From the
Ubiquitination of red blood cell (RBC) proteins was investigated
by encapsulation of
Ubiquitin conjugation
was ATP-dependent ( K
Ubiquitin (Ub)
The best understood role of protein ubiquitination is
certainly in protein degradation
(35) . In recent years,
however, there has been increasing evidence that the ligation of Ub to
proteins is implicated in a great variety of biological processes,
including cell cycle control
(8) , the modulation of receptor
function
(36) , and heat-shock response
(11) . In
reticulocytes, most of ubiquitin is conjugated to endogenous proteins.
An active ubiquitin and ATP-dependent proteolytic system in this cell
is responsible for the transition from the reticulocyte stage to the
mature erythrocyte stage
(22) . Mature erythrocytes no longer
have an active ubiquitin-dependent proteolytic system, essentially
because of the maturation-dependent decay of some of the E2s
responsible for Ub conjugation
(23) . However, in mature
erythrocytes the total ubiquitin content is similar to that found in
reticulocytes
(22) , and 30% of this is still conjugated to
endogenous proteins. Mature erythrocytes thus represent an exceptional
cell model to investigate protein ubiquitination independently of their
commitment to degradation.
Based on these considerations, we started
a study aimed at identifying the natural substrates of ubiquitination
in mature erythrocytes. The experimental approaches selected aimed at
investigating ubiquitination under conditions as close as possible to
those found in intact cells. Toward this end we used an encapsulation
procedure based on hypotonic hemolysis and isotonic resealing
(37) for the entrapment of
Why only this
membrane protein is ubiquitinated in mature erythrocytes is presently
unknown. From the stoichiometry of ubiquitin conjugation we calculated
that only a small fraction (1.71 ± 0.02%) of total
In conclusion, a new physiological substrate for protein
ubiquitination has been found. Others
(12, 41) have
previously reported the ubiquitination of cytoskeletal proteins. In the
erythrocyte,
Human RBC were submitted to a procedure
of hypotonic dialysis, isotonic resealing and reannealling as reported
under ``Experimental Procedures'' using 30 µg of
We gratefully thank A. Haas (Medical College of
Wisconsin, Milwaukee, WI) for helpful discussion and suggestions, J.
Callis (University of California, Davis, CA) for the generous supply of
anti-ubiquitin antibodies. We are very thankful to P. S. Low (Purdue
University, West Lafayette, IN) for teaching one of us (D. Corsi)
several methods used in studies involving the erythrocyte membrane.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-ubiquitin into human erythrocytes
using a procedure of hypotonic dialysis, isotonic resealing, and
reannealing. Incubation (37 °C, up to 2 h) of
I-ubiquitin-loaded cells resulted in the recovery of
I-ubiquitin with the cytosolic proteins (9.22 ±
0.4 µg/ml RBC) and conjugated to membrane proteins (2.18 ±
0.05 µg/ml RBC). This conjugation was time-dependent, and the
predominant membrane protein band that became labeled showed an
apparent molecular mass of 240 kDa on SDS-polyacrylamide gel
electrophoresis (PAGE). Western blotting experiments with three
different anti-ubiquitin antibodies revealed that this protein is also
ubiquitinated in vivo. Cell-free experiments have shown that
fraction II (a DEAE-bound protein fraction eluted by 0.5
M KCl) prepared from both mature erythrocytes and reticulocytes is
able to conjugate ubiquitin to this protein.
0.09 m
M),
time-dependent, and fraction II-dependent (8 ± 0.5 pmol of
I-ubiquitin/h/mg of fraction II). Isolation of the major
RBC membrane protein that is ubiquitinated was obtained by using
biotinylated ubiquitin. Membrane proteins, once ubiquitinated with this
derivative, were extracted and purified by affinity chromatography on
immobilized avidin. The major components retained by the column were
two peptides of molecular masses 220 and 240 kDa. Both proteins are
recognized by a monoclonal anti-spectrin antibody, but only the 240-kDa
component is detected by streptavidin peroxidase conjugate. That indeed
the ubiquitinated membrane protein of 240-kDa is
-spectrin was
confirmed by immunoaffinity chromatography using
I-ubiquitin and a monoclonal anti-spectrin antibody.
Antigen-antibody complexes were purified by protein A chromatography
and analyzed by SDS-PAGE and autoradiography. Again two bands of 240
and 220 kDa were eluted (
- and
-spectrin), but only one band
corresponding to the electrophoretic mobility of
-spectrin was
detected by autoradiography. Thus,
-spectrin is a substrate for
the ATP-dependent ubiquitination system, suggesting that the
cytoskeleton is covalently modified by ubiquitination both in
reticulocytes and mature RBC.
(
)
is a polypeptide of
molecular mass 8565 Da, highly conserved, and found both free and
covalently conjugated to other proteins in all eukaryotic cells
(1) . The covalent attachment of Ub to protein substrates occurs
by three enzymes called E1, E2, and E3
(2, 3, 4) . A protein can be conjugated with one
or more ubiquitin moieties
(5) . In multi-Ub chains successive
Ub are linked by an isopeptide bond involving the side chain of Lys-48
and the carboxyl group of Gly-76. The Ub conjugates may be: 1) degraded
by an ATP-dependent protease releasing amino acids and intact
ubiquitin, or 2) the Ub moiety may be removed by Ub-protein
isopeptidases releasing free intact protein and intact ubiquitin.
Moreover, isopeptidases may release free Ub from proteolytic breakdown
products
(5) . Usually the formation of polyubiquitin chains is
necessary but not obligatory
(6, 7) for protein
breakdown, although multiply ubiquitinated conjugates appear to have a
kinetic advantage in degradation. Until now ubiquitin has been found to
be conjugated with histones
(1) , suggesting a role in
transcriptional regulation, to cyclin
(8) which is involved in
cell cycle regulation, and to different proteins in the cytoplasm and
endoplasmic reticulum
(9) where ubiquitin is involved in
nonlysosomal proteolysis. Furthermore, ubiquitin has also been found to
be bound to several receptors, including the homing receptor
(10) , the growth hormone receptor, immunoglobulin E receptor,
and the platelet-derived growth factor receptor
(11) . Other
evidence includes the ubiquitination of microtubule-associated proteins
(12) and involvement in heat-shock response
(10) and
viral coat proteins
(13) . Thus, the best known function of Ub
is the
(14) marking of proteins destined
(15) for
selective elimination, but protein ubiquitination must have other
regulatory roles too. It is becoming increasingly evident (see above)
that the ligation of Ub to proteins is implicated in the control of a
great variety of biological processes
(16) . In particular
monoubiquitination may serve such a role
(17, 18) . In
fact the ligation of a single or few Ub molecules to proteins appears
to be implicated in the direct altering of the structure and function
of conjugates, affecting their interaction with other cellular
components
(19) and accounting for a nonproteolytic role for Ub
and for the existence of metabolically stable Ub-protein conjugates
(20, 21) without a certain known role(s). Most of our
knowledge about the Ub pathway comes from the characterization of this
pathway in rabbit reticulocytes or from in vitro studies using
cell-free rabbit reticulocyte extracts
(22) . Reticulocytes
contain both a stable and labile pool of Ub conjugates, whereas
erythrocytes contain a pool of conjugates that is relatively stable
(23) . Maturation of reticulocytes to erythrocytes, and
subsequent erythrocyte aging involves significant cellular remodeling
(22) . During this maturation the activities of a large number
of pathways are lost by selective turnover of key enzymes. Mature
erythrocytes have virtually no Ub- and ATP-dependent protein
degradation, but substantial levels of Ub-protein conjugates remain
(24) . Comparisons of conjugate pools within these two types of
cells showed that 83% in reticulocytes and 31% in erythrocytes of total
intracellular Ub exist covalently bound to target proteins. The absence
of energy-dependent proteolysis in mature erythrocytes is not a
consequence of loss in active Ub or a consequence of limited substrate
availability
(23) , rather it mainly results from the decay in
the level of some components required for conjugation and subsequent
breakdown such as active E2s
(20, 24) . The
highly active reticulocyte Ub system is excellent for studying the Ub
pathways and for identifying Ub conjugates in vitro (25) . Thus, mature erythrocytes represent an ideal system
for investigating protein ubiquitination when this process is not
followed by protein degradation. This fact, and the availability of a
procedure for the encapsulation of labeled ubiquitin without affecting
the main cellular functions, allowed us to investigate ubiquitin
conjugation in intact mature erythrocytes providing clear evidence for
the conjugation of Ub to a previously unidentified substrate, namely
the cytoskeletal protein
-spectrin. The functional role of this
process is not yet clear; however, spectrin represents the major
ubiquitinatable substrate in mature red cells.
Materials
Ubiquitin, chloramine T, monoclonal
antibody to human spectrin (clone SB-SP1), and anti-ubiquitin were
obtained from Sigma. Reticulocytes and erythrocyte fractions were
prepared as previously reported
(2) . The lysate was
fractionated on a DEAE-52 column. The unadsorbed material was
designated fraction I and contained ubiquitin, whereas proteins
adsorbed into the resin and eluted by 0.5
M KCl at pH 7.9 were
fraction II
(2) . 1 m
M PMSF, 1 µ
M leupeptin, 1 µ
M pepstatin, 0.5 m
M diisopropyl fluorophosphate, and 20% (v/v) glycerol were present
in the chromatographic buffer used in the preparation of fraction II.
N-Hydroxysuccinimide biotin, Coomassie Plus protein assay
reagent, and immobilized avidin (1-ml prepacked columns) were from
Pierce. Immobilized protein A was from Repligen. The ECL Western
blotting detection reagents and iodine-125 (100 mCi/ml) were from
Amersham International (Milan, Italy).
Ubiquitin Derivatives
Ubiquitin was radiolabeled
with carrier-free NaI (Amersham Corp.) by the
chloramine-T method as described in Ref. 26. The specific activity
obtained was 1.7
10
cpm/g. Ubiquitin (1 mg/ml) was
biotinylated by N-hydroxysuccinimide biotin (Pierce) following
the manufacturer's protocol.
Human erythrocytes were washed twice in 10
m
M Hepes containing 140 m
M NaCl, 5 m
M glucose, pH 7.4 (buffer A) to remove leukocytes and platelets and
were resuspended at 70% hematocrit. These cells were dialyzed for 45
min using a tube with a cutoff of 12-14 kDa against 50 volumes of
10 m
M NaHI-Ubiquitin Encapsulation in
Erythrocytes
PO
containing 20 m
M glucose, 4 m
M MgCl
, 3 m
M reduced
glutathione, 2 m
M ATP, pH 7.4. The osmolarity of this buffer
was 58 mOsm. After this time the dialyzed erythrocytes were transferred
to a tube with a cutoff of 3 kDa and 30 µg/ml of
I-ubiquitin were added. Dialysis was continued for a
further 15 min. All these procedures were performed at 4 °C.
Resealing of the erythrocytes was obtained by adding 0.1 volume of 5
m
M adenine, 100 m
M inosine, 2 m
M ATP, 100
m
M glucose, 100 m
M sodium pyruvate, 4 m
M MgCl
, 0.194
M NaCl, 1.6
M KCl, 35
m
M NaH
PO
, pH 7.4, per volume of
dialyzed erythrocytes and by incubation at 37 °C for 20 min.
Resealed cells were then washed three times in buffer A and used for
the experiments reported under ``Results.'' The resealed RBC
had normal glycolytic rates (2.8 ± 0.2 µmol of lactate/h/ml
of RBC) and normal ATP concentrations (1.4 ± 0.1 m
M).
Determinations were performed as described previously
(27) .
Preparation of Erythrocyte Membranes and Membrane
Proteins
Erythrocyte membranes were prepared by hemolysis in 5
m
M NaHPO
, 1 m
M PMSF, pH 8.0,
and washed until white. The cytoskeleton was prepared as described in
Ref. 28 with slight modifications. Triton X-100 was 0.5% (v/v), and
urea extraction was omitted. Spectrin dimers were prepared by
extraction of human erythrocyte membranes in low ionic strength buffer,
at 37 °C, pH 9.5, as described in Ref. 29, and gel filtration
(30) . Spectrin concentration was obtained by using Centriprep
concentrators from Amicon with a cutoff of 30-kDa.
Electrophoresis and Western
Blotting
SDS-polyacrylamide gel electrophoresis (PAGE) was
carried out using a mini-gel apparatus (Bio-Rad), unless otherwise
indicated, according to Laemmli
(31) . Samples were boiled at
100 °C for 5 min in sample buffer containing 4% mercaptoethanol.
Staining was with Coomassie Blue R-250 or with silver (Bio-Rad). The
gels were then fixed, dried, and autoradiographed with Kodak X-Omat AR
film at -70 °C in the presence of Du Pont Lighting Plus
intensifying screens. Exposure times were adjusted so that signal was
within the linear response range of the film. Quantitative
determinations were performed by direct counting the bands excised from
the dried gels in a Beckman 5500 -counter. Molecular mass
standards used were 200 kDa, 116 kDa, 97 kDa, 66 kDa, 45 kDa, from
Bio-Rad. The amount of ubiquitin bound was calculated from the specific
radioactivity of
I-Ub as described in Ref. 32.
Alternatively, the exposed films were scanned with an LKB Ultroscan XL
enhanced laser densitometer and the densitometric values compared with
those obtained by known amounts of
I-Ub. In some
experiments the gels were electroblotted according to Towbin et al. (33) . Blots involving biotinylated ubiquitin were
developed using streptavidin horseradish peroxidase conjugate (1:3,000
dilution, Amersham Life Science) and ECL. Blots involving native Ub
were first incubated with affinity-purified rabbit polyclonal anti-Ub
antibodies 1:1,000 dilution (provided by A. Haas, Medical College of
Wisconsin, Milwaukee, WI) or with anti-Ub antibodies (provided by J.
Callis, University of California, Davis, CA) and then with goat
anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad) 1:3,000
dilution or with protein A-horseradish peroxidase conjugate (1:1,000
dilution) was used as a secondary antibody. Blots involving native
spectrin were first incubated with monoclonal antibody anti-spectrin
(Sigma) and then with goat anti-mouse horseradish peroxidase conjugate
(Bio-Rad) 1:3,000 dilution. Enhanced chemiluminescence (ECL) was used
as detection system.
Assay of Ubiquitin Conjugation
The conjugation of
ubiquitin to red cell membranes was assayed by incubation of rabbit or
human red cell membranes with I-Ub in the presence of
fraction II. The reaction mixture contained (unless otherwise
indicated), in a final volume of 150 µl, 75 m
M Tris-HCl,
pH 7.5, 5 m
M MgCl
, 3 m
M dithiothreitol, 3
m
M ATP, 10 m
M creatine phosphate, 10 µg of
creatine phosphokinase, 60 µg of fraction II, 0.8
10
cpm of
I-Ub, 0.5 m
M diisopropyl
fluorophosphate, 1 m
M PMSF, 1
M leupeptin, 1
M pepstatin, and 50 µg of membranes. At time 0, 60, and 120 min
of incubation at 37 °C aliquots of the reaction mixture (45 µl)
were removed and the membranes pelleted by centrifugation in an
Eppendorf microcentrifuge at 16,000
g for 15 min at 4
°C. The supernatants were discharged (unreacted
I-Ub
and other components of the reaction mixture), whereas the pellets were
resuspended in 150 µl of PBS, pH 7.4, containing 1 m
M PMSF
and centrifuged again. The final membrane pellet was boiled for 5 min
in Laemmli sample buffer
(31) containing 2% (v/v)
mercaptoethanol. Control samples without ATP and the ATP regenerating
system were incubated as above. All samples were then electrophoresed
in SDS-polyacrylamide gels, fixed, dried, and autoradiographed.
Isolation of Ubiquitinated Proteins from Red Cell
Membranes
Human erythrocyte membranes were incubated in the
presence of biotinylated ubiquitin and the other components of the
reaction mixture as described under ``Assay of Ubiquitin
Conjugation.'' Crude spectrin was extracted (see above) and
concentrated to a final volume of 2 ml. This protein solution was
loaded onto a column of immobilized avidin (Pierce), equilibrated,
washed, and eluted according to the manufacturer's suggestions.
Eluted proteins were then precipitated by adding 1 volume of cold 20%
(w/v) trichloroacetic acid in methanol. After 20 min at 0 °C the
solution was centrifuged at 16,000 g for 20 min and
the protein pellet resuspended in sample buffer, pH 9, and used in the
electrophoretic and Western blotting procedures. As an alternative
procedure for the identification of ubiquitinated membrane proteins the
erythrocyte membranes were incubated with
I-Ub and then
spectrin was extracted as above. A monoclonal antibody against spectrin
(clone SB-SP1, Sigma) was added to the spectrin solution (66 µg of
IgG/ml of spectrin solution) and incubated for 3 h at room temperature.
Antigen-antibody complexes were isolated by adding 0.25 ml of
immobilized protein A and agitated overnight at 4 °C. The resin
suspension was packed into a small column, the column was washed with
20 volumes of 10 m
M Tris-HCl, pH 7.5, containing 0.3
N NaCl and then eluted using 150 µl of hot (80 °C) sample
buffer. Eluted samples were then used for electrophoresis and
autoradiography.
Other Determinations
Protein concentration was
determined by the method of Bradford
(34) using bovine serum
albumin as a standard. Alternatively, protein concentration was
determined spectrophotometrically at 280 nm. Scanning of autoradiograph
was by an LKB laser scan densitometer.
Encapsulation of
Human red blood cells were submitted to a
procedure of hypotonic dialysis, isotonic resealing, and reannealing to
encapsulate I-Ubiquitin in Human
Red Blood Cells
I-Ub. The first dialysis step (see
``Experimental Procedures'' for details) was performed using
a dialysis tube with a 12-kDa cutoff that allowed the removal of
unbound ubiquitin (70% of total Ub, Ref. 32), whereas the second
dialysis was done in a dialysis tube with a cutoff of 3 kDa in the
presence of
I-Ub (30 µg/ml RBC; 1.7 10
cpm/g) to encapsulate the labeled protein. At the end of the
procedure 11.4 ± 0.6 µg of
I-Ub were
encapsulated into each ml of packed RBC (38% of the
I-Ub
used). Preparation of the ``soluble fraction'' and of the
membranes from this sample showed that 89% of encapsulated
I-Ub was in the cytosol while 6% was membrane bound
(). One unexpected finding was the evidence of bound
ubiquitin to the RBC membranes. The experiments reported above were
repeated preparing the cytosol and the membranes after 0, 30, 60, and
120 min of incubation at 37 °C of the intact RBC loaded with
I-Ub. As shown in Fig. 1 there was a time-dependent
incorporation of
I-Ub into a membrane protein of 240 kDa
with an electrophoretic mobility similar to
-spectrin. This
protein appears to be the major ubiquitinatable substrate in mature
RBC. Quantitative evaluation of
I-Ub bound to membrane
protein after 2-h incubation was performed by
counting of the
membrane fraction and showed 80 ± 3 pg of Ub/g of membrane
protein. Since the conjugation of ubiquitin is usually an ATP-dependent
process, we have also measured the ATP concentration of resealed RBC at
the same time points. After 2-h incubation the cells still contained
70% of the ATP present at time 0 (1.4 m
M).
Ubiquitin Is Bound to Erythrocyte Membrane Proteins in
Vivo
In an attempt to investigate whether the conjugation of
I-Ub to membrane protein is a natural event or is caused
by the procedure of encapsulation used, we tested (by Western blotting)
the presence of ubiquitin conjugates on human and rabbit erythrocyte
membranes. Three polyclonal anti-ubiquitin antibodies were used. One
antibody was from a commercial source (Sigma) and two others were
kindly obtained from A. Haas (Medical College of Wisconsin) and J.
Callis (University of California at Davis). All three antibodies
recognize different protein bands. However, after the absorption of
these antibodies with free ubiquitin, only a membrane protein of
molecular mass 240 kDa appears to be specifically detected. The results
obtained with one of these antibodies (A. Haas' antibody) are
shown in Fig. 2. Qualitatively similar results were obtained with
the other antibodies. Thus, it must be concluded that the staining with
anti-ubiquitin antibodies is specific and that ubiquitin in vivo is normally conjugated to the same membrane protein of 240 kDa
detected by loading intact erythrocytes with
I-Ub.
Figure 2:
Immunological detection of ubiquitin
conjugates in human and rabbit erythrocyte membranes. Rabbit ( odd
numbers) and human ( even numbers) erythrocyte membrane
proteins (16 µg/line) were separated by SDS-PAGE in a 6%
polyacrylamide gel and stained with Coomassie Blue ( A) or
electroblotted as in Ref. 33 and probed with an anti-ubiquitin antibody
provided by A. Haas (Medical College of Wisconsin). B, D, and
F, fast green-stained nitrocellulose; C,
anti-ubiquitin antibody (diluted 1:1,000)- and goat anti-rabbit IgG
peroxidase-conjugated (Bio-Rad, diluted 1:5,000); detection was by
enhanced ECL. E, as in C except that the first
antibody was adsorbed with an excess of ubiquitin (21 mg/ml of antibody
solution); G, as in C but without the first antibody.
The arrow indicates the only protein band which appears to be
recognized specifically by the anti-ubiquitin
antibody.
In Vitro Conjugation of Ubiquitin to Erythrocyte Membrane
Proteins
The conjugation of ubiquitin to the 240-kDa membrane
protein was characterized in a cell-free system consisting of
erythrocyte membranes and fraction II as a source of conjugating
enzymes. First of all, since several proteins present in fraction II
can be substrates for ubiquitin conjugation, we developed a new Ub
conjugation assay. In our assay the incubation mixture was centrifuged
at each time point, the pelleted membranes were washed, resuspended in
sample buffer, and analyzed by SDS-PAGE and autoradiography (or by
Western blotting and ECL using a streptavidin-peroxidase conjugate to
detect biotinylated ubiquitin when this was used instead of
I-Ub). This system allows the unequivocal detection of a
major component in the membrane pellet that is ubiquitinated in an
ATP-dependent and time-dependent process (Fig. 3). Determination
of initial rates provided values of 8 ± 0.5 pmol of Ub
conjugated per h/mg of fraction II and show an optimum pH value of 7.6
(Fig. 3 B).
I-Ub conjugation depends on the
concentration of the acceptor substrate (membrane protein), shows
saturable hyperbolic kinetics with a K
of 0.22
± 0.02 mg of membrane protein/ml of reaction mixture, and also
depends on the concentration of ubiquitin with a K
of 0.15 ± 0.03 µ
M (not shown). Fraction II
prepared from both rabbit reticulocytes or human erythrocytes are able
to conjugate
I-Ub to the 240-kDa membrane protein at a
similar rate (7 ± 0.5 pmol of Ub conjugated per h/mg of fraction
II from reticulocytes and 8 ± 0.5 pmol of Ub conjugated per h/mg
Fraction II from erythrocytes).
Figure 3:I-Ubiquitin conjugation to
human erythrocyte membranes in a cell-free system. Human erythrocyte
membranes were incubated at 37 °C in the presence of human
erythrocyte fraction II, ATP, and the other components of the reaction
mixture as indicated under ``Experimental Procedures.'' At
different time points, aliquots of the reaction mixture were removed
and centrifuged in an Eppendorf microcentrifuge. The membrane pellets
were washed, resuspended in Laemmli sample buffer (31), separated by
SDS-PAGE, and analyzed by autoradiography. Each line received the
equivalent of 12 µg of membrane protein. The gel was then sliced
and the band corresponding to the 240-kDa protein that appears to be
ubiquitinated was counted in a
-counter. A, time
dependence of
I-Ub conjugation. B, pH
dependence.
Biotinylated Ubiquitin Is Conjugated to the 240-kDa
Membrane Protein
Biotinylated ubiquitin was also tested as a
substrate for protein ubiquitination essentially for two reasons,
i.e. first to validate I-Ub as a tracer for
ubiquitin conjugation and second for the experiments that will be
reported below. In these experiments detection was by streptavidin
peroxidase and ECL after Western blotting. As shown in
Fig. 4
biotinylated ubiquitin is conjugated to the same 240-kDa
membrane protein both in human and rabbit membranes in an ATP-dependent
process. Particularly when using rabbit erythrocytes few membrane bands
of lower molecular mass are recognized by the second reagent
(streptavidin peroxidase) but could be easily distinguished from the
specific ubiquitin conjugates (data not shown), since these also appear
in the absence of biotinylated ubiquitin and independently of ATP and
incubation time.
Figure 4:
Biotinylated ubiquitin conjugation to
rabbit ( lanes 1) and human ( lanes 2 and 3)
erythrocyte membranes. All incubations were performed for 1 h at 37
°C with or without ATP as indicated. In lanes 3 biotinylated Ub and Fraction II were omitted. A lane (labeled
UB) containing only biotinylated ubiquitin is also shown (0.5
µg/lane). Detection of biotinylated ubiquitin conjugates was
obtained by streptavidin-peroxidase and ECL.
Kinetics of Formation and Decay of Ubiquitin
Conjugates
The formation and decay of ubiquitin conjugates with
protein membranes were examined by using both I-ubiquitin
and biotinylated ubiquitin. Human erythrocyte membranes were first
incubated with
I-Ub in the presence of fraction II and
ATP. Samples were taken at different incubation times and processed for
SDS-PAGE as above. After 1 h at 37 °C the membranes from this first
incubation were collected by centrifugation, washed in PBS, and
submitted to a second incubation in the presence of biotinylated
ubiquitin, new fraction II, and ATP. Further samples were taken at
different intervals and processed in duplicate for SDS-PAGE. Half of
the gel was dried and autoradiographed, and the second half was
transferred by Western blotting on a nitrocellulose membrane that was
processed for the detection of ubiquitinated proteins by streptavidin
peroxidase and ECL. As shown in Fig. 5,
I-Ub is
incorporated into the 240-kDa membrane protein in a time-dependent
process. This
I-Ub-protein complex dissociates upon
incubation in the presence of biotinylated ubiquitin that becomes
incorporated by replacing the iodinate derivative. Direct counting in a
-counter of the gel slices corresponding to the 240-kDa membrane
protein provided values for ubiquitin incorporation of 8 pmol/h/mg of
fraction II and decay values of 6.3 pmol/h/mg of fraction II. Similar
results were obtained with fraction II from reticulocytes or
erythrocytes. Thus, the conjugation of ubiquitin to the erythrocyte
membrane 240-kDa protein is a dynamic process.
Figure 5:
Kinetic formation and decay of
I-ubiquitin conjugates. Human erythrocyte membranes were
incubated in the presence of
I-Ub, fraction II, and ATP.
Samples were taken at different time intervals during 1-h incubation at
37 °C and processed for SDS-PAGE and autoradiography. The membranes
from the remaining part of the reaction mixture were washed and
re-incubated with fresh fraction II, ATP, and biotinylated ubiquitin.
Samples were taken at different times in duplicate and processed for
SDS-PAGE and autoradiography or SDS-PAGE and Western blotting. In this
case detection was with streptavidin-peroxidase and ECL. The left
part of the figure show a scheme of the experiment with results.
The right part shows the quantitative values of
I-Ub incorporation in the 240-kDa membrane protein before
(time 0-60 min) and after (time 60-120 min) the replacement
of
I-Ub with biotinylated ubiquitin. These values are the
mean of three determinations that agreed within 10%. All values were
normalized for the effective amount of the 240-kDa membrane protein
loaded onto the gel as determined by laser scan
densitometry.
The apparent molecular masses of -Spectrin Is the Ubiquitinated Erythrocyte Membrane
Protein
- and
-spectrin are 240 and 220 kDa, respectively (although cDNA-deduced
amino acid sequences provide values of 280 and 260 kDa).
-Spectrin
is present on the erythrocyte membrane cytoskeleton in 200,000
copies/cell and represents one of the most abundant membrane proteins
(12.5% of total protein). Based on these considerations and on the data
reported above, it seems reasonable to suggest that
-spectrin may
be the protein substrate for ubiquitination on the erythrocyte
membrane. However, to obtain direct evidence for this, we used two
approaches. In one case we ubiquitinated the erythrocyte membranes with
biotinylated ubiquitin. The membranes were then washed and solubilized
in a low ionic strength buffer at 37 °C. The solubilized proteins
were chromatographed onto an avidin column that retains biotinylated
proteins. The column was eluted with 8
M guanidiniun chloride,
and the eluted proteins were separated by SDS-PAGE and analyzed by
Western blotting using streptavidin-peroxidase or a monoclonal antibody
against spectrin (both
- and
-spectrin are recognized by this
antibody, although the affinity for
-spectrin is higher). As shown
in Fig. 6, two proteins with electrophoretic mobilities corresponding
to
- and
-spectrin and a minor band corresponding to 4.1 are
retained by the avidin column. The first two major protein bands have
been identified as spectrin by a monoclonal anti-spectrin antibody.
Only
-spectrin is also detected by streptavidin-peroxidase. In
another experiment, ubiquitination was obtained using
I-ubiquitin. Membranes were then washed and crude
spectrin was extracted as above. The solubilized proteins were
concentrated by ultrafiltration and incubated for 3 h with a monoclonal
anti-spectrin antibody at room temperature. Antigen-antibody complexes
were purified by chromatography on immobilized protein A. After
extensive washing the column was eluted with hot (80 °C) Laemmli
sample buffer
(31) and analyzed by SDS-PAGE and
autoradiography. As shown in Fig. 7, four proteins (240, 220, 80,
and 43 kDa) are immunoprecipitated by the anti-spectrin antibody but
only
-spectrin is ubiquitinated.
Figure 7:
Isolation of I-ubiquitinated
spectrin. Human erythrocyte membranes (0.4 mg) were incubated with
I-Ub (20 µg) in the presence of ATP (3.5 m
M)
and fraction II (300 µg) in a final volume of 1,350 µl, for 1 h
at 37 °C. The membranes were then pelleted, washed, and crude
spectrin extracted by a low ionic strength buffer. The sample was
concentrated to 0.8 ml by Centricon (Amicon) ultrafiltration, and 78
µg of anti-spectrin monoclonal antibody (Sigma) was added in a
final volume of 1.2 ml containing 10 m
M Tris-HCl, pH 7.5.
After 3 h at room temperature with gentle agitation, 250 µl of
immobilized protein A (Pierce) (equilibrated in 10 m
M Tris-HCl, pH 7.5, containing 0.3% bovine serum albumin) were added
overnight at 4 °C. The suspension was loaded onto a small plastic
column, washed with 10 m
M Tris-HCl, pH 7.5, containing 0.3
M NaCl, and eluted by Laemmli sample buffer (150 µl at 80
°C). The eluate was then separated by SDS-PAGE. Detection was with
Coomassie stain and autoradiography. Lanes 1, erythrocyte
membranes; lanes 2, protein A
eluate.
I-Ub in mature human
erythrocytes. This procedure allows the internalization of molecules
into erythrocytes without modifying their normal biochemical,
immunological, and morphological properties
(35) . By this
approach we found that most of
I-Ub that become
conjugated in mature erythrocytes is bound to a 240-kDa membrane
protein. In a cell-free system this conjugation was shown to be
ATP-dependent, to show an optimum at physiological pH values, and is
catalyzed by enzymes present into fraction II prepared both from
reticulocytes and mature erythrocytes. Furthermore, ubiquitination of
the 240-kDa membrane protein is a dynamic process with relatively fast
rates of formation (8 pmol of Ub/h/mg of fraction II) and decay (6.3
pmol/h/mg of fraction II) of bound ubiquitin. By different methods the
240-kDa protein has been identified as
-spectrin.
-spectrin is ubiquitinated into mature erythrocytes. This limited
ubiquitination does not depend on the presence of
-spectrin in the
membrane, cytoskeleton, or as a complex with
-spectrin/band
4.1/actin. In fact, similar fractions of ubiquitinated
-spectrin
were found in all cases except on crude spectrin extracts (not shown).
Furthermore, based on the electrophoretic mobility of ubiquitinated
-spectrin, only few (one or two) molecules of ubiquitin are likely
be incorporated per molecule of
-spectrin. In reticulocytes
-spectrin is usually synthesized 2-3-fold in excess of
-spectrin
(38) . The molecules not involved in the
formation of
-spectrin dimers are rapidly degraded. No data
are currently available on the mechanism(s) responsible for this
degradation. However, in mature erythrocytes protein synthesis is not
longer active, and free
-spectrin is not present. Furthermore, we
have clearly shown that ubiquitination occurs on
-spectrin when
already assembled in dimers and tetramers in the cytoskeleton. This
ubiquitination occurs at similar rates in erythrocyte and reticulocyte
lysates (see ``Results''), whereas the ubiquitin-conjugating
enzymes responsible for the ubiquitin-dependent proteolysis are active
only in reticulocytes
(23) . Thus,
-spectrin ubiquitination
is catalyzed by one of the E2s that are usually utilized for
functions others then the marking of proteins for degradation. The
principal function of the spectrin skeleton in erythrocytes is to
provide structural support to the lipid bilayer
(39) . Thus, all
mechanisms that modulate protein interactions in the erythrocyte
membrane skeleton could influence cell deformability and stability.
Among the factors so far reported to regulate membrane skeletal
organization are Ca
and
-spectrin
phosphorylation
(40) . It could be speculated that
ubiquitination of
-spectrin may play a similar role in the control
of cell deformability. However, no experimental data are yet available.
-spectrin ubiquitination does not yet have a role;
however, occurring in mature cells, it may serve for functions other
than ATP-dependent proteolysis. The results reported in this paper
along with those of other investigators
(12) should prove
valuable in defining a further role(s) of ubiquitination in cell
deformability.
Table:
Intracellular distribution of I-Ub
encapsulated into human RBC
I-Ub/ml of RBC (1.7
10
cpm/µg). The
cells were then incubated at 37 °C for 1 h and the amount of
radioactivity determined in the cytosol and membrane fractions. All
values are the mean ± S.D. of five experiments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.