Proteolytic Cleavage Sites of Band 3 Protein in Alkali-Treated Membranes: Fidelity of Hydropathy Prediction for Band 3 Protein

Abstract

To assess the fidelity of hydropathy prediction for band 3 protein, we determined the cleavage sites of the protein and the portions of the protein tightly bound to the membrane lipid bilayer by means of in situ proteolytic digestion. For the removal of all anticipated hydrophilic connector loops from membranes, we had to denature the band 3 protein molecule in situ by alkali treatment. When the alkali-treated membranes were digested with trypsin, chymotrypsin, and pepsin, the majority of the anticipated transmembrane portions remained in the membrane fraction. However, five anticipated transmembrane portions were released into the supernatant fraction. Thus, the first, second, third, sixth and tenth anticipated transmembrane portions, in accordance with the hydropathy prediction, were released into the supernatant with the proteolytic digestion method. This indicates that these anticipated transmembrane portions are not bound with the boundary lipids although the hydrophobicity of these portions is comparable to that of the portions experimentally remaining in the membrane fraction. It is conceivable that the membrane peptide portions of band 3 protein could be classified into at least two categories, i.e. one bound to the boundary lipids and the other free from the boundary lipids. Approximately 90% of the transmembrane domain of the band 3 protein are recovered in either the supernatant fraction or the membrane fraction. The fidelity of hydropathy prediction for polytopic membrane proteins and the nature of the membrane embedded peptide portions are discussed.

Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface

“…Flexibility in this funnel region would presumably be undesirable by slowing both the rates of anion binding and release from AE, with concomitant reduction in the optimal rate of AE function. Region 656-663 may lie beneath the surface of the membrane, forming part of a TM with open structure, as has been suggested from proteolytic studies [33]. This interpretation would be consistent with the observed restricted dynamics.…”

Assessment of Topogenic Functions of Anticipated Transmembrane Segments of Human Band 3

“…A protein chemical analysis has already demonstrated that segments TM1 to TM3 of band 3 are stabilized in the membrane via an interaction between the peptide segments but not via protein-lipid interaction, because these segments were readily extracted from the erythrocyte membrane by alkali denaturation and subsequent protease treatment (16). These alkali-extractable transmembrane segments should be surrounded by other transmembrane segments that interact directly with the membrane lipids.…”

“…Band 3 is a major multispanning membrane protein in erythrocytes, and its topology has been extensively studied from various approaches (16,17). It consists of two domains: (a) an amino-terminal cytoplasmic domain of about 400 amino acid residues that bind to the cytoskeleton to regulate the cell shape of the erythrocytes, and (b) the carboxyl-terminal half, which is a transmembrane domain responsible for the anion-exchanging activity.…”

Novel Topology in C-terminal Region of the Human Plasma Membrane Anion Exchanger, AE1

“…This stretch was also identified as the senescence antigen of aged erythrocytes (17). Some proteolytic sites in the AE1 C-terminal region were accessible only after treatment of erythrocytes with high concentrations of sodium hydroxide, suggesting that those regions normally are folded into the AE1 structure (18). In addition, carbonic anhydrase II binds to the LDADD motif in the cytoplasmic C-terminal tail to facilitate HCO 3 Ϫ transport activity (19).…”