TEMPORAL SYNTHESIS OF BAND-3 OLIGOMERS DURING TERMINAL MATURATION OF MOUSE ERYTHROBLASTS - DIMERS AND TETRAMERS EXIST IN THE MEMBRANE AS PREFORMED STABLE SPECIES

Band 3, the anion transport protein of the erythrocyte membrane, exist s in the membrane as a mixture of dimers (B3D) and tetramers (B3T). Th e dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their in teraction with ankyrin. In this report we have examined the temporal s ynthesis and assembly of band 3 oligomers into the plasma membrane dur ing red cell maturation. The oligomeric state of newly synthesized ban d 3 in early and late erythroblasts was analyzed by size-exclusion hig h-pressure liquid chromatography of band 3 extracts derived by mild ex traction of plasma membranes with the nonionic detergent C12E8 (octaet hylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present pre dominantly as tetramers, whereas at the late stages of erythroid matur ation, it is present exclusively as dimers. To examine whether the dim ers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthes ized during erythroblast maturation was examined by pulse-chase analys is. We showed that the newly synthesized band 3 dimers and tetramers a re stable and that there is no interconversion between these species i n erythroblast membranes. Pulse-chase analysis followed by cellular fr actionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contr ast, in late erythroblasts the newly synthesized band 3 dimers move ra pidly to the plasma membrane fraction but then recycle between the pla sma membrane and microsomal fractions. Fluorescence photobleaching rec overy studies showed that significant fractions of B3T and B3D are lat erally mobile in early and late erythroblast plasma membranes, respect ively, suggesting that many B3T-ankyrin complexes are unattached to th e membrane skeleton in early erythroblasts and that the membrane skele ton has yet to become tightly organized in late erythroblasts. We post ulate that in early erythroblasts, band 3 tetramers are transported th rough microsomes and stably incorporated into the plasma membrane. How ever, when ankyrin synthesis is downregulated in late erythroblasts, i t appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal prot eins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins. (C) 1998 by The American Society of Hematology.