary fragments were translated separately in the cell-free system and the purified microsomes were then mixed, co-immunoprecipitation was observed only if the membranes were first fused using polyethylene glycol. This shows that co-immunoprecipitation results from specific interactions within the membrane and is not an artefact of detergent solubilization or immunoprecipitation. We demonstrate that band 3 assembly can occur within the membrane after translation, insertion and initial folding of the individual fragments have been completed. We conclude that most band 3 fragments contain the necessary information to fold in the membrane and adopt a structure that is sufficiently similar to the native protein that it permits correct assembly with its complementary partner.
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Research Article|
May 15 1998
Complementation studies with co-expressed fragments of human red cell band 3 (AE1): the assembly of the anion-transport domain in Xenopus oocytes and a cell-free translation system
Jonathan D. GROVES;
Jonathan D. GROVES
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
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Lin WANG;
Lin WANG
1
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
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Michael J. A. TANNER
Michael J. A. TANNER
2
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
2To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
November 06 1997
Revision Received:
February 02 1998
Accepted:
February 25 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 332 (1): 161–171.
Article history
Received:
November 06 1997
Revision Received:
February 02 1998
Accepted:
February 25 1998
Citation
Jonathan D. GROVES, Lin WANG, Michael J. A. TANNER; Complementation studies with co-expressed fragments of human red cell band 3 (AE1): the assembly of the anion-transport domain in Xenopus oocytes and a cell-free translation system. Biochem J 15 May 1998; 332 (1): 161–171. doi: https://doi.org/10.1042/bj3320161
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