title: Critical band 3 multiprotein complex interactions establish early during human erythropoiesis.
abstract: Band 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RhAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RhAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RhAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RhAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.
authors.affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, U.K.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Satchwell
authors.affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, U.K.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Bell
authors.affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, U.K.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Pellegrin
authors.affiliation: Bristol Institute for Transfusion Sciences, NHS Blood and Transplant, Bristol, U.K.
authors.forename: Sabine
authors.initials: S
authors.lastname: Kupzig
authors.affiliation: Bristol Institute for Transfusion Sciences, NHS Blood and Transplant, Bristol, U.K.
authors.forename: Kay
authors.initials: K
authors.lastname: Ridgwell
authors.affiliation: Bristol Institute for Transfusion Sciences, NHS Blood and Transplant, Bristol, U.K.
authors.forename: Geoff
authors.initials: G
authors.lastname: Daniels
authors.affiliation: Bristol Institute for Transfusion Sciences, NHS Blood and Transplant, Bristol, U.K.
authors.forename: David J
authors.initials: DJ
authors.lastname: Anstee
authors.affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, U.K.|Bristol Institute for Transfusion Sciences, NHS Blood and Transplant, Bristol, U.K.|Department of Hematopoiesis, Sanquin Research, Amsterdam, The Netherlands.
authors.forename: Emile
authors.initials: E
authors.lastname: van den Akker
authors.affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, U.K.|Bristol Institute for Transfusion Sciences, NHS Blood and Transplant, Bristol, U.K.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Toye
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004920:Erythropoiesis / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D034741:RNA, Small Interfering; D012156:Reticulocytes / Q000166:cytology / Q000378:metabolism*
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C498523:RHAG protein, human; D034741:RNA, Small Interfering; C044791:erythrocyte membrane band 4.2 protein; D010447:Peptide Hydrolases