Characterization of Glycolytic Enzyme Interactions with Murine Erythrocyte Membranes in Wild type and Membrane Protein Knockout Mice Short title for the running head: GLYCOLYTIC ENZYME INTERACTIONS WITH ERYTHROCYTE MEMBRANES

Previous research has shown that glycolytic enzymes (GEs) exist as multi-enzyme 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 α-spectrin, ankyrin, protein 4.2, protein 4.1, βadducin 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. Introduction The glycolytic enzymes (GEs), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, phosphofructokinase (PFK), lactate dehydrogenase (LDH) and pyruvate kinase (PK) have been shown to organize into multi-enzyme complexes on the inner surface of the human erythrocyte membrane . Because assembly of these complexes is sensitive to physiological stimuli such as band 3 phosphorylation and hemoglobin (Hb) deoxygenation, it has been suggested that regulation of GE assembly may serve an important biologic function, perhaps in regulation of glucose metabolism. Indeed, association of the complexes on the membrane has been observed to correlate with a shift in glucose metabolism from glycolysis to the pentose phosphate pathway 2, . The specific docking sites of GEs on band 3 have been mapped to tandem similar sequences (6-DDYED-10 and 19-EEYED-23) near the NH2-terminus of the polypeptide . However, these NH2-terminal sequences are not well conserved in other mammalian species including the mouse (Fig. 1), raising the question whether GE assembly into complexes is a feature of all mammalian red cells or a unique characteristic of human erythrocytes. Preliminary evidence in favor of GE binding to nonhuman erythrocyte membranes has come from staining data of Ercolani et al. 7 and Weber et al. 8 who show that antibodies to GAPDH label the membrane in both rat and mouse erythrocytes, respectively. However, no membrane staining of any other GEs has ever been reported in nonhuman erythrocytes, and the tyrosines with flanking acidic residues (Y8 and Y21) that are phosphorylated and in human band 3 and regulate GE association with the membrane are absent in murine band 3. To determine the generality of GE assembly on erythrocyte membranes, we undertook to characterize the membrane binding sites, if any, of GAPDH, aldolase, PFK, LDH and PK on murine erythrocyte membranes. In the present study, we demonstrate that i) GEs are For personal use only. on October 31, 2017. by guest www.bloodjournal.org From