Temporal Synthesis of Band 3 Oligomers During Terminal Maturation of
Mouse Erythroblasts. Dimers and Tetramers Exist in the Membrane as
Preformed Stable Species
Manjit Hanspal,
David E. Golan,
Yva Smockova,
Scott J. Yi,
Michael
R. Cho,
Shih-Chun Liu, and
Jiri Palek
From the Department of Biomedical Research, St Elizabeth's Medical
Center, Tufts University School of Medicine, Boston; and the
Departments of Biological Chemistry and Molecular Pharmacology and of
Medicine, Harvard Medical School, Hematology Division, Brigham and
Women's Hospital, Boston, MA.
Band 3, the anion transport protein of the erythrocyte membrane,
exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the
free mobile band 3 fraction. The tetramers are linked to the skeleton
by their interaction with ankyrin. In this report we have examined the
temporal synthesis and assembly of band 3 oligomers into the plasma
membrane during red cell maturation. The oligomeric state of newly
synthesized band 3 in early and late erythroblasts was analyzed by
size-exclusion high-pressure liquid chromatography of band
3 extracts derived by mild extraction of plasma membranes with the
nonionic detergent C12E8 (octaethylene glycol
n-dodecyl monoether). This analysis revealed that at the early
erythroblast stage, the newly synthesized band 3 is present
predominantly as tetramers, whereas at the late stages of erythroid
maturation, it is present exclusively as dimers. To examine whether the
dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these
species in erythroblast membranes. Pulse-chase analysis followed by
cellular fractionation 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 contrast, in late erythroblasts the newly synthesized band
3 dimers move rapidly to the plasma membrane fraction but then recycle
between the plasma membrane and microsomal fractions. Fluorescence
photobleaching recovery studies showed that significant fractions of
B3T and B3D are laterally mobile in early and late erythroblast plasma
membranes, respectively, suggesting that many B3T-ankyrin complexes are
unattached to the membrane skeleton in early erythroblasts and that the
membrane skeleton has yet to become tightly organized in late
erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated
into the plasma membrane. However, when ankyrin synthesis is
downregulated in late erythroblasts, it 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 proteins in stabilizing integral
membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 329-338
© 1998 by The American Society of Hematology.
