Functions of extracellular lysine residues in the human erythrocyte anion transport protein.

The extracellular lysine residues in the human erythrocyte anion transport protein (band 3) have been investigated using chemical modification with the impermeant homobifunctional active ester bis(sulfosuccinimidyl)-suberate (BSSS). This agent forms covalent intra- and intermolecular cross-links in human band 3 in intact cells (Staros and Kakkad. 1983. J. Membr. Biol. 74:247). We have found that the intermolecular cross-link has no detectable effect on the anion transport function of band 3. The intramolecular cross-link, however, causes major changes in the characteristics of the anion transport. These functional alterations are caused by the modification of lysine residues at the stilbene disulfonate binding site. BSSS pretreatment at pH 7.4 irreversibly inhibits Cl-Br exchange by at least 90% when the transport is assayed at extracellular pH above 8. In the same BSSS-pretreated cells, however, the Cl-Br exchange rate is activated by lowering the pH of the flux medium (intracellular pH fixed at 7). The flux is maximal at pH 5-6; a further lowering of the extracellular pH inhibits the anion exchange. This acid-activated Cl-Br exchange in the BSSS-treated cells is mediated by band 3, as indicated by phenylglyoxal and phloretin inhibition of the flux. Thus, the BSSS pretreatment has little effect on the maximal Cl-Br exchange flux catalyzed by band 3, but it shifts the alkaline branch of its extracellular pH dependence by approximately 5 pH units. BSSS also eliminates the self-inhibition of Cl-halide exchange by high extracellular Br or I concentrations. These results indicate that the BSSS-modified lysines do not participate directly in anion translocation, but that one of the lysines normally provides a positive charge that is necessary for substrate anion binding. This positive charge is removed by the BSSS treatment but can be replaced by lowering the extracellular pH. The results also provide insight regarding the halide selectivity of the maximal rate of chloride-halide exchange: the native selectivity (Br much greater than I) is nearly abolished by BSSS treatment, which suggests that the selectivity results from the very strong binding of iodide to an outward-facing modifier site.

Localization of a site of intermolecular cross-linking in human red blood cell band 3 protein.

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.

Erythrocyte band 3 protein: evidence for multiple membrane-crossing segments in the 17 000-dalton chymotryptic fragment.

We have investigated the topology of the band 3 protein of the human erythrocyte membrane by a combination of chemical labeling and proteolytic cleavage. The N-terminal third of the membrane-bound domain of band 3 is a 17 000-dalton chymotryptic fragment that is known to traverse the membrane an odd number of times. At least three lysine residues on this fragment can be labeled by reductive methylation of intact cells, under conditions that cause labeling of exofacial, but not intracellular, lysine residues. One of the labeled lysines is the one that reacts with anionic aryl isothiocyanates, and another is very close to the C terminus of the fragment. Both these are on the C-terminal 11-kilodalton CNBr peptide. The third labeled lysine is on the 6-kilodalton N-terminal CNBr peptide, which had not been previously known to have an extracellular site. Control experiments using a stilbenedisulfonate derivative demonstrate that the labeled 6-kilodalton CNBr peptide is not a degradation product of the 11-kilodalton CNBr fragment. Also, the exofacial lysine on the 6-kilodalton peptide can be labeled by reductive methylation even when the stilbenedisulfonate site is occupied by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate, which blocks band 3 mediated transport of BH4 into the cells. This is further indication that the labeled lysine is accessible from the extracellular water. These data are the first direct evidence that the 17-kilodalton chymotryptic fragment spans the membrane more than once.