(Received for publication, July 11, 1996, and in revised form, October 14, 1996)
From the G. Fornaini Institute of Biological
Chemistry, University of Urbino, Via Saffi 2, 61029 Urbino, Italy
and § INSERM U409, Faculté de Médecine Bichat,
75870 Paris cedex 18, France
Previously, we demonstrated that -spectrin is
a substrate for the ubiquitin system and that this conjugation is a
dynamic process (Corsi, D., Galluzzi, L., Crinelli, R., and Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935). In this study,
we mapped the sites of ubiquitination on erythrocyte
-spectrin. A
peptide map of digested
-spectrin, previously submitted to in
vitro 125I-ubiquitin conjugation, revealed the
presence of four distinct labeled bands with Mr
40,000, 36,000, 29,000, and 25,500. Western blotting experiments using
antibodies against each
-spectrin domain revealed that only IgG
anti-
III domain recognized the 125I-labeled ubiquitin
peptide of 29 kDa, whereas the IgG anti-
V domain recognized the
Mr 40,000 125I-ubiquitin-labeled
peptide. The other two labeled bands of Mr 36,000 and Mr 25,500 were identified as tetra
and tri multiubiquitin chains. Ubiquitination of the
III and
V
domains was further confirmed by anti-
-spectrin domain
immunoaffinity chromatography. Endoprotease Lys C-digested spectrin
conjugated previously to 125I-ubiquitin was incubated with
antibodies against each trypsin-resistant domain of
-spectrin. Gamma
counting of the radiolabeled antigen-antibody complexes purified by
protein A chromatography showed labeling in the IgG anti-
III and
anti-
V complexes alone. Domain
III is not associated with any
known function, whereas domain
V contains the nucleation site for
the association of the
and
chains. Ubiquitination of the latter
domain suggests a role for ubiquitin in the modulation of the
stability, deformability, and viscoelastic properties of the
erythrocyte membrane.
Ubiquitin (Ub),1 a 76-amino acid
protein, has been found both free and covalently bound to target
proteins via an isopeptide linkage between the carboxyl group of the
terminal glycine moiety of Ub and free amino groups on the target
protein (1). Rabbit reticulocyte fraction II (2) (protein adsorbed to
DEAE 52-cellulose and eluted with 0.5 M KCl) contains the
enzymatic system (E1, E2 s, E3 s) that is involved in ubiquitin
conjugation. Usually, a protein can have one or more sites for
ubiquitin, in which one ubiquitin or multiubiquitin chains can be
linked. In the latter case, one Ub is linked to the lysine 48 of
another ubiquitin bound to the substrate (3). Ub-conjugated proteins
can either be degraded to small peptides by a large 26 S
ATP-dependent protease complex, or the Ub moiety can be
removed by Ub isopeptidases, releasing ubiquitin and the intact protein
(4). Protein ubiquitination is a posttranslational process involved not
only in protein degradation but also in other cellular functions. In
fact, many studies have reported the in vivo presence of
several stable Ub-protein conjugates that are not subject to
degradation (5, 6). Moreover, another linkage in which ubiquitin is
linked to a previously bound Ub involves lysine 63 of Ub, and these
chains serve nonproteolytic functions (7). In general, ubiquitin is
involved in different cellular processes such as transcriptional
regulation (8), cell cycle regulation (9), stress responses (10), and
modulation of the immune response (11). In particular, in the last
decade Ub has been found to be bound to many specific substrates such as lysozyme (12), phytochrome (13), actin (14), histone (15),
calmodulin (16), cAMP-dependent protein kinase (17), p53
(18), ABC-transporter Ste 6 protein (19), c-jun (20), transducin (21),
T-cell antigen receptor (22), nuclear factor-
B1 precursor (23),
inhibitor of
B-
(24), platelet-derived growth factor-
, and
receptors, and epidermal growth factor, colony-stimulating factor-I,
fibroblast growth factor (25), cystic fibrosis transmembrane conductance regulator (26), and estrogen receptors (27), as well as
others.
Recently, we demonstrated that red blood cell -spectrin is also a
specific substrate for ubiquitination (28).
- and
-spectrin are
the major protein constituents of the red blood cell membrane skeleton
and contribute to about 25% of total red cell membrane proteins. This
membrane skeleton provides support for the overlying lipid bilayer and
contributes to the viscoelastic properties and deformability of the
membrane (29).
The spectrin molecule is composed of two subunits with apparent
molecular masses of 240 kDa (-spectrin) and 220 kDa (
-spectrin), intertwined side-to-side to form a heterodimer. The two
and
subunits of spectrin are different not only in molecular mass but also
in constitution. In fact, the
-spectrin chain consists of series of
22 repeats of 106 residues, whereas the
chain consists of 17 repeats of 106 residues (30). The tryptic digestion of spectrin gave
evidence of a linear disposition of trypsin-resistant domains (31).
Five domains were defined on the
chain (
I to
V from the N
terminus) and four on
chain (
I to
IV from the C terminus).
The heterodimer
-
assembly requires a specific nucleation site
located in the
V and
IV domains (32). A self-association between
the N-terminal region of the
chain and the C-terminal region of the
chain is involved in the formation of a tetramer, which is the
predominant form in the erythrocyte (33).
Moreover, spectrin forms noncovalent associations with other proteins of the cytoskeleton, such as band 2.1 (34), band 4.1, and actin (35). Other proteins such as adducin, tropomyosin, tropomodulin, and dematin function as accessory proteins of spectrin-actin junctions and are probably involved in the stabilization of spectrin-actin complexes (36). Furthermore, spectrin and protein 4.1 interact through phosphatidylserine with the inner leaflet of the lipid bilayer (37).
In an attempt to gain insight into the potential biological role of
-spectrin ubiquitination, we searched for the site(s) of
ubiquitination present on
-spectrin. The data reported in this study
show that ubiquitination occurs on the
III and
V domains of
-spectrin, suggesting that at least ubiquitination of the
V
domain can play a role in cytoskeleton stability mediated by the
-
-spectrin nucleation site.
Ubiquitin, chloramine T, and many biochemical reagents were obtained from Sigma. Reticulocyte fractions were prepared as reported previously (28). Immobilized protein A was obtained from Pierce. The ECL Western blotting detection reagents, Hybond N nitrocellulose and carrier-free Na125I, were from Amersham Corp. Endoprotease Lys C was from Boehringer Mannheim.
Ubiquitin LabelingReductive methylation of ubiquitin was carried out as described by Hershko and Heller (38). Native ubiquitin and methylated ubiquitin (meUb) were radiolabeled with carrier-free Na125I (Amersham Corp.) by the chloramine-T method (39). The specific activity obtained was 9400 cpm/pmol of ubiquitin for 125I-Ub and 9000 cpm/pmol of ubiquitin for 125I-meUb.
Electrophoresis and Western BlottingSDS-polyacrylamide gel electrophoresis (PAGE) was carried out according to the method of Laemmli (40) as reported previously by Corsi et al. (28).The molecular mass standards used were 94, 66, 45, 31, 21, and 14 kDa (Pharmacia Biotech Inc.). Thirty-five µg of sample protein were loaded for each lane, unless otherwise indicated.
The gels were electroblotted according to Towbin et al. (41)
using Hybond N nitrocellulose. Blots involving 125I-Ub
spectrin peptides were first dried and then exposed to obtain an
autoradiographic film of the nitrocellulose. After membrane rehydration, the different lanes were cut and incubated with different polyclonal IgG against each -spectrin domain (42).
Goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad) was used at a 1:3000 dilution as a second antibody. Enhanced chemiluminescence (ECL; Amersham Corp.) was used as the detection system.
Assay of Ubiquitin ConjugationHuman red blood cell membranes were prepared from healthy volunteers according to Corsi et al. (28). The conjugation of 125I-Ub to red cell membrane proteins was assayed as described previously (28) using 5 µM 125I-Ub or 125I-meUb (final concentration) for each incubation mixture and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) as antiproteolytic agent. The conjugation of 125I-Ub to brain fodrin was assayed in a similar way.
Crude Spectrin ExtractionAfter 120 min of incubation at
37 °C to permit 125I-Ub conjugation, the reaction
mixture was centrifuged in an Eppendorf microcentrifuge at 16,000 × g for 15 min at 4 °C. The supernatant was removed, and
the pelleted membranes were washed twice with phosphate-buffered saline, pH 7.4, containing 1 mM PMSF. The membranes were
then washed an additional two times with a low ionic strength buffer containing 0.3 mM Na2HPO4, pH 8.5, 0.1 mM EDTA, 1 mM PMSF, and 0.2 mM
AEBSF (extraction buffer) (43). The pellet thus obtained was
resuspended in a small volume of the extraction buffer (1 ml/5 mg of
membrane protein) and incubated at 37 °C for 30 min. The samples
were then centrifuged at 200,000 × g in a SW 65 rotor for 30 min at 4 °C, and the supernatant was collected. This
fraction, primarily 125I-Ub--spectrin,
-spectrin, and
-spectrin chains, actin, and traces of band 4.1, was referred to as
"crude spectrin." Total protein was adjusted to 1 mg/ml with
extraction buffer. Before protein determination, crude spectrin was
stored on ice at 4 °C overnight in 50 mM NaCl and 5 mM Na2HPO4, pH 8.0 (final
concentration).
Crude
spectrin extracted as above was dialyzed overnight at 4 °C using
dialysis tubing with a molecular weight cut-off of Mr 3500 against 2000 volumes of 10 mM Tris-HCl, pH 8.5, and 1 mM EDTA to eliminate
PMSF and AEBSF. Five µg of endoprotease Lys C were resuspended in 50 µl of H2O and added 1:10 (µl/µg of protein sample) to
crude spectrin. The digestion was for 1.5 or 3 h at 28 °C and
was stopped by adding 0.2 mM AEBSF and sample buffer 1:1
(v/v), after which the sample was boiled for 5 min. Thirty-five µg of
protein sample were used for each lane of SDS-PAGE, and the relative
autoradiogram of the gel was obtained. Quantitative determinations of
radioiodinated proteins were performed by direct counting of the
excised bands using a Beckman 5500 gamma counter. One hundred and
seventy-five µg of spectrin digestion products (35 µg for five
lanes) were used for Western blotting analysis. The nitrocellulose was
used first to obtain the autoradiograms and subsequently was cut in
five lanes and probed with IgG specific for each of the five different
domains of -spectrin.
125I-Ub
was conjugated to human red blood cell membranes for 2 h at
37 °C in the presence of rabbit reticulocyte fraction II as
described by Corsi et al. (28). Spectrin extraction and
digestion were performed as described above. The digestion was stopped
with 0.2 mM AEBSF, 1% (w/v) SDS, and boiling for 10 min.
The sample was diluted 100-fold with 10 mM Tris-HCl, pH
7.5, divided into 5 aliquots, and then separately incubated with
different antibodies against each domain of -spectrin (
I to
V)
previously buffered in 10 mM Tris-HCl, pH 7.5 (final
concentration). A 5.5-fold excess of specific anti-
-spectrin-domain
IgG (mol/mol) was used to form the antigen-antibody complex. The
antigen-antibody mixtures were left for 2 h at room temperature
and then overnight at 4 °C with gentle agitation. Five ml of
immobilized protein A were equilibrated with 10 volumes of 10 mM Tris-HCl, pH 7.5, then with 3 volumes of the same buffer
plus 1 mg/ml bovine serum albumin, and finally with 10 volumes of 10 mM Tris-HCl, pH 7.5. The protein A suspension was divided
into 1-ml aliquots, each of which was incubated overnight at 4 °C
with the different mixtures of antigen-antibody complexes formed as
above. The 5 aliquots of protein A suspension with bound antigen-IgG
complexes were packed in five different columns and washed with 10 volumes of 10 mM Tris-HCl, pH 7.5, until protein determination at 280 nm was zero. Five different eluates were obtained
from the columns with 0.2 M glycine, pH 2, and then counted in a gamma counter.
- and
-fodrin from bovine brain were purified as reported by Davis and
Bennet (44) and kindly provided by Dr. B. Geny (INSERM U 322, Paris,
France). The purified fodrin was left at 0 °C in 1 M
NaBr, 10 mM Tris-HCl, pH 8.2, 1 mM EGTA, 15 mM Na4P2O7, 1 mM NaN3, 1 mM dithiothrietol, and
dialyzed against 1000 volumes of 50 mM Tris-HCl, pH 7.5, 0.2 mM AEBSF at 4 °C for 5 h immediately prior to
use.
Protein concentrations were determined by the method of Bradford (45) using bovine serum albumin as standard or spectrophotometrically at 280 nm.
-Spectrin
is one of the most abundant membrane proteins (12.5%) in the
erythrocyte membrane (29). It is known that it can be digested with
trypsin into five trypsin-resistant domains of different molecular
weights known as
I,
II,
III,
IV, and
V (31). In an
attempt to identify the site(s) of ubiquitination on membrane
-spectrin, we first performed an in vitro ubiquitination assay using human erythrocyte membrane fraction II as a source of
ubiquitin-conjugating enzymes and 125I-Ub. After two
washing steps, crude spectrin was extracted as described under
"Experimental Procedures." In a first approach, extracted crude
spectrin was digested with trypsin at 1 mg/ml, 1:50 (µl/µg of crude
spectrin). Unfortunately, trypsin digestion of
125I-ubiquitinated spectrin produces many bands with low
labeling radioactivity. Furthermore, trypsin was also found to be able to digest 125I-Ub itself (46) and to cut polyubiquitin
chains (data not shown). Thus, spectrin peptide patterns were produced
using endoprotease Lys C instead of trypsin, and all of the data
reported hereafter in this report were obtained with this proteolytic
enzyme. The production of digested peptides was
time-dependent. Endoprotease Lys C did not cut ubiquitin
and produced four highly ubiquitinated bands of low molecular weight
(Mr 40,000, 36,000, 29,000, and 25,500) as
detected by autoradiography (Fig. 1B, lanes 1 and 2). Quantitative determinations obtained by gamma
counting of excised radiolabeled bands showed that the
Mr 36,000 and 25,500 peptides had an associated
radioactivity four and three times higher than the
Mr 40,000 and 29,000 bands (Fig.
1C).
Identification of
To
identify the site(s) of ubiquitination on erythrocyte -spectrin, we
performed the experiment represented in Fig.
2A. The conjugation of 125I-Ub to
-spectrin was obtained in a cell-free system using fraction II as a
source of ubiquitin-conjugating enzymes and spectrin extraction from
the membrane was performed as described above.
125I-Ub-
-spectrin was submitted to endoprotease Lys C
digestion. One fraction of the sample (35 µg) was analyzed by
SDS-PAGE and autoradiographed, whereas another fraction of the sample
was divided into five aliquots (each of 35 µg), processed for
SDS-PAGE on five different lanes, and Western blotted. The
nitrocellulose membrane was dried and autoradiographed (Fig. 2B,
odd numbers). The five lanes were then cut and probed separately
using five different antibodies against each domain of
-spectrin
(Fig. 2B, even numbers). The autoradiograms of the
nitrocellulose membranes and the films obtained by ECL were then
overlapped to observe the relative positions of the antibody-recognized
peptides compared to those of the radiolabeled peptides. The
autoradiogram of the gel (Fig. 2B, lane B) shows four
different radioiodinated bands with molecular weights of
Mr 40,000, 36,000, 29,000, and 25,500, as shown
previously in Fig. 1B, lanes 1 and 2. The
radioiodinated peptide of Mr 40,000 (Fig.
2B, a) was specifically recognized by IgG anti-
V (Fig.
2B, lanes 9 and 10), whereas the second
radioiodinated peptide of Mr 29,000 (Fig.
2B, c) was recognized by IgG anti-
III (Fig. 2B,
lanes 5 and 6). The two strongly radioiodinated
peptides of Mr 36,000 and 25,500 present in the
autoradiograms were not recognized by any IgG. According to the minimal
stoichiometry, one ubiquitin molecule is bound to each of the two bands
of Mr 40,000 and 29,000; therefore, the
molecular weights of the unconjugated peptides recognized by IgG
anti-
III and anti-
V are most likely Mr
31,000 and 20,000, respectively.
Identification of -spectrin ubiquitin
binding sites. A, scheme of the procedure used. Erythrocyte
membranes (900 µg) were incubated with 125I-Ub (5 µM) in the presence of ATP (3.5 mM) and
fraction II (800 µg) in a final volume of 3.1 ml of incubation
mixture. The membranes were then centrifuged at 16,000 × g and washed twice with phosphate-buffered saline, pH 7.4, containing 1 mM PMSF and 0.2 mM AEBSF to
eliminate fraction II proteins and unbound 125I-Ub. Crude
spectrin was extracted with a low ionic strength buffer. Extracted
crude spectrin was then dialyzed to eliminate antiproteolytic agents,
and endoprotease Lys C was added. After 3 h of digestion, polypeptides of different molecular weights were obtained. A portion of
the digestion products of crude spectrin was separated by SDS-PAGE and
analyzed by autoradiography. Another part of the digestion products was
divided into five aliquots and used for Western blotting analysis. An
autoradiogram of the nitrocellulose membranes was obtained, after which each of the
five nitrocellulose membrane lanes was probed with an antibody against
each domain of
-spectrin. Goat anti-rabbit IgG horseradish
peroxidase conjugate (Goat anti-rabbit HRP conjugate) was
used as second antibody and ECL as the detection system. Overlapping of
the autoradiograms and films obtained by ECL was used to determine
which antibody was able to recognize the radiolabeled polypeptides.
B, immunochemical identification of
-spectrin
ubiquitin-binding sites. One fraction (35 µg) of 125I-ubiquitinated
-spectrin digest was processed for
SDS-PAGE, stained with Coomassie Blue (lane A) and
autoradiographed (lane B). The other fraction was divided
into five aliquots (each of 35 µg), processed for SDS-PAGE, and
electroblotted onto five different nitrocellulose membranes.
Nitrocellulose membranes were dried and autoradiographed (odd
numbers). Then each of them was probed with a specific antibody
against each of the five
-spectrin domains (even
numbers). Dilution of the first antibody was: IgG anti-
I domain, 1:7,500; IgG anti-
II domain, 1:10,000; IgG anti-
III domain, 1:5,000; IgG anti-
IV domain, 1:40,000; and IgG anti-
V domain 1:7,500. The autoradiograms and the films obtained by ECL were
overlapped to identify the ubiquitinated peptides. a,
radioiodinated peptide of Mr 40,000 specifically
recognized by IgG anti-
V (lanes 9 and 10).
c, radioiodinated peptide of Mr
29,000 recognized by IgG anti-
III (lanes 5 and
6).
Presence of Polyubiquitin Chains in
An experiment identical to that described above
(Fig. 2A) was performed using 125I-meUb instead
of 125I-Ub. This particular ubiquitin derivative, although
a substrate for the ubiquitin-conjugating system, is unable to form
multiubiquitin chains (38). After SDS-PAGE, the digested
125I-meUb--spectrin was processed for Western blotting
analysis as in the experiment described above (Fig. 2A). In
the autoradiogram (Fig. 3, lane B), the two
bands of Mr 40,000 and 29,000 were present as in
the experiment with 125I-Ub (Fig. 1B, lanes 1 and 2). An additional band of Mr
38,000 was also found. The IgG against
V domain recognized the
ubiquitinated peptide of Mr 40,000 (Fig. 3,
a), whereas the IgG anti-
III recognized the ubiquitinated
peptides of Mr 29,000 (Fig. 3, c) and
Mr 38,000 (Fig. 3, b), confirming the
data obtained with 125I-ubiquitin. Moreover, the
Mr 38,000 peptide plus the peptide of
Mr 29,000 contained the same radioactivity found
in the Mr 29,000 band of the previous experiment
in which 125I ubiquitin was used.
Coomassie Blue staining of digested spectrin did not reveal any
difference using either the 125I-Ub or
125I-meUb derivatives in the conjugation assay. However,
some differences in the Mr 40,000 range were
evident in the films obtained by probing the membranes with IgG
anti-III and anti-
IV (Fig. 2B, lanes 6-8; Fig. 3,
lanes 6-8). These minor differences were not further investigated and could be due to different conformations of
-spectrin when Ub chains are bound, as well as to the relative
proteolytic susceptibility of multiubiquitinated versus
monoubiquitinated
-spectrin. Interestingly, when
125I-meUb was used, the two radioiodinated bands of
Mr 36,000 and 25,500 were no longer present.
Thus, it must be concluded that when using fraction II from rabbit
reticulocytes, the cytoskeletal protein
-spectrin is
multiubiquitinated, at least in vitro, and that the peptides
of Mr 36,000 and 25,500 correspond to tetra and
tri multiubiquitin chains.
To
directly demonstrate that ubiquitin is bound to the III and
V
domains of
-spectrin, we used a second approach, as described in the
scheme of Fig. 4A. Endoprotease Lys
C-digested spectrin was divided into five identical aliquots, each of
which was incubated with IgG against each domain of
-spectrin. Five
columns of immobilized protein A were used to retain IgG. Free
ubiquitin and digested peptides not recognized by IgG were removed
during the washing steps of the columns. The proteins retained and
eluted from the protein A columns were collected and counted in a gamma
counter. As shown in Fig. 4B, eluates from columns receiving
IgG anti-
I and anti-
II domains showed a very low radioactivity,
probably due to some nonspecific interaction between free
125I-Ub and the column. Eluate from the column with bound
IgG anti-
IV domain showed a higher radioactivity than the latter
eluates, whereas only the eluates from columns with bound IgG
anti-
III and anti-
V domains showed a strong radioactivity. This
experiment provides direct proof that the ubiquitinated peptides of
-spectrin are specifically recognized by IgG anti-
III domain and
anti-
V domain of
-spectrin. Thus, it must be concluded that
ubiquitin binding sites of
-spectrin are present in these two
domains.
Ubiquitination of Non-Erythroid Spectrin-like Protein
Brain spectrin (fodrin) was used as substrate for an in vitro conjugation assay using 125I-Ub in the presence of rabbit reticulocyte fraction II. Conjugation was stopped at 120 min with sample buffer, and the sample was boiled for 5 min and electrophoresed in SDS-polyacrylamide gels, stained, dried, and autoradiographed. No radioactive bands were found at the expected molecular weight of fodrin (Mr 260,000 and 225,000), indicating that this protein is not ubiquitinated, at least in vitro.
Spectrin is the principal component of the erythrocyte membrane
skeleton and plays a dominant role in determining such mechanical properties of the erythrocyte as elasticity and deformability (47).
Membrane equilibrium depends on the structural integrity of the
skeletal proteins and on normal molecular interactions between the
cytoskeletal proteins and membrane. Moreover, the binding of cytosolic
components such as enzymes and hemoglobin to cytoskeletal proteins can
play a role in membrane stability. Among the factors that may serve as
regulators of cytoskeletal organization is protein
phosphorylation-dephosphorylation (36). -Spectrin has been reported
to be a substrate for cytosol and membranous casein kinases. In
particular, this chain contains a cluster of six phosphorylation sites.
Phosphorylation of spectrin has been shown not to affect either
dimer-dimer associations (48) or spectrin binding to ankyrin in
vitro (49). However, phosphorylation affects spectrin
inextractability from "inside-out" vesicles (50) and modulates the
mechanical function and stability of the intact membrane structure
(51). Other mediators, such as Ca2+ and calmodulin, can
also regulate membrane stability (52). Interestingly, free calmodulin
can be ubiquitinated in a Ca2+-dependent manner
and subsequently degraded, a process which could act as a control
mechanism for all free calmodulin in excess (53). Recently, we
described and characterized a new posttranslational modification of
erythrocyte
-spectrin in which ubiquitin binds covalently to the
-spectrin chain (28).
In this report, using different approaches, we demonstrate the
existence of two binding sites for ubiquitin on -spectrin. Digestion
with endoprotease Lys C of ubiquitinated spectrin revealed the presence
of two 125I-Ub peptides of Mr 40,000 and 29,000. As shown by Western blotting, these radiolabeled peptides
were recognized by polyclonal IgG anti-
V domain and anti-
III
domain, respectively, indicating that these two peptides are the sites
of ubiquitination on red blood cell
-spectrin. It could be
speculated that
-spectrin ubiquitination on domains III and V may
play a role in membrane stability as found for other mediators of red
blood cell membrane. The
III domain is not associated to any known
function, whereas the
V domain contains the nucleation site for
association with the
chain (32) and is involved in Ca2+
binding (54) and, with the
IV domain, participates in the interaction with actin and protein 4.1 (55). Interestingly, the
V
domain is involved in the ubiquitination of
-spectrin and contains
repeats 20 through 22, which exhibit atypical features. In fact, there
is insertion of several amino acids into the repeats 20 and 21. Moreover, repeat 22 has a reduced homology to a typical spectrin
repeat. Moreover, the nucleation site present in the
V domain is not
only responsible for the initial
-
spectrin binding but also
controls the side-to-side register of the many homologous repeats in
both subunits. An unusual feature of the nucleation regions is that
three of the repeats (two in the
and one in the
subunits) have
an eight-residue insertion in the normal 106-residue repeat unit (32).
These eight-residue insertions, which contain a lysine residue, might
confer unique conformational properties upon the nucleation site, and
the ubiquitination of domain
V might play a role in this context.
The erythroblast-to-erythrocyte maturation process is accompanied by
changes in the composition and properties of the plasma membrane.
Furthermore, mature erythrocytes are incapable of ubiquitin/26 S
proteosome-dependent degradation (56). Thus, ubiquitination
of
-spectrin could play two different roles during erythrocyte
maturation. In erythroblasts, the amount of
-spectrin synthesized
exceeds by more than 3-fold the amount assembled on the membrane, and
the excess unassembled peptides are rapidly degraded (57). It could be
speculated that the binding of ubiquitin to
-spectrin in
erythroblasts involves subsequent degradation. It is important to note
that the sequence of
-spectrin contains a glutamic residue in the
first position that may act as a secondary destabilizing residue
according to the N-end rule (58) when spectrin is in the unassembled
form. The second determinant, a specific internal lysine residue, could
be the first lysine located at position 5 or 15 (domain I) of the
-spectrin sequence. Excess hemoglobin subunits are subject to an
analogous targeted degradation in thalassemia (59). Because, as shown
in this report, ubiquitination occurs in mature red blood cells on the
III and
V domains, and thus quite far from the
I domain
(moreover, these cells are incapable of ubiquitin/26 S
proteosome-dependent degradation), the ubiquitination
process of assembled
-spectrin probably has a different role in
these cells than in erythroblasts. Ubiquitin itself and/or
multiubiquitin chains could have a potential function as
conformation-perturbing devices when conjugated to cytoskeletal proteins, given their orientational flexibility and reversibility (60).
Thus, we suggest that the ubiquitination of
-spectrin in mature
erythrocytes should be considered a new posttranslational event with a
regulatory role in spectrin function rather than a signal for
-spectrin degradation. In fact, ubiquitination is a dynamic process
(28), the linkage is covalent, and a significant amount (3% of the
total
-spectrin chain) is continuously ubiquitinated in
erythrocytes. As reported previously for globin-spectrin complex formation during erythrocyte senescence (61), such an amount could
account for a significant change in membrane deformability. We also
investigated whether other proteins belonging to the spectrin superfamily are ubiquitinated. Brain
-fodrin was not found to be a
substrate for ubiquitin in an in vitro assay. This
non-erythroid spectrin and erythroid
-spectrin have very similar
sequences (54% identity) throughout their entire length (62), but
interestingly, fodrin at its C-terminal has an atypical sequence of 150 residues in repeat 22 (
V domain), and the identity of the 37 residues at the very C-terminal is less than 10%. Moreover, fodrin
differs considerably from erythrocyte
-spectrin in repeats 11 and 12 (
III domain) and possesses a calmodulin-binding site in the latter repeat that is absent in
-spectrin (63). Thus, erythrocyte
-spectrin and brain fodrin differ mainly in the domains found to be
susceptible to ubiquitination. It would be interesting to examine
whether
-actinin, another protein of the spectrin superfamily, is
ubiquitinated. In particular, repeats 20 through 22, together with the
nonrepeat C terminus of
-spectrin, are highly homologous with the C
terminus of
-actinin (64). Preliminary studies now in progress in
our laboratory indicate that
-actinin is ubiquitinated, at least
in vivo. To date, no information is available on the function of the
III domain of
-spectrin, but because the carboxyl terminus of the
-spectrin subunits (
V domain) is involved in the
binding of the
-spectrin chain to form the
-
dimer and is an
important site for many mediators in red blood cells, the ubiquitination of this cytoskeletal protein may be of physiological significance.