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FRACTIONATION ANALYSIS OF THE ENDOSOMAL COMPARTMENT DURING RAT RETICULOCYTE MATURATION
Cell Biology International 2002, Vol. 26, No. 8, 669–678 doi:10.1006/cbir.2002.0917, available online at http://www.idealibrary.com on
FRACTIONATION ANALYSIS OF THE ENDOSOMAL COMPARTMENT DURING RAT RETICULOCYTE MATURATION VALE u RIE DARDALHON1, CHARLES GE u MINARD2, HUBERT REGGIO2, MICHEL VIDAL2* and JOSETTE SAINTE-MARIE2 1
UMR 5535 CNRS, IGM CNRS, Route de Mende, 2UMR 5539 CNRS, Universite´ Montpellier II, cc 107, 34095 Montpellier Ce´dex 05, France Received 28 June 2001; accepted 11 November 2001
A subcellular fractionation procedure was developed to isolate the different endosomal compartments present during reticulocyte maturation. After reticulocyte lysis and removal of excess haemoglobin by gel chromatography, membrane vesicles were separated over a discontinuous sucrose gradient (10–40%). Two fractions were isolated: P1 at the 25–35% sucrose interface and P2 at the 17–25% sucrose interface. These fractions were morphologically characterized by electron microscopy and the distribution of endocytic markers in the fractions was detected by Western blot. Moreover, this fractionation technique was used to study the effect of 3-methyladenine (3-MA), an autophagy inhibitor, on reticulocyte maturation. The presence of 3-MA during in vitro maturation of reticulocytes induced a decrease in exosome secretion, as measured by the amount of transferrin receptor (TfR) released in the extracellular medium. The subcellular fractionation results suggested that multivesicular endosome formation from early endosomes is the step affected by 3-MA. 2002 Elsevier Science Ltd. All rights reserved. K: 3-methyladenine; multivesicular endosomes; rab proteins; reticulocyte; subcellular fractionation; transferrin receptor. A: CEP, crude endosomal preparation; 3-MA, 3-methyladenine; TfR, transferrin receptor; MVE, multivesicular endosome; Hsc70, heat shock cognate 70 kDa protein.
INTRODUCTION Transferrin receptors (TfR), which promote efficient iron uptake, disappear from the reticulocyte surface during maturation into erythrocyte (Pan and Johnstone, 1983; Vidal et al., 1989). Indeed, during this final differentiation stage, reticulocytes release small vesicles termed exosomes that contain some plasma membrane proteins (e.g. TfR, acetylcholinesterase, glucose and nucleoside transporters) (Johnstone et al., 1987). On the other hand, the anion channel, which is excluded from exosomes, is highly maintained on the erythrocyte surface (Johnstone et al., 1987). Membrane proteins are selectively sorted during budding and pinching off of the endosomal membrane into the luminal space, leading to the formation of multivesicular structures (Pan et al., 1985; Johnstone, *To whom correspondence should be addressed. Tel: (33) 467-144-777; Fax: (33) 467-144-286; E-mail: [email protected] 1065–6995/02/$-see front matter
1992). Exosomes are released in the extracellular medium when these multivesicular endosomes (MVE) fuse with the plasma membrane. However, the molecular basis of the process leading to exosome secretion is still poorly understood. Intracellular processes involved in MVE formation in reticulocytes is thus of special interest for understanding red blood cell maturation. We developed a subcellular fractionation procedure to characterize the reticulocyte endocytic compartment: early endosomes and MVE. Indeed, MVE have similarities (chronologically, morphologically, etc.) with late endosomes found in other cell types, and a wide variety of endosome isolation methods have been described (Beaumelle and Hopkins, 1989; Gorvel et al., 1991; Courtoy, 1993; Aniento et al., 1996). We used a procedure described by Nun˜ez (Nun˜ez et al., 1990) to obtain a crude endosomal preparation from rat reticulocytes, and we further analysed this vesicle pool by 2002 Elsevier Science Ltd. All rights reserved.
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sucrose gradient centrifugation. The morphological and biochemical data obtained here show that, while MVE are endocytic structures issued from early endosomes, very low amounts of the typical markers of late endosomes (i.e. rab7, CI-M6P) could be found associated with MVE, in agreement with the non-degradative characteristics of the exosomal pathway. Moreover, this fractionation approach was used to study the effect of 3-MA on exosome secretion. 3-MA is a well-known inhibitor of autophagy (Seglen and Gordon, 1982), a process that resembles MVE formation. We show here that 3-MA substantially decreases exosome secretion during in vitro reticulocyte maturation. Modification of the distribution of markers (i.e. TfR, Rab5, Hsc70) in subcellular fractions suggests that 3-MA could affect MVE formation.
MATERIALS AND METHODS Materials Mouse monoclonal anti-TfR (MAB 1451) was from Chemicon International Inc. (Temecula, CA, U.S.A.). Rat monoclonal anti-Hsc70 (SPA-815) was obtained from StressGen Biotechnologies (Victoria, Canada). Rabbit polyclonal anti-Rab4 (sc-312) and anti-Rab5A (sc-309) and goat polyclonal anti-Rab7 (sc-6563) came from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, U.S.A.). A goat polyclonal anti-human cation independent mannose-6 phosphate receptor (CI-M6PR) was kindly provided by Franc¸oise Vignon (U540 INSERM, Montpellier, France). This antibody crossreacts with rat-CI-M6PR (not shown). Peroxidase-conjugated donkey anti-species IgG were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, U.S.A.). 3-Methyladenine was from Sigma (St Louis, MO, U.S.A.), Bio-Gel A-5m was obtained from BioRad Laboratories (Hercules, CA, U.S.A.), ECL reagents and protein A-Sepharose were from Amersham Pharmacia Biotech (Pistacaway, NJ, U.S.A.). Isolation, labelling and fractionation of reticulocytes Reticulocyte production in Sprague–Dawley white rats was induced by phenylhydrazine as previously described (Vidal et al., 1997). After removing the buffy coat, red blood cells (reticulocyte percentage generally >70%) were washed three times with phosphate-buffered saline (PBS) and incubated
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in RPMI medium plus BSA (5 mg/ml) for 4 h at 37C, in the presence of 20 g/ml of human transferrin radio-iodinated as previously described (Sainte-Marie et al., 1997). When indicated, 3-Methyladenine (5 mM, 10 mM and 15 mM) was added during this incubation. The medium was then removed, followed by extensive washing with cold PBS, and cell-associated radioactivity was determined in a counter (Packard Cobra). Surface-bound transferrin was receptor-dissociated by incubating the radiolabelled cells in PBS-10% rat serum for 1 h at 4C, cells were then pelleted and the released radioactivity was counted. Endocytic vesicles were obtained by a modified version of the method of Nun˜ez (Nun˜ez et al., 1990). Briefly, radiolabelled cells were resuspended in 1 vol of isolation buffer (10 mM Hepes/Tris, pH 7.2, 1 mM EGTA, 1 mM MgSO4, 3 mM NaN3, 50 mM NaCl, 50 mM KCl, 0.2 mM PMSF) in the presence of a cocktail of anti-proteases (Roche, Meylan, France). After 15 min on ice, the cells were lysed by freeze thawing in an acetone/dry ice mixture (20 min). The thawing step was carried out at 4C. After centrifugation (5 min1000 g), pelleted unbroken cells were submitted to a second freezingthawing cycle. The pooled supernatants were centrifuged for 20 min20,000 g to discard mitochondria and reticulocyte plasma membranes. The supernatant was loaded on a Bio-Gel A-5m column equilibrated with isolation buffer. The vesicle-containing fractions detected by 125 I-transferrin-associated radioactivity and by turbidity were pooled, loaded on top of 2 ml of sucrose solution (40% sucrose w/w, 3 mM imidazole, pH 7.4, 0.5 mM EDTA) and centrifuged at 100,000 g for 2 h in a SW 40.1 rotor (Beckman) to obtain an endocytic vesicle enriched layer that we called the crude endosomal preparation (CEP). The vesicle pool was adjusted to 10% sucrose and loaded on top of a step gradient consisting of 40% (0.5 ml)/35% (2 ml)/25% (3 ml)/17% (3 ml)/10% (3.5 ml) w/w, and centrifuged at 100,000 g for 2 h at 4C in a SW 40.1 rotor. Fractions (300 l) were collected from the bottom of the tube and (i) counted for radioactivity, (ii) assayed for protein content by the method of Bradford (Bradford, 1976), and (iii) the sucrose density was determined by refractometry. Western analysis of the different fractions The proteins of indicated fractions were separated by 10% SDS-PAGE (7.5% or 12% SDS-PAGE for CI-M6PR and Rab proteins detection, respectively) according to Laemmli (Laemmli, 1970) and
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electrophoretically transferred to PVDF membrane (Immobilon-P, Millipore) as described by Towbin (Towbin et al., 1979). Immunoblotting was performed using peroxidase-conjugated antibodies and the ECL procedure. Western blot quantification was carried out by densitometry scanning using ImageQuaNT (Molecular Dynamics) image analysis software.
Exosomes and reticulocyte membrane preparation Exosomes were collected by sequential centrifugations from the supernatant of reticulocyte subcultures (Vidal et al., 1989). Briefly, blood samples were centrifuged at 1000 g for 10 min at 4C to remove plasma. After three washings with Hanks’ buffer, the cells (3% final packed cell volume) were cultured in RPMI 1640 supplemented with 5 mM adenosine, 10 mM inosine, 5 mM glutamine and 3% (v/v) fetal calf serum (FCS). After 48 h incubation at 37C in a CO2 incubator, cells were pelleted and the supernatant centrifuged for 20 min at 20,000 g to remove mitochondria and cellular fragments. The supernatant was ultracentrifuged for 90 min at 40,000 g, and the vesicle pellet was resuspended in phosphate-buffered saline (PBS). Reticulocyte membranes were prepared according to (Vidal and Stahl, 1993). Briefly, packed reticulocytes were lysed by vigorous agitation in 40 vol of ice-cold hypotonic buffer (0.15 mM EDTA, 5 mM phosphate buffer, pH 7.4). The lysate was centrifuged for 5 min at 19,000 g and the membranes were rocked from the bottom of the tube and washed twice more with PBS.
Electron microscopy The fractions were fixed in 2.5% glutaraldehyde for 24 h at 4C, and centrifuged at 100,000 g for 1.5 h after 1:20 dilution with PBS. The membrane pellet was again fixed in 2% glutaraldehyde for 3 h at 4C, washed by centrifugation for 30 min at 10,000 g and postfixed in 1% osmium. The pellet was bulk stained with 1% magnesium uranyl acetate, dehydrated in graded ethanol and embedded in epon. Sections were observed under a Hitachi 7000–100 electron microscope (Hitachi Scientific Instruments, Dusseldorf, Germany). Colloidal gold-transferrin was prepared by the citrate method (Slot and Geuze, 1981). Colloidal gold-Tf uptake and preparation of samples for electron microscopy was carried out as described (Harding et al., 1983).
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Association of Hsc70 with immobilized TfR Protein A-Sepharose resuspended in 100 mM potassium phosphate (pH 8), was incubated with anti-TfR (0.2 mg IgG per ml of packed gel) for 1 h at room temperature. The Sepharose gel was washed twice with PBS and resuspended 1:1 (v:v) in TG buffer (10 mM Tris/HCl, pH 7.6, 50 mM NaCl, 10% glycerol (w/v)). Reticulocyte ghosts, exosomes, crude endosomal preparation, P1 or P2 subcellular fractions were solubilized in 20 mM sodium phosphate buffer containing 1% TX-100, and incubated with protein A-Sepharose at 4C overnight with gentle agitation. The Sepharose pellet was then washed four times with TG buffer. Exogenous Hsc70 was provided to immobilize TfR in the form of membrane-free lysate, obtained from a 1:5 dilution of reticulocytes in 5 mM phosphate buffer, pH 7.4, containing 0.1 mM PMSF. Prior to use, MgCl2 and CaCl2 (2 mM) and KCl and urea (67 mM) were added. The samples were then incubated at 30C for 30 min, gently resuspended every 5 min, followed by five washes with TG buffer. The retained proteins were eluted off the beads with Laemmli loading buffer and analysed by SDS-PAGE and Western blot. After hybridization, the immunoblots were quantified with the ImageQuaNT software program (cf. above) and normalized. Data are means of four immunoprecipitation experiments. RESULTS Preparation of a crude endosomal fraction The MVE compartment was labelled by incubating the cells with 125I-Tf for 4 h at 37C. Surface-bound 125 I-Tf (about 30% total labelling, in agreement with (Vidal et al., 1989)), was eliminated as described in the Materials and Methods. Purification yields during the various fractionation steps were then calculated from this intracellular labelling level. Among other lysis procedures tested, freezing thawing was found to be the most efficient for obtaining intracellular vesicles while avoiding vesicle shedding from the plasma membrane. Moreover, the gel chromatography step was crucial to get rid of most of the haemoglobin and obtain reproducible preparation yields. The mean intracellular-associated 125I-Tf yields from lysis, exclusion column and ‘pelleting’ steps were: 30 5, 65 4.5 and 95 5% (n=13), respectively, with an overall yield of 17–18.5%. Protein recovery in the crude endosomal preparation was 1.2–1.5 mg for 10 ml packed cell volume. Note that
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this protein yield is very similar to one obtained previously (Nun˜ez et al., 1990). Two experiments were carried out to assess plasma-membrane contamination of intracellular vesicles. A control fractionation experiment was performed on reticulocytes incubated with 125I-Tf at 4C, precluding any intracellular labelling. In this case, the overall 125I-Tf recovery yield was <1% (two experiments, data not shown) demonstrating that endocytic vesicles isolated by this procedure were not significantly contaminated by the plasma membrane. Moreover, proteins in the plasma membrane and crude endosomal preparation were separated by SDS-PAGE and Coomassie-blue stained. The protein content of endocytic vesicle and plasma membrane fractions was very different (Vidal and Stahl, 1993 and Fig. 3). Note that plasma membrane proteins with MW 240 kDa, 95 kDa and 43 kDa (probably corresponding to spectrin, anion channel and actin, respectively) were not detected in the crude endosomal preparation.
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Fig. 1. Profile of the endosomal preparation after discontinuous sucrose gradient separation. A sample (about 220 g) of crude endosomal preparation was loaded on top of a sucrose gradient (as described in the Materials and Methods), centrifuged at 100,000 g for 2 h at 4C. Fractions (300 l) were collected from the bottom of the tube (fraction 1). The protein content () (g/fraction), 125I-Tf radioactivity ( ) (cpm/ fraction) were determined. This profile is representative of the fractioning of all experiments.
Sucrose gradient analysis of the endosomal compartment After centrifugation on sucrose step gradient (see Materials and Methods), the 125I-Tf distribution pattern displayed extensive labelling in fractions 5–7 (P1) and 15–17 (P2), recovered respectively at the 25/35% and 17/25% sucrose interface. The protein content of the fractions revealed another peak consisting of fractions 25–28 (P3), recovered at the 10/17% sucrose interface, while a very low amount of 125I-Tf was co-localized with these fractions (Fig. 1). Moreover, 56.5 5% and 57 2% of the total protein content and 125I-Tf amount loaded in the gradients, respectively, were recovered in the pooled fractions. Several previous studies found that early endosomes were enriched at the 25/35% sucrose interface and endosomal carrier vesicles and/or late endosomes at the 17/25% sucrose interface (Chavrier et al., 1990; Gorvel et al., 1991; Aniento et al., 1996; Seemann et al., 1996). Fractions pooled from the three interfaces were characterized by electron microscopy. Fraction P1 appeared as vesicular (100–200 nm diameter) and tubular structures (Fig. 2(a)), typical of early endosome preparations (Beaumelle and Hopkins, 1989; Gorvel et al., 1991). Conversely, fraction P2 contained predominantly larger structures (250–500 nm) with a multivesicular appearance (Fig. 2(b)). The electron microscopy data obtained from the P3 fraction did not show any structure resembling
those from P1 or P2, but showed a few diluted vesicles and small shares of membrane (data not shown). The protein composition of these peaks was compared (Fig. 3). The three fractions showed some differences despite having very similar Coomassie staining patterns. In particular, three proteins (asterisks) appeared to be enriched in P2 compared to P1. On the other hand, major proteins found in P1 and P2 (approx. 87 kDa and 75 kDa) were either absent or much less abundant in P3, and conversely some proteins present in the crude endosomal preparation (arrows) were found in P3 but not in P1 and P2. Taken together, these biochemical and morphologic data strongly suggested that fractions P1 and P2 could correspond to early endosomes and MVEs respectively, while P3 would be cytosolic proteins. Characterization of fractions by Western blot The presence of endocytic markers was assessed by Western blot to further characterize fractions obtained from sucrose gradient analysis. TfR is a protein marker often used to discriminate between early and late endosomal compartments. Indeed, TfR usually cycles between the plasma membrane and early endosomes without reaching prelysosomal compartments. However, during reticulocyte
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Fig. 2. Electron microscopy of P1 and P2 fractions. P1 and P2 fractions were treated as described in the Materials and Methods. A: Structures in P1. The arrows indicate small vesicles typical of early endosome preparations (100–200 nm diameter). B: Structures in P2. The arrow indicates a typical MVE structure (250–500 nm diameter).
maturation, TfR is sorted from this recycling pathway, accumulates in MVE and is released in association with exosomes (Pan and Johnstone, 1983). TfR was indeed detected by immunoblot in both P1 and P2 fractions but with significantly (P>0.01) different ratios (Table 1). 32%3% of the total TfR (P1+P2+P3) was detected in the P1 fraction, while the signal amounted to 50%5% in P2. Note that this was in perfect agreement with the 125I-Tf
Fig. 3. Electrophoresis profile of plasma membrane, crude endosomal preparation, and fractions P1, P2 and P3. Proteins of the indicated fractions (10 g of each sample) were separated by 10% SDS-PAGE according to Laemmli, and then Coomassie-blue stained. The molecular mass (kDa) standards are indicated to the left. Asterisks (*) indicate the main proteins in P2 that were in minority or absent in P1. Arrows indicate proteins in CEP and P3 that were in minority or absent in P1 and P2.
counts (37%2.3% and 53 %2% in P1 and P2, respectively). Low molecular weight GTPases of the Rab family were shown to be associated with different compartments involved in vesicular traffic. Among them, Rab4 and Rab5 were demonstrated to be localized in sorting endosomes (Chavrier et al., 1990; Gorvel et al., 1991; van der Sluijs et al., 1991), Rab5 and Rab11 in recycling endosomes (Ullrich et al., 1996), while Rab 7 was found exclusively in late endosomes (Chavrier et al., 1990). Moreover, Rab4 and Rab5 have a different subcellular distribution in reticulocytes (Vidal and Stahl, 1993). Rab5 was found to be mainly membrane associated, enriched in plasma membrane as compared to endocytic vesicles and exosomes, while Rab4 was more uniformly distributed in the different fractions. We thus looked at the distribution and/or localization of Rab4, Rab5 and Rab7 in P1, P2 and P3 fractions. As shown in Figure 4(a), Rab5 was detected in the crude endosomal preparation and its distribution was mainly in P2 (17%4% in P1 vs 63%6% in P2) (Table 1). Rab4 was detected in the crude endosomal preparation with an almost complete distribution in the P2 fraction (Fig. 4(b) and Table 1). A 1 M KCl wash was used to strip Rab proteins from the membrane vesicles. As shown in Figure 4(a), Rab5 detection in the P1 fraction was almost abolished after the KCl wash, contrary to the P2 fraction. Western blotting the TfR in the same fractions demonstrated that KCl treatment did not affect transmembrane protein
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Table 1 Distribution of endocytic markers at the three peaks Control
Protein 125 I-Tf TfR Hsc70 Clathrin Rab4 Rab5 Rab7 CI-M6PR
3-MA
P1
P2
P3
P1
P2
P3
274 372 323 335 104 — 174 — —
334 532 505 484 145 100 636 100 100
404 101.5 184 206 767 — 125 — —
2610 53 4 52 7 60 5* n.d. n.d. 4510* n.d. n.d.
26 5 33 2 28 5* 20 5* n.d. n.d. 2810 n.d. n.d.
4112 14 2 11 5 18.5*10 73 5 n.d. 2615* n.d. n.d.
The sum of markers in P1+P2+P3 was set at 100%. The distribution of markers at each peak was expressed (meanSEM, n=7 for control and n=3 for 3-MA) as a function of this sum. ( —: not detectable, n.d.: not determined, *P<0.05).
recovery (Fig. 4(c)). This is in perfect agreement with the topology of Rab proteins in a multivesicular compartment such as MVE. Rab proteins bound to the cytosolic leaflet of endosomal membranes and were thus inaccessible for KCl treatment when located in the lumen of internal MVE vesicles (cf. Fig. 8). Similarly, KCl treatment of the P2 fraction did not decrease Rab4 detection (Fig. 4(b)). We confirmed the efficient fractionation of the endosomal compartment using this procedure by looking at the distribution of classical late endosomal markers such as Rab7 and CI-M6PR. As expected (Gorvel et al., 1991; Aniento et al., 1996), both markers were found to be only associated with the P2 fraction, however in very low amounts (data not shown), in agreement with a prelysosomal compartment present only as remnant traces. We studied the distribution of the chaperone protein Hsc70 in the different fractions. Indeed, Hsc70 was found in a molar ratio with TfR in exosomes and suggested to be involved in TfR sorting into exosomes (Davis et al., 1986). Moreover, Hsc70 was demonstrated to interact with the cytosolic domain of the exosomal TfR, in agreement with this hypothesis (Mathew et al., 1995). Hsc70 could thus be a potential MVE-specific marker (vs early endosomes) in reticulocytes. As shown in Figure 4(d) (and Table 1), Hsc70 was indeed detected in the crude endosomal preparation and mainly in the P2 fraction (48%4%), but the chaperone protein was also found to be associated with the P1 fraction (33%5%). Moreover, a KCl wash did not significantly change the distribution of the chaperone within the two fractions.
Interactions of Hsc70 with TfR of different membrane origins were then studied. TfR was immunoprecipitated from exosomes, plasma membrane or the crude endosomal preparation as described in the Materials and Methods. Depending on its membrane origin, the TfR immobilized on proteinA-sepharose did not bring down the same amount of Hsc70 (Fig. 5). In the absence of TfR, no Hsc70 was found to be associated with the protein A-Sepharose pellet (not shown). Immunoblots of several experiments were scanned, quantified and the Hsc70/TfR ratio was determined. Data were normalized with a ratio equal to 1 for exosomes. In agreement with (Mathew et al., 1995), Table 2 shows that the amount of Hsc70 retained by TfR decreased as a function of the isolated membrane: exosomes>crude endosome preparation>plasma membrane. The TfR immobilization results from P1 or P2 fractions did not allow us to conclude definitively in favour of a preferential Hsc70 association with TfR from either fraction (Table 2). 3-methyladenine affects exosome release Autophagic vacuole formation was reported to be involved in organelle removal (e.g. mitochondria) during in vitro reticulocyte maturation (Gronowicz et al., 1984). As shown in Figure 6, autophagic structures can be localized in continuity with, or at least close to, colloidal gold-Tf labelled MVE. Since 3-methyladenine was shown to almost completely prevent the formation of new autophagic vacuoles (Punnonen and Reunanen, 1990) and inhibit transport between late endosomes and lysosomes (Punnonen et al., 1994), we tested the effect of 3-MA on reticulocyte maturation. We first
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Fig. 5. Association of Hsc70 to TfR immunoprecipitated from exosomes, the crude endosomal preparation and reticulocyte plasma membranes. TfR was immunoprecipitated and immobilized as described in the Materials and Methods. In each sample, the same quantity (0.2 ml) of membrane-free reticulocyte lysate was added, incubated for 30 min at 37C and the experiment was carried out as described in the Materials and Methods. After SDS-PAGE separation (10%), proteins were transferred on PVDF membranes and sequentially revealed by anti-TfR (upper panel) and anti-Hsc70 (lower panel) antibodies.
Table 2. Hsc70 binding to immobilized TfR from different membrane fractions TfR origin Exosome Crude endosomal preparation Plasma membrane
Ratio Hsc70/TfR 1 0.700.09 0.420.05
TfR was immunoprecipitated from different membrane origins and Hsc70 binding was assessed as described in the Materials and Methods. MeanSEM (n=4). Fig. 4. Immunodetection of Rab5, Rab4, TfR and Hsc70 in the CEP and fractions P1, P2 before or after KCl treatment. Proteins (20 g) of subcellular fractions samples (before and after KCl treatment) were separated by SDS-PAGE (10% for TfR and Hsc70 isolation, 12% for Rab4 and Rab5) and transferred to PVDF membranes, as described in the Materials and Methods. Immunoblotting was performed using antibodies against (a) Rab5, (b) Rab4, (c) TfR, (d) Hsc70, peroxidase-conjugated antibodies and the ECL procedure.
verified, in agreement with (Punnonen et al., 1994), that 3-MA did not modify either the binding or the internalization of radiolabelled transferrin in reticulocytes (not shown). The addition of 3-MA during in vitro reticulocyte maturation largely decreased exosome secretion, as assessed by the amount of TfR associated with the vesicle pellet (Fig. 7). Quantification of Western blots indicated a decrease of about 66% in TfR released in the presence of 15 mM 3-MA. Subcellular fractionation of reticulocytes incubated with 3-MA was then carried out. We did not obtain evidence of a
significant morphological effect of 3-MA on P1 and P2 fractions compared to fractions from untreated cells (data not shown), indicating that 3-MA does not interfere with fractionation of the compartments. However, as shown in Table 1, the distribution of the different markers was significantly affected by the presence of 3-MA. We observed a 1.5- to 2-fold decrease of 125I-Tf, TfR, Hsc70 and Rab5 amounts in the P2 fraction, with a concomitant increase in P1. This highly suggested that 3-MA inhibited the formation of MVE from early endosomes.
DISCUSSION Receptor-mediated endocytosis of transferrin has been largely described in many cell types. The fate of TfR in reticulocytes during maturation into erythrocytes is somewhat more original
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Fig. 6. Electronic microscopy of reticulocyte internal vesicles labelled with colloidal gold-transferrin. Reticulocytes incubated with colloidal-gold transferrin for 30 min at 37C were treated as described in the Materials and Methods. The arrow shows continuity between autophagic structures and typically colloidal gold-Tf labelled MVE.
Fig. 7. Immunodetection of TfR in exosomes after maturation in the absence (Ctrl) or presence of 3-methyladenine. Exosomes from rat reticulocytes were collected after overnight incubation in the presence or absence of 3-MA at the indicated concentration, as described in the Materials and Methods. An aliquot of exosomes was then subjected to SDS-PAGE, transferred to PVDF membranes and blotted with an anti-TfR.
(Johnstone, 1992), but is now well documented. The multivesicular endosomal compartment that leads to exosome release by fusion with the plasma membrane, although morphologically well described, is still poorly characterized from a biochemical point of view. The aim of this study was to develop a fractionation procedure that would allow the isolation and characterization of vesicles of the reticulocyte endosomal compartment. The mild conditions used in the fractionation procedure led to a low lysis yield (around 20% of 125I-Tf in the crude endosomal preparation). However, the high enrichment of the radiolabelled marker in the crude endosomal preparation (approx. 250-fold) and in the P1 and P2 fractions (approx. 500-fold) highlighted the purity of the endocytic compartment. The sucrose density gradient fractionation allowed isolation of two kinds of vesicular compartment. The P1 fraction was pooled from
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Fig. 8. Schematic diagram of reticulocyte internal vesicles. Corresponding subcellular fractions obtained in this study are indicated to the left. The white arrow indicates the probable transport step affected by 3-MA. Partially unfolded TfR: uTfR.
fractions collected at the 25/35% sucrose interface that is reported to be enriched in early endosomes (Gorvel et al., 1991). In complete agreement with this, electron microscopy observations showed that P1 was constituted of vesicles that were typical (morphology and size) of those found in early endosome compartments. Conversely, the P2 fraction collected at the 17/25% sucrose interface corresponding to late endosomes (Gorvel et al., 1991), was enriched with multivesicular structures, as seen by EM. Besides the morphological characteristics, biochemical data confirmed the efficiency of the fractionation procedure. First, the two peripheral Rab proteins (Rab4, Rab5) were resistant to a KCl wash of the P2 fraction, demonstrating their inaccessibility to medium, as supported by their removal in multivesicular structures (Fig. 8), before being secreted through exosomes (Vidal and Stahl, 1993). Secondly, although in low amount due to vanishing of the lysosomal pathway in reticulocytes, late endosome markers (Rab7, CI-M6P) were only detected in the lighter membrane fraction (P2), in agreement with the literature (Gorvel et al., 1991; Aniento et al., 1996). As shown in the diagram in Figure 8, the P1 fraction probably corresponded to the early endosomal compartment of reticulocytes, mainly constituted of small vesicles and tubules (Harding et al., 1983), while P2 consisted of structures such as MVE that are involved in exosome release (Harding et al., 1983; Davis et al., 1986). TfR was found to be enriched in the P2 fraction (relative to P1), reflecting the exosomal sorting function of the MVE compartment. There was a similar significant enrichment of 125I-Tf in P2, in
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agreement with EM studies using colloidal gold-Tf (Harding et al., 1983 and Fig. 6), and TfR released in exosomes was still able to bind its ligand (Orr et al., 1987). Although enriched in the P2 fraction Hsc70, was also present in significant amounts in P1. It would be difficult to come to a definitive conclusion on this point relative to the TfR sorting mechanism, since Hsc70 could be associated with other membrane components. However, data collected through Western blot quantification of Hsc70 (Table 1) were in line with the results of in vitro interactions between Hsc70 and TfR of different membrane origins (Mathew et al., 1995 and Table 2). All of these data are consistent with an EE maturation process which would lead to MVE formation. Markers of the early endosomal compartment such as Rab4 and Rab5 were present in MVE while classical markers of late endosomes such as Rab7 and CI-M6P were present in trace amounts. This is in perfect agreement with the fact that exosome formation occurs in a non-hydrolytic compartment, as demonstrated by the integrity and activity of exosomal proteins (e.g. TfR, acetylcholinesterase, nucleoside transporter). Moreover, the fact that Hsc70 was found to be associated with the early endosomal compartment (P1 fraction) and that Hsc70 bound to TfR along a PM
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treatment confirmed that 3-MA decreased exosome release through MVE formation. This is in close agreement with data showing that the late steps of endocytic transport were affected by 3-MA (Punnonen et al., 1994). It was more recently shown that the effect of 3-MA on autophagy was due to inhibition of phosphatidylinositol 3-kinase (PI3K) (Blommaart et al., 1997; Petiot et al., 2000). Otherwise, it is known that PI3K is involved in several vesicular traffic steps (Reaves et al., 1996; Davidson, 1995) and in the morphogenesis of multivesicular bodies (Fernandez-Borja et al., 1999). We are thus currently examining the possibility that PI3K could be involved in the exosome release process through the MVE formation step. ACKNOWLEDGEMENTS This work was supported by the ‘Centre National de la Recherche Scientifique’, by the ‘Universite´ Montpellier II’, and by Grant 9580 from the ‘Association pour la Recherche sur le Cancer’ (to M.V.). REFERENCES A F, G F, P RG, G J, 1996. An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes. J Cell Biol 133: 29–41. B BD, H CR, 1989. High-yield isolation of functionally competent endosomes from mouse lymphocytes. Biochem J 264: 137–149. B EF, K U, S JP, VS H, M AJ, 1997. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243: 240–246. B M, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254. C P, P RG, H HP, S K, Z M, 1990. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell 62: 317–329. C JP, 1993. Analytical subcellular fractionation of endosomal compartments in rat hepatocytes. In: Bergeron JM, Harnes JR, ed. Subcellular Biochem. vol. 19. New York, Plenum Press. 29–68. D HW, 1995. Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes. J Cell Biol 130: 797–805. D JQ, D D, J RM, B V, 1986. Selective externalization of an ATP-binding protein structurally related to the clathrin-uncoating ATPase/heat
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shock protein in vesicles containing terminal transferrin receptors during reticulocyte maturation. J Biol Chem 261: 15368–15371. F-B M, W R, C J, J H, D N, D S, N J, 1999. Multivesicular body morphogenesis requires phosphatidyl-inositol 3-kinase activity. Curr Biol 14: 55–58. G´ C, N F, J RM, V M, 2001. Characteristics of the interaction between Hsc70 and the transferrin receptor in exosomes released during reticulocyte maturation. J Biol Chem 276: 9910–9916. G JP, C P, Z M, G J, 1991. rab5 controls early endosome fusion in vitro. Cell 64: 915–925. G G, S H, S TL, 1984. Maturation of the reticulocyte in vitro. J Cell Sci 71: 177–197. H C, H J, S P, 1983. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol 97: 329–339. J RM, 1992. The Jeanne Manery-Fisher Memorial Lecture 1991. Maturation of reticulocytes: formation of exosomes as a mechanism for shedding membrane proteins. Biochem Cell Biol 70: 179–190. J RM, A M, H JR, O L, T C, 1987. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 262: 9412–9420. L UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. M A, B A, J RM, 1995. Hsp70 is closely associated with the transferrin receptor in exosomes from maturing reticulocytes. Biochem J 308: 823–830. N˜ MT, G V, W JA, G J, 1990. Mobilization of iron from endocytic vesicles. The effects of acidification and reduction. J Biol Chem 265: 6688–6692. O L, A M, J RM, 1987. Externalization of membrane-bound activities during sheep reticulocyte maturation is temperature and ATP dependent. Biochem Cell Biol 65: 1080–1090. P BT, J RM, 1983. Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33: 967–977. P BT, T K, W C, A M, J RM, 1985. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol 101: 942–948. P A, O-D E, B EF, M AJ, C P, 2000. Distinct classes of phosphatidylinositol 3 -kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 275: 992–998.
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P EL, R H, 1990. Effects of vinblastine, leucine, and histidine, and 3-methyladenine on autophagy in Ehrlich ascites cells. Exp Mol Pathol 52: 87–97. P EL, M VS, R H, 1994. 3-Methyladenine inhibits transport from late endosomes to lysosomes in cultured rat and mouse fibroblasts. Eur J Cell Biol 65: 14–25. R BJ, B NA, M BM, L JP, 1996. The effect of wortmannin on the localisation of lysosomal type I integral membrane glycoproteins suggests a role for phosphoinositide 3-kinase activity in regulating membrane traffic late in the endocytic pathway. J Cell Sci 109: 749–762. S-M J, L V, P´ EI, F J, P JR, B¨ A, 1997. Transferrin receptor functions as a signal-transduction molecule for its own recycling via increases in the internal Ca2+ concentration. Eur J Biochem 250: 689–697. S J, W K, O M, P RG, G V, 1996. The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain. Mol Biol Cell 7: 1359–1374. S PO, G PB, 1982. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA 79: 1889–1892. S JW, G HJ, 1981. Sizing of protein A-colloidal gold probes for immunoelectron microscopy. J Cell Biol 90: 533–536. T H, S T, G J, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354. U O, R S, U´ S, Z M, P RG, 1996. Rab11 regulates recycling through the pericentriolar recycling endosome. J Cell Biol 135: 913–924. V S P, H M, Z A, T A, G B, M I, 1991. The small GTP-binding protein rab4 is associated with early endosomes. Proc Natl Acad Sci USA 88: 6313–9317. V MJ, S PD, 1993. The small GTP-binding proteins Rab4 and ARF are associated with released exosomes during reticulocyte maturation. Eur J Cell Biol 60: 261–267. V M, M P, H D, 1997. Aggregation reroutes molecules from a recycling to a vesicle-mediated secretion pathway during reticulocyte maturation. J Cell Sci 110: 1867–1877. V M, S-M J, P J, B¨ A, 1989. Asymmetric distribution of phospholipids in the membrane of vesicles released during in vitro maturation of guinea pig reticulocytes: evidence precluding a role for ‘aminophospholipid translocase’. J Cell Physiol 140: 455–462.
FRACTIONATION ANALYSIS OF THE ENDOSOMAL COMPARTMENT DURING RAT RETICULOCYTE MATURATION VALE u RIE DARDALHON1, CHARLES GE u MINARD2, HUBERT REGGIO2, MICHEL VIDAL2* and JOSETTE SAINTE-MARIE2 1
UMR 5535 CNRS, IGM CNRS, Route de Mende, 2UMR 5539 CNRS, Universite´ Montpellier II, cc 107, 34095 Montpellier Ce´dex 05, France Received 28 June 2001; accepted 11 November 2001
A subcellular fractionation procedure was developed to isolate the different endosomal compartments present during reticulocyte maturation. After reticulocyte lysis and removal of excess haemoglobin by gel chromatography, membrane vesicles were separated over a discontinuous sucrose gradient (10–40%). Two fractions were isolated: P1 at the 25–35% sucrose interface and P2 at the 17–25% sucrose interface. These fractions were morphologically characterized by electron microscopy and the distribution of endocytic markers in the fractions was detected by Western blot. Moreover, this fractionation technique was used to study the effect of 3-methyladenine (3-MA), an autophagy inhibitor, on reticulocyte maturation. The presence of 3-MA during in vitro maturation of reticulocytes induced a decrease in exosome secretion, as measured by the amount of transferrin receptor (TfR) released in the extracellular medium. The subcellular fractionation results suggested that multivesicular endosome formation from early endosomes is the step affected by 3-MA. 2002 Elsevier Science Ltd. All rights reserved. K: 3-methyladenine; multivesicular endosomes; rab proteins; reticulocyte; subcellular fractionation; transferrin receptor. A: CEP, crude endosomal preparation; 3-MA, 3-methyladenine; TfR, transferrin receptor; MVE, multivesicular endosome; Hsc70, heat shock cognate 70 kDa protein.
INTRODUCTION Transferrin receptors (TfR), which promote efficient iron uptake, disappear from the reticulocyte surface during maturation into erythrocyte (Pan and Johnstone, 1983; Vidal et al., 1989). Indeed, during this final differentiation stage, reticulocytes release small vesicles termed exosomes that contain some plasma membrane proteins (e.g. TfR, acetylcholinesterase, glucose and nucleoside transporters) (Johnstone et al., 1987). On the other hand, the anion channel, which is excluded from exosomes, is highly maintained on the erythrocyte surface (Johnstone et al., 1987). Membrane proteins are selectively sorted during budding and pinching off of the endosomal membrane into the luminal space, leading to the formation of multivesicular structures (Pan et al., 1985; Johnstone, *To whom correspondence should be addressed. Tel: (33) 467-144-777; Fax: (33) 467-144-286; E-mail: [email protected] 1065–6995/02/$-see front matter
1992). Exosomes are released in the extracellular medium when these multivesicular endosomes (MVE) fuse with the plasma membrane. However, the molecular basis of the process leading to exosome secretion is still poorly understood. Intracellular processes involved in MVE formation in reticulocytes is thus of special interest for understanding red blood cell maturation. We developed a subcellular fractionation procedure to characterize the reticulocyte endocytic compartment: early endosomes and MVE. Indeed, MVE have similarities (chronologically, morphologically, etc.) with late endosomes found in other cell types, and a wide variety of endosome isolation methods have been described (Beaumelle and Hopkins, 1989; Gorvel et al., 1991; Courtoy, 1993; Aniento et al., 1996). We used a procedure described by Nun˜ez (Nun˜ez et al., 1990) to obtain a crude endosomal preparation from rat reticulocytes, and we further analysed this vesicle pool by 2002 Elsevier Science Ltd. All rights reserved.
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sucrose gradient centrifugation. The morphological and biochemical data obtained here show that, while MVE are endocytic structures issued from early endosomes, very low amounts of the typical markers of late endosomes (i.e. rab7, CI-M6P) could be found associated with MVE, in agreement with the non-degradative characteristics of the exosomal pathway. Moreover, this fractionation approach was used to study the effect of 3-MA on exosome secretion. 3-MA is a well-known inhibitor of autophagy (Seglen and Gordon, 1982), a process that resembles MVE formation. We show here that 3-MA substantially decreases exosome secretion during in vitro reticulocyte maturation. Modification of the distribution of markers (i.e. TfR, Rab5, Hsc70) in subcellular fractions suggests that 3-MA could affect MVE formation.
MATERIALS AND METHODS Materials Mouse monoclonal anti-TfR (MAB 1451) was from Chemicon International Inc. (Temecula, CA, U.S.A.). Rat monoclonal anti-Hsc70 (SPA-815) was obtained from StressGen Biotechnologies (Victoria, Canada). Rabbit polyclonal anti-Rab4 (sc-312) and anti-Rab5A (sc-309) and goat polyclonal anti-Rab7 (sc-6563) came from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, U.S.A.). A goat polyclonal anti-human cation independent mannose-6 phosphate receptor (CI-M6PR) was kindly provided by Franc¸oise Vignon (U540 INSERM, Montpellier, France). This antibody crossreacts with rat-CI-M6PR (not shown). Peroxidase-conjugated donkey anti-species IgG were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, U.S.A.). 3-Methyladenine was from Sigma (St Louis, MO, U.S.A.), Bio-Gel A-5m was obtained from BioRad Laboratories (Hercules, CA, U.S.A.), ECL reagents and protein A-Sepharose were from Amersham Pharmacia Biotech (Pistacaway, NJ, U.S.A.). Isolation, labelling and fractionation of reticulocytes Reticulocyte production in Sprague–Dawley white rats was induced by phenylhydrazine as previously described (Vidal et al., 1997). After removing the buffy coat, red blood cells (reticulocyte percentage generally >70%) were washed three times with phosphate-buffered saline (PBS) and incubated
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in RPMI medium plus BSA (5 mg/ml) for 4 h at 37C, in the presence of 20 g/ml of human transferrin radio-iodinated as previously described (Sainte-Marie et al., 1997). When indicated, 3-Methyladenine (5 mM, 10 mM and 15 mM) was added during this incubation. The medium was then removed, followed by extensive washing with cold PBS, and cell-associated radioactivity was determined in a counter (Packard Cobra). Surface-bound transferrin was receptor-dissociated by incubating the radiolabelled cells in PBS-10% rat serum for 1 h at 4C, cells were then pelleted and the released radioactivity was counted. Endocytic vesicles were obtained by a modified version of the method of Nun˜ez (Nun˜ez et al., 1990). Briefly, radiolabelled cells were resuspended in 1 vol of isolation buffer (10 mM Hepes/Tris, pH 7.2, 1 mM EGTA, 1 mM MgSO4, 3 mM NaN3, 50 mM NaCl, 50 mM KCl, 0.2 mM PMSF) in the presence of a cocktail of anti-proteases (Roche, Meylan, France). After 15 min on ice, the cells were lysed by freeze thawing in an acetone/dry ice mixture (20 min). The thawing step was carried out at 4C. After centrifugation (5 min1000 g), pelleted unbroken cells were submitted to a second freezingthawing cycle. The pooled supernatants were centrifuged for 20 min20,000 g to discard mitochondria and reticulocyte plasma membranes. The supernatant was loaded on a Bio-Gel A-5m column equilibrated with isolation buffer. The vesicle-containing fractions detected by 125 I-transferrin-associated radioactivity and by turbidity were pooled, loaded on top of 2 ml of sucrose solution (40% sucrose w/w, 3 mM imidazole, pH 7.4, 0.5 mM EDTA) and centrifuged at 100,000 g for 2 h in a SW 40.1 rotor (Beckman) to obtain an endocytic vesicle enriched layer that we called the crude endosomal preparation (CEP). The vesicle pool was adjusted to 10% sucrose and loaded on top of a step gradient consisting of 40% (0.5 ml)/35% (2 ml)/25% (3 ml)/17% (3 ml)/10% (3.5 ml) w/w, and centrifuged at 100,000 g for 2 h at 4C in a SW 40.1 rotor. Fractions (300 l) were collected from the bottom of the tube and (i) counted for radioactivity, (ii) assayed for protein content by the method of Bradford (Bradford, 1976), and (iii) the sucrose density was determined by refractometry. Western analysis of the different fractions The proteins of indicated fractions were separated by 10% SDS-PAGE (7.5% or 12% SDS-PAGE for CI-M6PR and Rab proteins detection, respectively) according to Laemmli (Laemmli, 1970) and
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electrophoretically transferred to PVDF membrane (Immobilon-P, Millipore) as described by Towbin (Towbin et al., 1979). Immunoblotting was performed using peroxidase-conjugated antibodies and the ECL procedure. Western blot quantification was carried out by densitometry scanning using ImageQuaNT (Molecular Dynamics) image analysis software.
Exosomes and reticulocyte membrane preparation Exosomes were collected by sequential centrifugations from the supernatant of reticulocyte subcultures (Vidal et al., 1989). Briefly, blood samples were centrifuged at 1000 g for 10 min at 4C to remove plasma. After three washings with Hanks’ buffer, the cells (3% final packed cell volume) were cultured in RPMI 1640 supplemented with 5 mM adenosine, 10 mM inosine, 5 mM glutamine and 3% (v/v) fetal calf serum (FCS). After 48 h incubation at 37C in a CO2 incubator, cells were pelleted and the supernatant centrifuged for 20 min at 20,000 g to remove mitochondria and cellular fragments. The supernatant was ultracentrifuged for 90 min at 40,000 g, and the vesicle pellet was resuspended in phosphate-buffered saline (PBS). Reticulocyte membranes were prepared according to (Vidal and Stahl, 1993). Briefly, packed reticulocytes were lysed by vigorous agitation in 40 vol of ice-cold hypotonic buffer (0.15 mM EDTA, 5 mM phosphate buffer, pH 7.4). The lysate was centrifuged for 5 min at 19,000 g and the membranes were rocked from the bottom of the tube and washed twice more with PBS.
Electron microscopy The fractions were fixed in 2.5% glutaraldehyde for 24 h at 4C, and centrifuged at 100,000 g for 1.5 h after 1:20 dilution with PBS. The membrane pellet was again fixed in 2% glutaraldehyde for 3 h at 4C, washed by centrifugation for 30 min at 10,000 g and postfixed in 1% osmium. The pellet was bulk stained with 1% magnesium uranyl acetate, dehydrated in graded ethanol and embedded in epon. Sections were observed under a Hitachi 7000–100 electron microscope (Hitachi Scientific Instruments, Dusseldorf, Germany). Colloidal gold-transferrin was prepared by the citrate method (Slot and Geuze, 1981). Colloidal gold-Tf uptake and preparation of samples for electron microscopy was carried out as described (Harding et al., 1983).
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Association of Hsc70 with immobilized TfR Protein A-Sepharose resuspended in 100 mM potassium phosphate (pH 8), was incubated with anti-TfR (0.2 mg IgG per ml of packed gel) for 1 h at room temperature. The Sepharose gel was washed twice with PBS and resuspended 1:1 (v:v) in TG buffer (10 mM Tris/HCl, pH 7.6, 50 mM NaCl, 10% glycerol (w/v)). Reticulocyte ghosts, exosomes, crude endosomal preparation, P1 or P2 subcellular fractions were solubilized in 20 mM sodium phosphate buffer containing 1% TX-100, and incubated with protein A-Sepharose at 4C overnight with gentle agitation. The Sepharose pellet was then washed four times with TG buffer. Exogenous Hsc70 was provided to immobilize TfR in the form of membrane-free lysate, obtained from a 1:5 dilution of reticulocytes in 5 mM phosphate buffer, pH 7.4, containing 0.1 mM PMSF. Prior to use, MgCl2 and CaCl2 (2 mM) and KCl and urea (67 mM) were added. The samples were then incubated at 30C for 30 min, gently resuspended every 5 min, followed by five washes with TG buffer. The retained proteins were eluted off the beads with Laemmli loading buffer and analysed by SDS-PAGE and Western blot. After hybridization, the immunoblots were quantified with the ImageQuaNT software program (cf. above) and normalized. Data are means of four immunoprecipitation experiments. RESULTS Preparation of a crude endosomal fraction The MVE compartment was labelled by incubating the cells with 125I-Tf for 4 h at 37C. Surface-bound 125 I-Tf (about 30% total labelling, in agreement with (Vidal et al., 1989)), was eliminated as described in the Materials and Methods. Purification yields during the various fractionation steps were then calculated from this intracellular labelling level. Among other lysis procedures tested, freezing thawing was found to be the most efficient for obtaining intracellular vesicles while avoiding vesicle shedding from the plasma membrane. Moreover, the gel chromatography step was crucial to get rid of most of the haemoglobin and obtain reproducible preparation yields. The mean intracellular-associated 125I-Tf yields from lysis, exclusion column and ‘pelleting’ steps were: 30 5, 65 4.5 and 95 5% (n=13), respectively, with an overall yield of 17–18.5%. Protein recovery in the crude endosomal preparation was 1.2–1.5 mg for 10 ml packed cell volume. Note that
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this protein yield is very similar to one obtained previously (Nun˜ez et al., 1990). Two experiments were carried out to assess plasma-membrane contamination of intracellular vesicles. A control fractionation experiment was performed on reticulocytes incubated with 125I-Tf at 4C, precluding any intracellular labelling. In this case, the overall 125I-Tf recovery yield was <1% (two experiments, data not shown) demonstrating that endocytic vesicles isolated by this procedure were not significantly contaminated by the plasma membrane. Moreover, proteins in the plasma membrane and crude endosomal preparation were separated by SDS-PAGE and Coomassie-blue stained. The protein content of endocytic vesicle and plasma membrane fractions was very different (Vidal and Stahl, 1993 and Fig. 3). Note that plasma membrane proteins with MW 240 kDa, 95 kDa and 43 kDa (probably corresponding to spectrin, anion channel and actin, respectively) were not detected in the crude endosomal preparation.
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Fig. 1. Profile of the endosomal preparation after discontinuous sucrose gradient separation. A sample (about 220 g) of crude endosomal preparation was loaded on top of a sucrose gradient (as described in the Materials and Methods), centrifuged at 100,000 g for 2 h at 4C. Fractions (300 l) were collected from the bottom of the tube (fraction 1). The protein content () (g/fraction), 125I-Tf radioactivity ( ) (cpm/ fraction) were determined. This profile is representative of the fractioning of all experiments.
Sucrose gradient analysis of the endosomal compartment After centrifugation on sucrose step gradient (see Materials and Methods), the 125I-Tf distribution pattern displayed extensive labelling in fractions 5–7 (P1) and 15–17 (P2), recovered respectively at the 25/35% and 17/25% sucrose interface. The protein content of the fractions revealed another peak consisting of fractions 25–28 (P3), recovered at the 10/17% sucrose interface, while a very low amount of 125I-Tf was co-localized with these fractions (Fig. 1). Moreover, 56.5 5% and 57 2% of the total protein content and 125I-Tf amount loaded in the gradients, respectively, were recovered in the pooled fractions. Several previous studies found that early endosomes were enriched at the 25/35% sucrose interface and endosomal carrier vesicles and/or late endosomes at the 17/25% sucrose interface (Chavrier et al., 1990; Gorvel et al., 1991; Aniento et al., 1996; Seemann et al., 1996). Fractions pooled from the three interfaces were characterized by electron microscopy. Fraction P1 appeared as vesicular (100–200 nm diameter) and tubular structures (Fig. 2(a)), typical of early endosome preparations (Beaumelle and Hopkins, 1989; Gorvel et al., 1991). Conversely, fraction P2 contained predominantly larger structures (250–500 nm) with a multivesicular appearance (Fig. 2(b)). The electron microscopy data obtained from the P3 fraction did not show any structure resembling
those from P1 or P2, but showed a few diluted vesicles and small shares of membrane (data not shown). The protein composition of these peaks was compared (Fig. 3). The three fractions showed some differences despite having very similar Coomassie staining patterns. In particular, three proteins (asterisks) appeared to be enriched in P2 compared to P1. On the other hand, major proteins found in P1 and P2 (approx. 87 kDa and 75 kDa) were either absent or much less abundant in P3, and conversely some proteins present in the crude endosomal preparation (arrows) were found in P3 but not in P1 and P2. Taken together, these biochemical and morphologic data strongly suggested that fractions P1 and P2 could correspond to early endosomes and MVEs respectively, while P3 would be cytosolic proteins. Characterization of fractions by Western blot The presence of endocytic markers was assessed by Western blot to further characterize fractions obtained from sucrose gradient analysis. TfR is a protein marker often used to discriminate between early and late endosomal compartments. Indeed, TfR usually cycles between the plasma membrane and early endosomes without reaching prelysosomal compartments. However, during reticulocyte
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Fig. 2. Electron microscopy of P1 and P2 fractions. P1 and P2 fractions were treated as described in the Materials and Methods. A: Structures in P1. The arrows indicate small vesicles typical of early endosome preparations (100–200 nm diameter). B: Structures in P2. The arrow indicates a typical MVE structure (250–500 nm diameter).
maturation, TfR is sorted from this recycling pathway, accumulates in MVE and is released in association with exosomes (Pan and Johnstone, 1983). TfR was indeed detected by immunoblot in both P1 and P2 fractions but with significantly (P>0.01) different ratios (Table 1). 32%3% of the total TfR (P1+P2+P3) was detected in the P1 fraction, while the signal amounted to 50%5% in P2. Note that this was in perfect agreement with the 125I-Tf
Fig. 3. Electrophoresis profile of plasma membrane, crude endosomal preparation, and fractions P1, P2 and P3. Proteins of the indicated fractions (10 g of each sample) were separated by 10% SDS-PAGE according to Laemmli, and then Coomassie-blue stained. The molecular mass (kDa) standards are indicated to the left. Asterisks (*) indicate the main proteins in P2 that were in minority or absent in P1. Arrows indicate proteins in CEP and P3 that were in minority or absent in P1 and P2.
counts (37%2.3% and 53 %2% in P1 and P2, respectively). Low molecular weight GTPases of the Rab family were shown to be associated with different compartments involved in vesicular traffic. Among them, Rab4 and Rab5 were demonstrated to be localized in sorting endosomes (Chavrier et al., 1990; Gorvel et al., 1991; van der Sluijs et al., 1991), Rab5 and Rab11 in recycling endosomes (Ullrich et al., 1996), while Rab 7 was found exclusively in late endosomes (Chavrier et al., 1990). Moreover, Rab4 and Rab5 have a different subcellular distribution in reticulocytes (Vidal and Stahl, 1993). Rab5 was found to be mainly membrane associated, enriched in plasma membrane as compared to endocytic vesicles and exosomes, while Rab4 was more uniformly distributed in the different fractions. We thus looked at the distribution and/or localization of Rab4, Rab5 and Rab7 in P1, P2 and P3 fractions. As shown in Figure 4(a), Rab5 was detected in the crude endosomal preparation and its distribution was mainly in P2 (17%4% in P1 vs 63%6% in P2) (Table 1). Rab4 was detected in the crude endosomal preparation with an almost complete distribution in the P2 fraction (Fig. 4(b) and Table 1). A 1 M KCl wash was used to strip Rab proteins from the membrane vesicles. As shown in Figure 4(a), Rab5 detection in the P1 fraction was almost abolished after the KCl wash, contrary to the P2 fraction. Western blotting the TfR in the same fractions demonstrated that KCl treatment did not affect transmembrane protein
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Table 1 Distribution of endocytic markers at the three peaks Control
Protein 125 I-Tf TfR Hsc70 Clathrin Rab4 Rab5 Rab7 CI-M6PR
3-MA
P1
P2
P3
P1
P2
P3
274 372 323 335 104 — 174 — —
334 532 505 484 145 100 636 100 100
404 101.5 184 206 767 — 125 — —
2610 53 4 52 7 60 5* n.d. n.d. 4510* n.d. n.d.
26 5 33 2 28 5* 20 5* n.d. n.d. 2810 n.d. n.d.
4112 14 2 11 5 18.5*10 73 5 n.d. 2615* n.d. n.d.
The sum of markers in P1+P2+P3 was set at 100%. The distribution of markers at each peak was expressed (meanSEM, n=7 for control and n=3 for 3-MA) as a function of this sum. ( —: not detectable, n.d.: not determined, *P<0.05).
recovery (Fig. 4(c)). This is in perfect agreement with the topology of Rab proteins in a multivesicular compartment such as MVE. Rab proteins bound to the cytosolic leaflet of endosomal membranes and were thus inaccessible for KCl treatment when located in the lumen of internal MVE vesicles (cf. Fig. 8). Similarly, KCl treatment of the P2 fraction did not decrease Rab4 detection (Fig. 4(b)). We confirmed the efficient fractionation of the endosomal compartment using this procedure by looking at the distribution of classical late endosomal markers such as Rab7 and CI-M6PR. As expected (Gorvel et al., 1991; Aniento et al., 1996), both markers were found to be only associated with the P2 fraction, however in very low amounts (data not shown), in agreement with a prelysosomal compartment present only as remnant traces. We studied the distribution of the chaperone protein Hsc70 in the different fractions. Indeed, Hsc70 was found in a molar ratio with TfR in exosomes and suggested to be involved in TfR sorting into exosomes (Davis et al., 1986). Moreover, Hsc70 was demonstrated to interact with the cytosolic domain of the exosomal TfR, in agreement with this hypothesis (Mathew et al., 1995). Hsc70 could thus be a potential MVE-specific marker (vs early endosomes) in reticulocytes. As shown in Figure 4(d) (and Table 1), Hsc70 was indeed detected in the crude endosomal preparation and mainly in the P2 fraction (48%4%), but the chaperone protein was also found to be associated with the P1 fraction (33%5%). Moreover, a KCl wash did not significantly change the distribution of the chaperone within the two fractions.
Interactions of Hsc70 with TfR of different membrane origins were then studied. TfR was immunoprecipitated from exosomes, plasma membrane or the crude endosomal preparation as described in the Materials and Methods. Depending on its membrane origin, the TfR immobilized on proteinA-sepharose did not bring down the same amount of Hsc70 (Fig. 5). In the absence of TfR, no Hsc70 was found to be associated with the protein A-Sepharose pellet (not shown). Immunoblots of several experiments were scanned, quantified and the Hsc70/TfR ratio was determined. Data were normalized with a ratio equal to 1 for exosomes. In agreement with (Mathew et al., 1995), Table 2 shows that the amount of Hsc70 retained by TfR decreased as a function of the isolated membrane: exosomes>crude endosome preparation>plasma membrane. The TfR immobilization results from P1 or P2 fractions did not allow us to conclude definitively in favour of a preferential Hsc70 association with TfR from either fraction (Table 2). 3-methyladenine affects exosome release Autophagic vacuole formation was reported to be involved in organelle removal (e.g. mitochondria) during in vitro reticulocyte maturation (Gronowicz et al., 1984). As shown in Figure 6, autophagic structures can be localized in continuity with, or at least close to, colloidal gold-Tf labelled MVE. Since 3-methyladenine was shown to almost completely prevent the formation of new autophagic vacuoles (Punnonen and Reunanen, 1990) and inhibit transport between late endosomes and lysosomes (Punnonen et al., 1994), we tested the effect of 3-MA on reticulocyte maturation. We first
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Fig. 5. Association of Hsc70 to TfR immunoprecipitated from exosomes, the crude endosomal preparation and reticulocyte plasma membranes. TfR was immunoprecipitated and immobilized as described in the Materials and Methods. In each sample, the same quantity (0.2 ml) of membrane-free reticulocyte lysate was added, incubated for 30 min at 37C and the experiment was carried out as described in the Materials and Methods. After SDS-PAGE separation (10%), proteins were transferred on PVDF membranes and sequentially revealed by anti-TfR (upper panel) and anti-Hsc70 (lower panel) antibodies.
Table 2. Hsc70 binding to immobilized TfR from different membrane fractions TfR origin Exosome Crude endosomal preparation Plasma membrane
Ratio Hsc70/TfR 1 0.700.09 0.420.05
TfR was immunoprecipitated from different membrane origins and Hsc70 binding was assessed as described in the Materials and Methods. MeanSEM (n=4). Fig. 4. Immunodetection of Rab5, Rab4, TfR and Hsc70 in the CEP and fractions P1, P2 before or after KCl treatment. Proteins (20 g) of subcellular fractions samples (before and after KCl treatment) were separated by SDS-PAGE (10% for TfR and Hsc70 isolation, 12% for Rab4 and Rab5) and transferred to PVDF membranes, as described in the Materials and Methods. Immunoblotting was performed using antibodies against (a) Rab5, (b) Rab4, (c) TfR, (d) Hsc70, peroxidase-conjugated antibodies and the ECL procedure.
verified, in agreement with (Punnonen et al., 1994), that 3-MA did not modify either the binding or the internalization of radiolabelled transferrin in reticulocytes (not shown). The addition of 3-MA during in vitro reticulocyte maturation largely decreased exosome secretion, as assessed by the amount of TfR associated with the vesicle pellet (Fig. 7). Quantification of Western blots indicated a decrease of about 66% in TfR released in the presence of 15 mM 3-MA. Subcellular fractionation of reticulocytes incubated with 3-MA was then carried out. We did not obtain evidence of a
significant morphological effect of 3-MA on P1 and P2 fractions compared to fractions from untreated cells (data not shown), indicating that 3-MA does not interfere with fractionation of the compartments. However, as shown in Table 1, the distribution of the different markers was significantly affected by the presence of 3-MA. We observed a 1.5- to 2-fold decrease of 125I-Tf, TfR, Hsc70 and Rab5 amounts in the P2 fraction, with a concomitant increase in P1. This highly suggested that 3-MA inhibited the formation of MVE from early endosomes.
DISCUSSION Receptor-mediated endocytosis of transferrin has been largely described in many cell types. The fate of TfR in reticulocytes during maturation into erythrocytes is somewhat more original
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Fig. 6. Electronic microscopy of reticulocyte internal vesicles labelled with colloidal gold-transferrin. Reticulocytes incubated with colloidal-gold transferrin for 30 min at 37C were treated as described in the Materials and Methods. The arrow shows continuity between autophagic structures and typically colloidal gold-Tf labelled MVE.
Fig. 7. Immunodetection of TfR in exosomes after maturation in the absence (Ctrl) or presence of 3-methyladenine. Exosomes from rat reticulocytes were collected after overnight incubation in the presence or absence of 3-MA at the indicated concentration, as described in the Materials and Methods. An aliquot of exosomes was then subjected to SDS-PAGE, transferred to PVDF membranes and blotted with an anti-TfR.
(Johnstone, 1992), but is now well documented. The multivesicular endosomal compartment that leads to exosome release by fusion with the plasma membrane, although morphologically well described, is still poorly characterized from a biochemical point of view. The aim of this study was to develop a fractionation procedure that would allow the isolation and characterization of vesicles of the reticulocyte endosomal compartment. The mild conditions used in the fractionation procedure led to a low lysis yield (around 20% of 125I-Tf in the crude endosomal preparation). However, the high enrichment of the radiolabelled marker in the crude endosomal preparation (approx. 250-fold) and in the P1 and P2 fractions (approx. 500-fold) highlighted the purity of the endocytic compartment. The sucrose density gradient fractionation allowed isolation of two kinds of vesicular compartment. The P1 fraction was pooled from
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Fig. 8. Schematic diagram of reticulocyte internal vesicles. Corresponding subcellular fractions obtained in this study are indicated to the left. The white arrow indicates the probable transport step affected by 3-MA. Partially unfolded TfR: uTfR.
fractions collected at the 25/35% sucrose interface that is reported to be enriched in early endosomes (Gorvel et al., 1991). In complete agreement with this, electron microscopy observations showed that P1 was constituted of vesicles that were typical (morphology and size) of those found in early endosome compartments. Conversely, the P2 fraction collected at the 17/25% sucrose interface corresponding to late endosomes (Gorvel et al., 1991), was enriched with multivesicular structures, as seen by EM. Besides the morphological characteristics, biochemical data confirmed the efficiency of the fractionation procedure. First, the two peripheral Rab proteins (Rab4, Rab5) were resistant to a KCl wash of the P2 fraction, demonstrating their inaccessibility to medium, as supported by their removal in multivesicular structures (Fig. 8), before being secreted through exosomes (Vidal and Stahl, 1993). Secondly, although in low amount due to vanishing of the lysosomal pathway in reticulocytes, late endosome markers (Rab7, CI-M6P) were only detected in the lighter membrane fraction (P2), in agreement with the literature (Gorvel et al., 1991; Aniento et al., 1996). As shown in the diagram in Figure 8, the P1 fraction probably corresponded to the early endosomal compartment of reticulocytes, mainly constituted of small vesicles and tubules (Harding et al., 1983), while P2 consisted of structures such as MVE that are involved in exosome release (Harding et al., 1983; Davis et al., 1986). TfR was found to be enriched in the P2 fraction (relative to P1), reflecting the exosomal sorting function of the MVE compartment. There was a similar significant enrichment of 125I-Tf in P2, in
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agreement with EM studies using colloidal gold-Tf (Harding et al., 1983 and Fig. 6), and TfR released in exosomes was still able to bind its ligand (Orr et al., 1987). Although enriched in the P2 fraction Hsc70, was also present in significant amounts in P1. It would be difficult to come to a definitive conclusion on this point relative to the TfR sorting mechanism, since Hsc70 could be associated with other membrane components. However, data collected through Western blot quantification of Hsc70 (Table 1) were in line with the results of in vitro interactions between Hsc70 and TfR of different membrane origins (Mathew et al., 1995 and Table 2). All of these data are consistent with an EE maturation process which would lead to MVE formation. Markers of the early endosomal compartment such as Rab4 and Rab5 were present in MVE while classical markers of late endosomes such as Rab7 and CI-M6P were present in trace amounts. This is in perfect agreement with the fact that exosome formation occurs in a non-hydrolytic compartment, as demonstrated by the integrity and activity of exosomal proteins (e.g. TfR, acetylcholinesterase, nucleoside transporter). Moreover, the fact that Hsc70 was found to be associated with the early endosomal compartment (P1 fraction) and that Hsc70 bound to TfR along a PM
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treatment confirmed that 3-MA decreased exosome release through MVE formation. This is in close agreement with data showing that the late steps of endocytic transport were affected by 3-MA (Punnonen et al., 1994). It was more recently shown that the effect of 3-MA on autophagy was due to inhibition of phosphatidylinositol 3-kinase (PI3K) (Blommaart et al., 1997; Petiot et al., 2000). Otherwise, it is known that PI3K is involved in several vesicular traffic steps (Reaves et al., 1996; Davidson, 1995) and in the morphogenesis of multivesicular bodies (Fernandez-Borja et al., 1999). We are thus currently examining the possibility that PI3K could be involved in the exosome release process through the MVE formation step. ACKNOWLEDGEMENTS This work was supported by the ‘Centre National de la Recherche Scientifique’, by the ‘Universite´ Montpellier II’, and by Grant 9580 from the ‘Association pour la Recherche sur le Cancer’ (to M.V.). REFERENCES A F, G F, P RG, G J, 1996. An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes. J Cell Biol 133: 29–41. B BD, H CR, 1989. High-yield isolation of functionally competent endosomes from mouse lymphocytes. Biochem J 264: 137–149. B EF, K U, S JP, VS H, M AJ, 1997. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243: 240–246. B M, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254. C P, P RG, H HP, S K, Z M, 1990. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell 62: 317–329. C JP, 1993. Analytical subcellular fractionation of endosomal compartments in rat hepatocytes. In: Bergeron JM, Harnes JR, ed. Subcellular Biochem. vol. 19. New York, Plenum Press. 29–68. D HW, 1995. Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes. J Cell Biol 130: 797–805. D JQ, D D, J RM, B V, 1986. Selective externalization of an ATP-binding protein structurally related to the clathrin-uncoating ATPase/heat
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