Abstract
Subunit interactions in the band 3 protein of the human red blood cell membrane have been examined by a combination of cross-linking, chemical labeling, and in situ proteolysis. In agreement with Staros (Staros, J. V. (1982) Biochemistry 21, 3950-3955), we find that the membrane-impermeant active ester bis(sulfosuccinimidyl) suberate (BSSS) cross-links band 3 in intact cells to a dimer, with no formation of higher oligomer. Combined cross-linking of the outer surface with BSSS and the cytoplasmic domain with Cu2+/o-phenanthroline does not produce significant covalent tetramer of band 3 (beyond that produced by Cu2+/o-phenanthroline alone). Therefore, the membrane domains and cytoplasmic domains of the same pair of subunits are cross-linked to each other. 4,4'-Diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) is known to form a covalent cross-link between complementary chymotryptic fragments (Mr 60,000 and 35,000). Edman degradation of band 3 from H2DIDS/chymotrypsin-treated cells shows that the H2DIDS cross-link is between fragments of the same subunit. In contrast, BSSS forms both intramolecular and intermolecular cross-links between complementary chymotryptic fragments. No intermolecular cross-links between two 35,000-dalton or two 60,000-dalton fragments are detectable. We have localized one end of the BSSS intermolecular cross-link to within 4 residues of the exofacial chymotrypsin cleavage site. The polypeptide sequence on each side of the site suggests that hydrophobic membrane-crossing segments emerge at the cell surface near the site of intermolecular cross-linking. This is the first detailed information available on the regions of the band 3 primary structure near the interface between subunits.
Highlights
SDuibiusnotithsiaorceyacnroodsish-lyidnrkoesdtitlobeenae-c2h,2o’thedri.sulfo nate (H2DIDS)is known to forma covalent cross-link between complementary chymotryptic fragments (Mr have been localized in theprimary structure of the membrane domain [7,8,9,12,13,14,15], placing restrictions on the possible ways the polypeptide can be foldedin themembrane
Edman degradation of band 3 measurements have shown that neighboring band 3protomers from H2DIDS/chymotrypsin-treatedcells shows that are closer to each other than would bepredicted if the protein the HzDIDS cross-link is between fragments of the were monomeric [23, 24]
The polypeptide sequence on each side of the site suggests that hydrophobic membrane-crossing segments emergaet the cell surface near thseite of intermolecular cross-linking
Summary
Human red blood cells were obtained from the Blood Donor Center interactions between cytoplasmic domains are between subor the Lipid Research Laboratory of the University ofIowa. A similar pattern isobserved if the band 3 a covalent cross-linkbetween 60,000- and 35,000-&ton chyhad been cross-linked to a dimer with BSSS prior to mem- motryptic fragments (Fig. 3) of the same subunit This asbrane isolation and oxidative cross-linking (Fig. 2, lower).In sumption is based on the fact that, in chymotrypsin-cleaved three such experiments, there was no more tetramer in the red cells, HzDIDS cross-links the two band 3 fragments to doubly cross-linked membranes than in those treated with each other quantitatively, but, in uncleavedcells,H,DIDS. “c branes were isolated, depleted of bands 1,2,5, and 6 [7],and incubated for 10 min at 23 in the presence or absence of 40 p~ CuSO,, 200 pM 1,~O-phenanthroline.The membranes were solubilized and the chymotrypsin cleavage site and shows directly that neither of the 2 lysines near the chymot ~ s i nsite is covalently attached to HzDIDS. Cellswere incubated with BSSS inthe presence or absence of 1 mM DNDS, an anion transport inhibitor that
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