title: Identification of the components of a glycolytic enzyme metabolon on the human red blood cell membrane.
abstract: Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as "label transfer" that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Estela
authors.initials: E
authors.lastname: Puchulu-Campanella
authors.forename: Haiyan
authors.initials: H
authors.lastname: Chu
authors.forename: David J
authors.initials: DJ
authors.lastname: Anstee
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Galan
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
authors.forename: Philip S
authors.initials: PS
authors.lastname: Low
mesh_terms: D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism*; D006019:Glycolysis; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D004798:Enzymes; D008565:Membrane Proteins