Characterization of glycolytic enzyme interactions with murine erythrocyte membranes in wild-type and membrane protein knockout mice

Abstract

Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking ␣-spectrin, ankyrin, protein 4.2, protein 4.1, ␤-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3. (Blood. 2008;112:3900-3906)

Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps

“…2) is consistent with a hydrophobic ATP compartment. Our results also raise the question of how the corrals that harbor the ATP pool relate to the known structures of the membrane-cytoskeletal complexes (22)(23)(24). The major RBC membrane complexes are of two types, a junctional complex and an ankyrin complex, with some overlap in the constituents of the two (25,26).…”

Molecular physiology and genetics of Na+-independent SLC4 anion exchangers

“…AE1 is a dimer or tetramer in the membrane, and a detergent-solubilized dimer. The extreme N-terminal sequence of human AE1 binds multiple glycolytic enzymes (Campanella et al, 2008;Chu and Low, 2006) and hemoglobin under the control of hemoglobin oxygenation. Other parts of the N-terminal cytoplasmic domain provide binding sites for the erythroid cytoskeletal proteins ankyrin-1 (Chang and Low, 2003;Stefanovic et al, 2007), protein 4.2 (Toye et al, 2005), the ERM protein 4.1R (Salomao et al, 2008), and integrin-linked kinase (Keskanokwong et al, 2007).…”

Regulation of membrane-cytoskeletal interactions by tyrosine phosphorylation of erythrocyte band 3

“…8 Staining was performed using polyclonal anti-band 3 antibody (raised in rabbit against purified human cdb3) and monoclonal anti-phosphotyrosine (py99; Santa Cruz Biotechnology). Band 3 was visualized with Cy2-conjugated antirabbit and anti-phosphotyrosine with Cy3-conjugated anti-mouse antibodies (Jackson ImmunoResearch Laboratories).…”

“…[1][2][3][4][5] One of the major targets of erythrocyte signaling appears to be the predominant membrane-spanning protein, band 3. Band 3 (AE1) catalyzes the exchange of anions (primarily HCO 3 Ϫ for Cl Ϫ ) across the erythrocyte membrane, 6 anchors the spectrin/ actin cytoskeleton to the lipid bilayer, 7 organizes and regulates a complex of glycolytic enzymes, 8,9 participates in control of erythrocyte lifespan, 10,11 nucleates several important membrane-spanning proteins, 12 and serves as a docking site for multiple peripheral membrane proteins, including protein 4.1, protein 4.2, and several kinases and phosphatases. [13][14][15][16] Not surprisingly, mutations in band 3 are frequently associated with various hemolytic diseases.…”