©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ubiquitin Is Conjugated to the Cytoskeletal Protein -Spectrin in Mature Erythrocytes (*)

Dario Corsi , Luca Galluzzi , Rita Crinelli , Mauro Magnani (§)

From the (1) Institute of Biological Chemistry ``G. Fornaini,'' University of Urbino, Via Saffi 2, 61029 Urbino, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ubiquitination of red blood cell (RBC) proteins was investigated by encapsulation of 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.

Ubiquitin conjugation was ATP-dependent ( K0.09 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.


INTRODUCTION

Ubiquitin (Ub)() 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.


EXPERIMENTAL PROCEDURES

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 1.710cpm/g. Ubiquitin (1 mg/ml) was biotinylated by N-hydroxysuccinimide biotin (Pierce) following the manufacturer's protocol.

I-Ubiquitin Encapsulation in Erythrocytes

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 NaHPOcontaining 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 NaHPO, 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, 0.810cpm 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.


RESULTS

Encapsulation of I-Ubiquitin in Human Red Blood Cells

Human red blood cells were submitted to a procedure of hypotonic dialysis, isotonic resealing, and reannealing to encapsulate 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 10cpm/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 Kof 0.22 ± 0.02 mg of membrane protein/ml of reaction mixture, and also depends on the concentration of ubiquitin with a Kof 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.



-Spectrin Is the Ubiquitinated Erythrocyte Membrane Protein

The apparent molecular masses of - 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.




DISCUSSION

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 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.

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 -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 Caand -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.

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, -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

Human RBC were submitted to a procedure of hypotonic dialysis, isotonic resealing and reannealling as reported under ``Experimental Procedures'' using 30 µg of I-Ub/ml of RBC (1.710cpm/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.



FOOTNOTES

*
This work was supported by Consiglio Nazionale delle Ricerche target projects ``Aging'' and ``Biotechnology and Biostrumentation.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Istituto di Chimica Biologica ``G. Fornaini,'' Universit degli Studi di Urbino, Via Saffi 2; 61029 Urbino, Italy. Tel.: 39-722-305211; Fax: 39-722-320188.

The abbreviations used are: Ub, ubiquitin; PMSF, phenylmethylsulfonyl fluoride; RBC, red blood cell; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Rechsteiner, M. (1988) Ubiquitin, Plenum Press, New York
  2. Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983) J. Biol. Chem. 258, 8206-8214 [Abstract/Free Full Text]
  3. Haas, A. L., and Bright, P. M. (1988) J. Biol. Chem. 263, 13258-13267 [Abstract/Free Full Text]
  4. Cook, W. J., Jeffrey, L. C., Sullivan, M. L., and Viestra, R. D. (1992) J. Biol. Chem. 267, 15116-15121 [Abstract/Free Full Text]
  5. Sullivan, M. L., Callis, J., and Viestra, R. D. (1990) Plant Physiol. ( Bethesda) 94, 710-716
  6. Hershko, A., and Heller, A. (1985) Biochem. Biophys. Res. Commun. 128, 1079-1086 [Medline] [Order article via Infotrieve]
  7. Johnson, E. S., Bartel, B., Seufert, W., and Varshavsky, A. (1992) EMBO J. 2, 497-505
  8. Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991) Nature 349, 132-138 [CrossRef][Medline] [Order article via Infotrieve]
  9. Sommer, T., and Jentsch, S. (1993) Nature 369, 176-179
  10. St. John, T., Gallatin, W. M., Siegelman, M., Smith, H. T., Fried, V. A., and Weissman, I. L. (1986) Science 231, 845-850 [Medline] [Order article via Infotrieve]
  11. Hershko, A. (1988) J. Biol. Chem. 263, 15237-15240 [Free Full Text]
  12. Murti, K. G. Smith, H. T., and. Fried, V. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3019-3023 [Abstract]
  13. Dunigan, D. D., Dietrgen, R. G., Schoelz, J. E., and Zaiten, M. (1988) Virology 165, 310-312 [Medline] [Order article via Infotrieve]
  14. Magnani, M., Crinelli, R., Corsi, D., and Serafini, G. (1992) Ann. N. Y. Acad. Sci. 673, 103-109 [Medline] [Order article via Infotrieve]
  15. Chau, V., Tobias, J. W., Bachmair, A., Marriot, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583 [Medline] [Order article via Infotrieve]
  16. Kong, S.-K., and Chock, P. B. (1992) J. Biol. Chem. 267, 14189-14192 [Abstract/Free Full Text]
  17. Hershko, A., and Ciechanover, A. (1992) Annu. Rev. Biochem. 61, 761-807 [CrossRef][Medline] [Order article via Infotrieve]
  18. Haas, A., Reback, P. M., Pratt, G., and Rechsteiner, M. (1990) J. Biol. Chem. 265, 21664-21669 [Abstract/Free Full Text]
  19. Fried, V. A., Smith, H. T., Hildebrandt, E., and Weiner, K. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3685-3689 [Abstract]
  20. Finley, D., and Chau, V. (1991) Annu. Rev. Cell Biol. 7, 25-69 [CrossRef]
  21. Mayer, R. J., Arnold, J., Lszl, L., Landon, M., and Lowe, J. (1991) Biochim. Biophys. Acta 1089, 141-157 [Medline] [Order article via Infotrieve]
  22. Haas, A. L.(1991) in Red Blood Cell Aging (Magnani, M., and De Flora, A., eds) pp. 191-205, Plenum Press, New York
  23. Haas, A. L., and Bright, P. M. (1985) J. Biol. Chem. 260, 12464-12473 [Abstract/Free Full Text]
  24. Pickart, C. M., and Vella, A. T. (1988) J. Biol. Chem. 263, 12028-12035 [Abstract/Free Full Text]
  25. Hershko, A., Ganoth, D., Pehrson, J., Palazzo, R. E., and Cohen, L. H. (1991) J. Biol. Chem. 266, 16376-16379 [Abstract/Free Full Text]
  26. Feber, S., and Ciechanover, A. (1986) J. Biol. Chem. 261, 3128-3134 [Abstract/Free Full Text]
  27. Rossi, L., Bianchi, M., and Magnani, M. (1992) Biotechnol. Appl. Biochem. 15, 207-216 [Medline] [Order article via Infotrieve]
  28. Sobue, K., and Muramoto, Y. (1981) Biochem. Biophys. Res. Commun. 100, 1063-1070 [Medline] [Order article via Infotrieve]
  29. Sato, S. E., and Ohnishi, S. J. (1983) Eur. J. Biochem. 130, 19-25 [Abstract]
  30. Morrow, J. S., Speicher, D. W., Knowles, W. J., Hsu, C. J., and Marchesi, V. T. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6592-6596 [Abstract]
  31. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  32. Haas, A. L., and Bright, P. M. (1988) J. Biol. Chem. 263, 13258-13267 [Abstract/Free Full Text]
  33. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  34. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  35. Rechsteiner, M. (1991) Cell 66, 615-618 [Medline] [Order article via Infotrieve]
  36. Paolini, R., and Kinet, J. P. (1993) EMBO J. 12, 779-786 [Abstract]
  37. Magnani, M., and De Loach, J. R. (1992) The Use of Resealed Erythrocytes as Carriers and Bioreactors, Plenum Press, New York
  38. Hanspal, M., and Palek, J. (1992) Semin. Hematol. 29, 305-319 [Medline] [Order article via Infotrieve]
  39. Cohen, C. M. (1983) Semin. Hematol. 20, 141-158 [Medline] [Order article via Infotrieve]
  40. Vann Bennett (1990) Physiol. Rev. 70, 1029-1054 [Free Full Text]
  41. Ball, E., Karlik, C. C., Beall, C. J., Saville, D. L., Sparrow, J. C., Bullard, B., and Fyrberg, E. A. (1987) Cell 51, 221-228 [Medline] [Order article via Infotrieve]

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