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Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 329-338
Temporal Synthesis of Band 3 Oligomers During Terminal Maturation of
Mouse Erythroblasts. Dimers and Tetramers Exist in the Membrane as
Preformed Stable Species
By
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.
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ABSTRACT |
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.
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INTRODUCTION |
BAND 3, THE ANION EXCHANGER, is the most
abundant integral membrane protein of the mammalian red blood cell
(RBC). The protein consists of two domains with distinct structure and
function. The C-terminal 56-kD membrane domain consists of multiple
hydrophobic segments that traverse the plasma membrane 12 to 14 times.
This domain of band 3 mediates the electroneutral exchange of
bicarbonate for chloride.1 The 41-kD N-terminal domain has
a net negative charge, projects into the cytoplasm, and anchors the
membrane skeleton to the membrane through its interactions with
ankyrin,2 protein 4.1,3 and protein
4.2.4 This domain also binds hemoglobin and several
glycolytic enzymes.5
Band 3 is thought to play a pivotal role in the assembly of the
membrane skeleton in developing erythroblasts. In vitro evidence indicates that the proper assembly of membrane skeleton components into
a stable supramolecular complex at the plasma membrane requires prelocalization of band 3 within the plasma membrane. Pulse-labeling and pulse-chase studies of avian6-8 and
mammalian9-12 erythroid precursors reveal that spectrin and
ankyrin are synthesized before band 3 in developing erythroblasts but
are assembled into a stable membrane skeleton only after the synthesis
of band 3 is initiated. However, recent findings of normal membrane
skeleton assembly in RBCs lacking band 3 challenge this interpretation
of the in vitro studies.13,14
Band 3 is self-associated and it exists in the red cell membrane as a
mixture of dimers (B3D) and tetramers (B3T). The dimers are not tightly
associated with the skeleton and constitute the free mobile band 3 fraction that can be removed from the membrane by mild
extraction.15 The tetramers are tightly associated with ankyrin with the stoichiometry of four band 3 copies to one
ankyrin.15 Recent studies using sedimentation equilibrium
analysis have shown that purified band 3 tetramers also bind to band
4.1 protein in solution. One band 3 tetramer can bind up to 4 (or even
up to 8) band 4.1 molecules.16 However, in an earlier study
from our laboratory15 using high-pressure liquid
chromatography (HPLC) fractionation of crude band 3 extracts, we
detected protein 4.1 only in the band 3 dimer fraction and not in the
tetramer fraction. Thus, at this stage it is not clear which species of
band 3 associates with protein 4.1 in situ.
Recent evidence suggests that the transport of band 3 to the plasma
membrane is facilitated by at least two chaperon-like proteins. The
first potential chaperon is glycophorin A, whose coexpression with band
3 in oocytes leads to accelerated band 3 expression in the oocyte
membrane.17 The second possible chaperon is ankyrin, as
suggested by recent studies in mouse erythroleukemia (MEL)
and embryonic kidney 293 cell lines.18 These studies
indicate that the band 3-ankyrin complex is detected in the pre-Golgi
compartment, suggesting that the formation of this complex may
facilitate band 3 delivery to the plasma membrane. Using nb/nb
mice as an ankyrin-deficient red cell model, recent studies from our
laboratory have shown that in severely ankyrin-deficient erythrocyte
membranes, band 3 is present exclusively as band 3 dimers. These
results suggest that ankyrin is required for the formation of stable
band 3 tetramers.19 However, it is not known whether
ankyrin binds to preformed band 3 tetramers or whether ankyrin can link
two preformed band 3 dimers into a stable ankyrin-band 3 tetramer
complex.
Here, we have examined the temporal relationship between the synthesis
of band 3 dimers and tetramers during terminal maturation of mouse
erythroid cells. We have also studied the rates of turnover of band 3 oligomers in situ in early (proerythroblasts and basophilic normoblasts) and late (polychromatophilic/orthochromatophilic normoblasts and reticulocytes) stages of erythroblast maturation. We
find that the synthesis of band 3 tetramers precedes that of dimers and
that the newly synthesized dimers and tetramers do not undergo
interconversion during the terminal maturation of erythroblasts.
Pulse-chase analysis indicates that, while newly synthesized band 3 tetramers are initially present in the microsomal fraction of early
erythroblasts and later incorporated stably into the plasma membrane
fraction, newly synthesized band 3 dimers appear rapidly in the plasma
membrane fraction of late erythroblasts and later recycle between
plasma membrane and microsomal compartments. Fluorescence
photobleaching recovery (FPR) studies show that ankyrin-linked band 3 tetramers are laterally mobile in early erythroblast plasma membranes
and that band 3 dimers are laterally mobile in late erythroblast
membranes.
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MATERIALS AND METHODS |
Isolation of cells.
Erythroid cells were obtained from spleens of mice infected with the
anemia-inducing strain of Friend virus (FVA) as described previously.20 Briefly, Balb/c mice were injected through
the tail vein with approximately 104 spleen focus-forming
units of FVA. After 2 weeks, spleens were obtained from the mice, and
early erythroblasts (proerythroblasts and basophilic normoblasts) were
isolated by unit gravity sedimentation over a linear gradient of 1% to
2% deionized bovine serum albumin (BSA).11
To obtain late erythroblasts, early erythroblasts were cultured in
vitro for 40 to 44 hours in 5% CO2/air at a density of 1 × 106 cells per mL.11 The cell culture
medium consisted of Iscove's modified Dulbecco's medium (IMDM;
GIBCO-BRL, Gaithersburg, MD) containing 30% fetal calf
serum (FCS; Hyclone, Logan, UT), 0.1% deionized BSA,
0.1 mmol/L thioglycerol, penicillin/streptomycin, and 0.2 U/mL erythropoietin (generously provided by Ortho Biotech, Raritan,
NJ). Cellular morphology was assessed by staining with Wright Giemsa
and benzidine-hematoxylin. After 44 to 48 hours of culture, a majority
of cells were at the polychromatophilic/orthochromatic normoblast stage
(late erythroblasts).
Metabolic labeling of cells.
Both early and late erythroblasts (1 × 108 cells for
each time point) were washed twice in methionine-free Dulbecco's
modified minimum essential medium (DMEM; GIBCO-BRL) and
resuspended in 10 mL of the medium containing 20% FCS prewarmed to
37°C for 15 minutes. They were then incubated for different lengths
of time with 300 µCi [35S]methionine (1,000 Ci/mmol;
ICN Biomedicals, Irvine, CA). For the pulse-chase experiment, further
incorporation of [35S]methionine was stopped by the
addition of unlabeled methionine (0.8 mmol/L), and the incubation was
then continued for different time periods. At the end of the labeling
period, 10 volumes of 155 mmol/L choline chloride and 5 mmol/L HEPES,
pH 7.1, were added, and the cells were obtained by centrifugation and
washed once with ice-cold saline buffer (150 mmol/L NaCl, 5 mmol/L KCl,
1 mmol/L MgCl2, and 1 mmol/L CaCl2).
Cell fractionation.
[35S]methionine-labeled early and late erythroblasts were
lysed in a buffer containing 5 mmol/L Tris-HCl, pH 8.0, 5 mmol/L MgCl2, 2 mmol/L EDTA, 200 mmol/L
tosyl-L-lysine chloromethyl ketone (TLCK), 200 mmol/L
tosylamide-2-phenyl-ethyl chloromethyl ketone (TPCK), and 2 mmol/L
phenylmethylsulfonyl fluoride (PMSF) and homogenized with a
Dounce homogenizer (Wheaton Scientific, Millville, NJ). The homogenate was made isotonic by adding appropriate volumes of 5 mol/L NaCl and then ultracentrifuged at 100,000g for 2 hours. The resulting pellet was resuspended in 1 mL of concanavalin A (Con A)
loading buffer (20 mmol/L Tris-HCl, pH 7.4, 300 mmol/L NaCl, 1 mmol/L
CaCl2, 1 mmol/L MgCl2, 1 mmol/L
MnCl2) and loaded onto a column made of Con A lectin
(Sigma, St Louis, MO) preequilibrated in this buffer. The
eluate containing microsomal vesicles was collected. Vesicles made from
plasma membranes bound to the column and were eluted with 2 mL of the Con A loading buffer containing 300 mmol/L  -methyl
mannoside.18
Spectrin extraction from late erythroblast plasma membranes.
Spectrin was extracted from [35S]methionin-labeled late
erythroblast membranes by incubation in equal volumes of a low ionic strength buffer containing 0.1 mmol/L NaPO4, 0.1 mmol/L
EDTA, 0.1 mmol/L PMSF, 0.1 mmol/L N--p-tosyl-L-lysine chloromethyl ketone HCl, and 0.1 mmol/L dithiothreitol, pH 8.0, at
37°C for 20 minutes. Spectrin-depleted membranes were obtained by
centrifugation at 150,000g for 35 minutes at 4°C.
Size exclusion HPLC of band 3 extracted from erythroblast membranes
by the nonionic detergent octaethylene glycol n-dodecyl monoether
(C12E8).
Plasma membranes prepared from early and late erythroblasts were
dissolved in 5 volumes (usually 50 µL of membranes were dissolved in
250 µL) of 0.5% C12E8 in hypotonic buffer (5 mmol/L NaPi, pH 7.4) at 4°C. After centrifugation at
150,000g for 30 minutes, the supernatants were analyzed by size
exclusion HPLC using a TSK-4000 SWXL column (7.8 × 300 mm; Tosohaas, Tokyo, Japan), as described previously.21
The standard elution buffer contained 0.01%
C12E8, 100 mmol/L NaCl, 5 mmol/L NaPi, pH 7.0.
Protein in HPLC fractions was concentrated by centrifugation of 0.5 to
2.0 mL aliquots in a 10,000 molecular weight cut-off ultracentrifugation unit (Millipore, Lexington, MA) at 5,000 rpm for 15 to 20 minutes. The concentrated supernatant was removed, dissolved in
sodium dodecyl sulfate (SDS) and analyzed by SDS-polyacrylamide gel
electrophoresis (PAGE) according to Laemmli.22
Immunoprecipitation.
Band 3 was immunoprecipitated from the HPLC peaks (B3T and B3D) and
from the microsomal and plasma membrane fractions obtained after Con A
column treatment. The samples were suspended in immunoprecipitation buffer (IPB) containing 10 mmol/L Tris, pH 7.5, 1% NP-40, 0.5 % sodium deoxycholate, 150 mmol/L NaCl, 5 mmol/L EDTA, 2 mg/mL BSA, 200 mmol/L TLCK, 200 mmol/L TPCK, and 2 mmol/L PMSF. The resulting extracts
were precleared by mixing 50 µL of protein A-sepharose CL-4B
(Pharmacia LKB Biotechnology Inc, Piscataway, NJ; 50 mg
of beads/mL of IPB) for 1 hour at 4°C and centrifuged to
remove sepharose beads. The supernatants were then immunoprecipitated with rabbit anti-mouse band 3 IgG, and the samples were incubated overnight at 4°C with gentle shaking. Thereafter, 100 µL of
protein A-sepharose CL-4B was added and the samples incubated for
another 3 hours at 4°C with gentle shaking. The immunoprecipitates
were washed successively with buffer 1 (IPB containing 0.1% SDS);
buffer 2 (IPB without BSA and NP-40); and buffer 3 (10 mmol/L Tris, 1 mmol/L EDTA). The final pellet was resuspended in 70 µL of SDS sample
buffer and boiled for 2 minutes. Beads were removed by centrifugation
and the supernatants were directly loaded on 10% SDS-polyacrylamide
Laemmli22 gels.
Endoglycosidase H digestion.
Early and late erythroblasts were metabolically labeled with
[35S]methionine for 30 minutes and then chased with cold
methionine for 0 or 4 hours. Cells were then lysed in an NP-40 buffer
(10 mmol/L Tris, pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 150 mmol/L NaCl, 5 mmol/L EDTA, 2 mg/mL BSA, 200 mmol/L TLCK, 200 mmol/L TPCK, and 2 mmol/L PMSF), centrifuged at low speed to remove nuclei, and the resulting extracts were immunoprecipitated with anti-band 3 antibody as described above. Half of the immunoprecipitated material
was incubated with 0.1 volume of endoglycosidase H (30 µg/mL) at
37°C for 16 to 18 hours, as described.23
FPR.
Band 3 lateral mobility was measured in plasma membranes of early and
late erythroblasts in culture. Briefly, 100 µL containing a 20%
suspension of erythroblasts in 140 mmol/L KCl, 15 mmol/L NaPO4, 10 mmol/L glucose, pH 7.4, was incubated with 40 µL of eosin-5-maleimide (Molecular Probes, Eugene, OR), 0.25 mg/mL, for 15 minutes at room temperature. Fluorescence SDS-polyacrylamide gel
scanning of membranes from identically labeled human erythroblasts has
shown that greater than 90% of the eosin fluorescence comigrates with
band 3, and that the band 3-deficient erythroleukemia cell line K562
does not label upon incubation with eosin-5-maleimide (unpublished observations, May 1989). FPR was used to
measure the lateral mobility of eosin-labeled band 3 in membranes of
early and late erythroblasts at 37°C.24
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RESULTS |
Newly synthesized band 3 is tetrameric in early erythroblasts and
dimeric in late erythroblasts.
To elucidate the role of band 3 oligomerization in the biogenesis of
the erythrocyte membrane skeleton during terminal maturation of mouse
erythroid cells, we examined the temporal synthesis and assembly of
band 3 dimers and tetramers. Erythroblasts at the early and late stages
of maturation were isolated from the spleens of mice infected with FVA.
Both early and late erythroblasts were metabolically labeled with
[35S]methionine for 60 minutes before isolating plasma
membranes, which were then subjected to a mild extraction of band 3 with the nonionic detergent C12E8. The
C12E8 extract was analyzed by size-exclusion
HPLC to determine the oligomeric states of band 3 (Fig 1). Using this analysis in mature
mouse erythrocytes, we previously showed15 that the
C12E8 extract contains only B3D, whereas the
tetrameric species remains associated with the
C12E8-insoluble membrane skeleton. In striking
contrast, the extracts of early erythroblasts contained predominantly
B3T with only a trace amount of B3D (Fig 1A). Autoradiograms showed
that the tetrameric peak containing band 3 and ankyrin was
radiolabeled, suggesting that in early erythroblasts the newly
synthesized band 3 exists predominantly as tetramer. Because the
spectrin-based skeleton is not yet assembled on the membrane of early
erythroblasts, a mild extraction of such membranes with
C12E8 releases band 3 tetramers, which are
inextractable from mature cells under these conditions. In contrast, in
late erythroblasts mild extraction of the membranes with
C12E8 released only B3D (Fig 1B), whereas B3T
could be released only when spectrin was removed before
C12E8 extraction (Fig 1C). Densitometric scans of gels corresponding to the tetramer fraction showed that the band
3-to-ankyrin stoichiometry was similar to that in mature red cells, ie,
four copies of band 3 for each copy of ankyrin. These results suggest
that at the late stages of erythroid maturation when the skeleton has
been nearly completely formed, band 3 extracted under mild conditions
is mainly dimeric whereas the tetrameric band 3 remains associated with
the spectrin-based skeleton. Furthermore, autoradiograms show that the
radiolabeled band 3 is detected only in the B3D peak and not the B3T
peak. Thus, 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.
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| Fig 1.
Size-exclusion HPLC of C12E8
extracts from (A) early erythroblast membranes, (B) late erythroblast
membranes, and (C) late erythroblast membranes after spectrin
extraction. Plasma membranes isolated from
[35S]methionine-labeled early and late erythroblasts were
dissolved in 0.5% C12E8 in hypotonic buffer at
4°C. Samples were centrifuged at 150,000g for 30 minutes
and the supernatants were analyzed by size-exclusion HPLC using a
TSK-4000 SWXL column. At the bottom of the HPLC profile are
shown the elution positions for the standard proteins: T,
thyroglobulin; F, ferritin; and A, aldolase. The void volume
(V0) was determined from the elution position of blue dextran 2000 (average molecular weight 2 × 106 daltons). The tetramer (B3T) and dimer (B3D) peaks were
examined by SDS-PAGE followed by autoradiography. Newly
synthesized band 3 exists as tetramer in early erythroblasts and
as dimer in late erythroblasts.
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Band 3 dimers and tetramers do not undergo interconversion during
erythroid maturation.
The above results show that band 3 dimers and tetramers are synthesized
at distinct stages of erythroid development. However, whether they
exist as preformed stable species or are interconvertible is not known.
To address this question, we used pulse-chase analysis to examine the
fate of newly synthesized band 3 species during terminal maturation of
erythroblasts. We took advantage of the fact that, at a given stage of
erythroid maturation, the newly synthesized band 3 exists predominantly
as either dimers or tetramers.
Early and late erythroblasts were metabolically labeled with
[35S]methionine, which predominantly labels band 3 tetramers at the early stage and dimers at the late stage of
maturation. The cells were then chased with unlabeled methionine; early
erythroblasts were chased for 48 hours, during which time the cells
matured to the late erythroblast stage, while late erythroblasts were chased for 2 or 16 hours. Thereafter, plasma membranes were isolated from both the unchased and chased cells and were subjected to mild
extraction of band 3 with the detergent C12E8.
The resulting pellets and supernatants were then either subjected to
size-exclusion HPLC or immunoprecipitated with anti-band 3 antibodies.
Using this approach, we first examined the stability of band 3 tetramers in early erythroblasts. The C12E8
supernatants of chased and unchased erythroblast membranes were
subjected to HPLC analysis. As shown in Fig
2A, before the chase HPLC analysis yielded a predominant peak
containing band 3 tetramers, whereas after the 48-hour chase band 3 was
eluted predominantly as dimers. Autoradiograms showed that the
tetramers isolated before the chase were radiolabeled whereas the
dimers isolated after the chase were unlabeled. These results suggest
that radiolabeled tetramers did not dissociate into dimers during the
48-hour chase period. Furthermore, the C12E8
pellets and supernatants of chased and unchased erythroblast membranes
were immunoprecipitated with anti-band 3 antibodies. As shown in Fig
2B, before the chase when the cells were at the early erythroblast
stage, the radiolabeled band 3 was present exclusively in the
supernatant as tetramers, whereas after the 48-hour chase when the
cells had matured to the late erythroblast stage, practically all of
the radiolabeled band 3 was recovered in the pellet that normally
contained skeletal-associated band 3 tetramers. On the other hand, in
the supernatant that typically contained dimers at this stage,
virtually no radiolabeled band 3 was detected, suggesting that the
tetramers synthesized in early erythroblasts were stable and that they
did not convert into dimers during maturation.
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| Fig 2.
Pulse-chase analysis of metabolically labeled early
erythroblasts. Early erythroblasts were labeled with
[35S]methionine for 60 minutes and then chased with
unlabeled methionine for 48 hours. Plasma membranes isolated from
unchased and chased cells were extracted with
C12E8. (A) The C12E8
extracts were subjected to size exclusion HPLC. (B)
C12E8-insoluble pellets (P) and -soluble supernatants (S) were immunoprecipitated with anti-band 3 antibodies. The immunoprecipitates were analyzed by SDS-PAGE and the gels were
processed for fluorography. Band 3 tetramers synthesized before the
chase were stable and did not convert into dimers during the 48-hour
chase period.
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Next, using a similar approach, we examined the stability of band 3 dimers in late erythroblasts. Following a 60-minute labeling, late
erythroblasts were chased for 0, 2, or 16 hours. HPLC analysis showed
that band 3 was present as dimers both before and after the chase
(Fig 3A). Immunoprecipitation of band 3 from the C12E8 pellets and supernatants
isolated from the plasma membranes of unchased and chased erythroblasts
showed that the amount of radiolabeled band 3 dimers in the supernatant
remained the same after the chase (Fig 3B). Moreover, no radiolabeled
band 3 was detected in the pellet that typically contained tetramers in
late erythroblasts, suggesting that band 3 dimers that had been labeled
before the chase were stable and were not able to convert into
tetramers during the chase period. Quantitation of radiolabeled band 3 confirmed that the newly synthesized dimers in late erythroblasts did
not turn over during the 16 hours of chase (Fig 3C).
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| Fig 3.
Pulse-chase analysis of metabolically labeled late
erythroblasts. Late erythroblasts were labeled with
[35S]methionine for 60 minutes and then chased with
unlabeled methionine for 16 hours. Plasma membranes isolated from
unchased and chased cells were extracted with
C12E8. (A) The C12E8
extracts were subjected to size exclusion HPLC. (B)
C12E8-insoluble pellets (P) and -soluble supernatants (S) were immunoprecipitated with anti-band 3 antibodies. The immunoprecipitates were analyzed by SDS-PAGE and the gels were
processed for fluorography. (C) The autoradiograms shown in B were
scanned and the area under each peak integrated (the autoradiograms for
the 2-hour chase period are not shown). Band 3 dimers synthesized
before the chase were stable and did not associate to form tetramers
during the 16-hour chase period.
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Newly synthesized band 3 dimers are transported more rapidly to the
plasma membrane than are band 3 tetramers, and are recycled between
plasma membrane and microsomal compartments.
To determine the efficiency of band 3 transport to the plasma membrane,
the rate of turnover of band 3 was measured in the microsomes and
plasma membranes of early and late erythroblasts. Early and late
erythroblasts were metabolically labeled with
[35S]methionine for 30 minutes followed by a chase with
unlabeled methionine for different periods of time. Thereafter, cells
were lysed to obtain microsomes and plasma membranes as described in Materials and Methods. Band 3 was then immunoprecipitated from these
fractions using anti-band 3 antibody.
As shown in Fig 4, in early erythroblasts
newly synthesized band 3 appeared initially in the microsomal fraction
and later in the plasma membrane fraction. In contrast, in late
erythroblasts newly synthesized band 3 moved rapidly to the plasma
membrane fraction and only later appeared in the microsomal fraction.
These results are consistent with the hypotheses that (1) band 3 tetramers are transported more slowly to the plasma membrane than are
band 3 dimers; (2) band 3 tetramers remain stably localized in the plasma membrane after insertion; and (3) band 3 dimers recycle between
plasma membrane and microsomal compartments after rapid insertion into
the plasma membrane. These results were confirmed by Endoglycosidase H
(Endo H) digestion of band 3 immunoprecipitated from
[35S]methionine-labeled early and late erythroblasts.
Susceptibility to Endo H reflects an absence of processing in the Golgi
and retention of the protein in the ER.25-27 Early and late
erythroblasts were labeled for 30 minutes and chased for 0 or 4 hours
followed by band 3 immunoprecipitation. Half of the immunoprecipitated
sample was treated with Endo H. As shown in
Fig 5,
[35S]methionine-labeled band 3 immunoprecipitated from
early erythroblasts was sensitive to Endo H if the cells were lysed
immediately following the pulse (Fig 5, lanes 1 and 2), but the protein
became Endo H-resistant after a 4-hour chase (Fig 5, lanes 3 and 4)
indicating its exit from the ER or the early Golgi compartment.
However, in late erythroblasts, a significant fraction of band 3 was
sensitive to Endo H after 4 hours of chase reflecting its movement from the plasma membrane back to the ER/Golgi. To determine the oligomeric state of the band 3 species that was retained in the microsomes of late
erythroblasts, size exclusion HPLC analysis was used; C12E8 extracts of microsomes isolated from
[35S]methionine-labeled late erythroblasts were analyzed
by HPLC. This analysis revealed the presence of only B3D (data not
shown), indicating that at the late erythroblast stage band 3 dimers
accumulated in microsomes. This accumulation was likely caused by
recycling of band 3 dimers between plasma membrane and microsomal
compartments in the late stages of erythroid maturation when ankyrin
synthesis was downregulated.
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| Fig 4.
Turnover of band 3 in the microsomal and plasma membrane
fractions of early and late erythroblasts. (A) Microsomes and plasma membranes were isolated from erythroblasts labeled with
[35S]methionine for 30 minutes and chased for different
time periods (0 to 120 minutes). Band 3 was immunoprecipitated using
anti-band 3 antibodies. The immunoprecipitates were analyzed by
SDS-PAGE and the gels were processed for fluorography. B3: Band 3. (B) The autoradiograms shown in (A) were scanned and the area under each
peak integrated.
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| Fig 5.
Endo H digestion of immunoprecipitated band 3 from early
and late erythroblasts. Early and late erythroblasts were either [35S]methionine-labeled for 30 minutes (lanes 1, 2, 5, and 6) or labeled for 30 minutes and chased for 4 hours (lanes 3, 4, 7, and 8). NP-40 extracts were immunoprecipitated with band 3 antibodies, and half of the samples were incubated with (lanes 2, 4, 6,and 8) or
without (lanes 1, 3, 5, and 7) Endo H before the separation of proteins
on an SDS gel.
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Significant fractions of band 3 tetramers and dimers are laterally
mobile in early and late erythroblast plasma membranes, respectively.
Lateral mobility measurements have been used to measure the extent of
interaction between band 3 oligomers and the membrane skeleton in
intact human and mouse erythrocytes. In normal mouse erythrocytes
approximately 90% of the band 3 molecules are laterally immobile,
suggesting the presence of tight binding and/or steric hindrance interactions between band 3 oligomers and the membrane skeleton in these cells.19 However, no studies of band 3 mobility have been performed in developing erythroblasts. To quantify
the development of binding and/or steric hindrance interactions
between band 3 molecules and the membrane skeleton in developing
erythroid cells, we used FPR to measure the lateral mobility of
eosin-labeled band 3 molecules in the plasma membrane of early and late
erythroblasts. Band 3 manifested a lateral diffusion coefficient of
(2.1 ± 1.1) × 10 10 cm2/s and a
fractional mobility of 36 ± 5% (mean ± SD; n = 9 cells) in
plasma membranes of early erythroblasts, showing that a
significant fraction of B3T is laterally mobile in these membranes. In
late erythroblast membranes band 3 had a lateral diffusion coefficient of (3.9 ± 2.6) x 10-10 cm2s-1
and a fractional mobility of 29 ± 11% (n = 13 cells),
suggesting that a significant fraction of B3D is laterally mobile in
these membranes.
| |
DISCUSSION |
This study addresses the role of band 3 oligomerization in the
biogenesis of the erythrocyte membrane skeleton during terminal maturation of mouse erythroid cells. We have used FVA cells at the
early and late stages of erythroid maturation. Although one study using
normal human bone marrow cells has suggested that transformed cells may
not recapitulate the sequence of protein synthesis in
vivo,28 FVA cells have been used extensively to study the
biogenesis of the erythroid membrane skeleton.11,29 In an
attempt to confirm the validity of the FVA model system for the present
studies, we compared the oligomerization state of newly synthesized
band 3 in late FVA erythroblasts with that in explanted late
erythroblasts from nb/nb mouse spleens. The nb/nb
erythroblast isolation procedure was identical to that
described.13 The newly synthesized band 3 was present
exclusively as dimers in both FVA and nb/nb late erythroblasts
(data not shown). Similar studies could not be performed in early
nb/nb erythroblasts because of the lack of a sufficient number
of cells.
Oligomeric states of band 3 were analyzed in the native erythroblast
membrane by mild extraction of band 3 with the nonionic detergent
C12E8 followed by size-exclusion HPLC. This
analysis revealed the presence of B3T in the extracts of early
erythroblast membranes and B3D in the extracts of late erythroblast and
mature mouse erythrocyte membranes. B3T can, however, be extracted from the membranes of late erythroblasts and mature erythrocytes when spectrin is removed before C12E8 extraction.
SDS-PAGE analysis shows that the tetrameric peak of early erythroblasts
contains band 3 and ankyrin, whereas the dimeric peak of late
erythroblasts contains only band 3. Thus, in the extracts of early
erythroblast membranes the predominant band 3 species is tetramer.
Because only small amounts of spectrin and ankyrin are incorporated
into a highly unstable membrane skeleton of these cells,11
mild extraction of early erythroblast membranes with
C12E8 releases B3T, a species that is
inextractable from mature cells under these conditions. In contrast, in
late erythroblasts where substantial amounts of spectrin and ankyrin
have already been assembled into a stable membrane
skeleton,11 mild extraction of membranes with
C12E8 releases only B3D whereas B3T remains
associated with the skeleton. HPLC analysis of band 3 extracts derived
from the plasma membranes of metabolically labeled early and late
erythroblasts shows that the newly synthesized band 3 exists as
tetramers in early and as dimers in late erythroblasts.
To examine the stability of the newly synthesized band 3 oligomers in
the plasma membrane, we have used pulse-chase analysis of metabolically
labeled erythroid precursors. Pulse-chase of early erythroblasts for 2 days, during which time the erythroblasts concomitantly matured to late
erythroblasts, shows that radiolabeled newly synthesized band 3 tetramers are stable and that they do not turn over or dissociate into
dimers. Radiolabeled tetramers, which are easily extractable by
C12E8 before the chase, become skeletal-associated and hence inextractable after the chase. Similarly, pulse-chase of late erythroblasts for up to 16 hours shows that the
newly synthesized band 3 dimers are stable. Moreover, radiolabeled dimers do not convert into tetramers, as shown by their extractability by C12E8 both before and after the chase. These
results show that band 3 dimers and tetramers exist in the membrane as
preformed stable species and that they do not undergo interconversion.
Recent evidence has suggested that band 3 is delivered to the plasma
membrane as a band 3-ankyrin complex, which is already formed in the
ER/pre-Golgi compartment. The formation of this complex has been shown
to be important for efficient processing of band 3 through the early
secretory compartments of the cell.18 Recent studies in
ankyrin-deficient nb/nb mouse red cells have shown, however,
that although ankyrin is required for the formation of stable band 3 tetramers, it is not absolutely required for delivery of band 3 to the
plasma membrane.19 These results support the notion that
band 3 is transported to the plasma membrane in both ankyrin-dependent
and -independent modes. Based on the temporal synthesis of band 3 oligomers, we propose that at early stages of erythroid maturation,
band 3 is delivered to the plasma membrane via an ankyrin-dependent
pathway in which ankyrin acts as a chaperon to transport B3T,
presumably as preformed B3T-ankyrin complexes, from the ER to the
plasma membrane. In contrast, at late erythroblast stages when ankyrin
synthesis is downregulated, band 3 is inserted as a dimer. In the
absence of ankyrin, the possibility that glycophorin A or another
chaperon-like protein transports band 3 dimers to the plasma membrane
remains to be investigated.
We have used a pulse-chase metabolic labeling design and Endo H
digestion to study the kinetics of band 3 transport to the plasma
membrane. In early erythroblasts, the newly synthesized band 3 appears
initially in the ER and later exits this compartment to become stably
incorporated in the plasma membrane. Because at this stage of erythroid
maturation the rate of ankyrin synthesis is greater than that of band
3,11 the kinetics of band 3 transport from ER to plasma
membrane presumably represents the efficiency of formation of
B3T-ankyrin complexes in the ER and of transport of these complexes to
the plasma membrane. In contrast, in late erythroblasts the newly
synthesized band 3 is transported rapidly to the plasma membrane and
only later accumulates in the ER as well as the plasma membrane.
Because at this stage of erythroid maturation ankyrin synthesis has
decreased markedly while band 3 dimers are being synthesized in
abundance, this process presumably represents the efficiency of
transport of "free" B3D to the plasma membrane. Although the late
appearance of band 3 in the ER could represent "backing up" of
band 3 along its biosynthetic pathway as the band 3 level in the plasma
membrane approaches saturation, we favor a model in which "free"
band 3 dimers continue to be transported rapidly to the plasma membrane
and then to recycle between the plasma membrane and ER compartments.
Many other integral membrane proteins have been shown to recycle
between plasma membrane and endosome compartments, including hormone
and autacoid receptors,30-35 receptors that internalize
extracellular ligands or present ligands that have been processed
intracellularly,36-40 adhesion molecules,41,42 and ion and water transporters.43-46 In many cases the
molecular mechanisms responsible for both retention of integral
proteins in the plasma membrane and recycling of such proteins between plasma membrane and endosome compartments are incompletely understood. Our data suggest that stable linkages to membrane skeletal proteins could serve both to stabilize the incorporation of integral proteins in
the plasma membrane and to prevent protein recycling to endosomes. With
specific reference to erythroid maturation, we postulate that band 3 tetramers are required at the plasma membrane of early erythroblasts to
mediate mechanical "coupling" between the plasma membrane and the
developing membrane skeleton, but that the full complement of anion
transporters is not required at the plasma membrane until the nearly
mature reticulocytes are released from the bone marrow into the
peripheral circulation. Only at this last stage of development is it
important to localize B3D as well as B3T to the plasma membrane of the
mature erythrocyte.
About 35% of band 3 molecules in the plasma membranes of early
erythroblasts are capable of lateral diffusion in the plane of the
membrane. This observation is consistent with the finding that much of
the band 3 in these membranes is extractable by the nonionic detergent
C12E8. It is notable, however, that the band 3 in early erythroblast membranes consists almost entirely of B3T, which
are laterally immobile in membranes of mature mouse red
cells.19 In mature red cells the major molecular mechanism that immobilizes ankyrin-linked B3T is high-affinity binding
interactions between ankyrin and the  chain of
spectrin.19 In early erythroblasts, however, the spectrin
skeleton is not yet assembled on the plasma membrane,9-12
so ankyrin-linked B3T are free to diffuse laterally. The diffusion rate
of the mobile fraction of band 3 molecules is similar to that of other
transmembrane proteins in models of developing erythroid
cells.47
About 30% of band 3 molecules in late erythroblast membranes are
capable of lateral diffusion, consistent with the result that some of
the band 3 in these membranes is extractable by
C12E8. Unlike the extractable band 3 in early
erythroblasts, which consists of B3T, the extractable band 3 in late
erythroblasts consists only of B3D. Presumably, by the late
erythroblast stage of development the B3T are immobilized by
high-affinity binding interactions between ankyrin and spectrin. It is
interesting, however, that a significant fraction of the B3D population
is capable of lateral diffusion in late erythroblasts whereas the
entire B3D population is laterally immobile in membranes of mature
mouse red cells.19 In mature red cells the major molecular
mechanism that immobilizes B3D is steric hindrance interactions between
the cytoplasmic domain of band 3 and the spectrin-based membrane
skeleton.19 In late erythroblasts, however, although the
spectrin-based skeleton is assembling on the membrane, it is apparently
not yet organized sufficiently to effect lateral immobilization of B3D.
In summary, although a significant fraction of band 3 molecules is
laterally mobile in early and late erythroblast membranes, the
laterally mobile molecules represent different molecular species in the two cases. In early erythroblasts, the absence of ankyrin binding sites
on spectrin allows the ankyrin-linked B3T fraction to diffuse laterally, whereas in late erythroblasts the relatively loose organization of the developing membrane skeleton allows the B3D fraction to diffuse laterally.
| |
FOOTNOTES |
Submitted September 2, 1997;
accepted March 6, 1998.
Supported by National Institutes of Health (NIH) Research Grants No.
HL37462, HL32854 and HL15157.
Presented in part in abstract form at the 37th Annual Meeting of the
American Society of Hematology in Seattle, WA, December 1-5, 1995.
Address reprint requests to Manjit Hanspal, PhD, Department of
Biomedical Research, St Elizabeth's Medical Center, Boston, MA 02135;
e-mail: mbh{at}tiac.net.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Hemant Thatte for helpful discussions and Donna
Marie Mironchuk for the art work.
| |
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Abstract
Introduction
Abstract