From the Department of Cell Biology, Utrecht
University Medical Centre and Institute of Biomembranes, Room
G02.525, Heidelberglaan 100, 3585 CX Utrecht, The Netherlands and
§ Kekule-Institut fuer Organische Chemie und Biochemie
der Universitaet Bonn, Gerhard-Domagk-Strasse 1, D-53121 Bonn,
Germany
Received for publication, July 26, 2002, and in revised form, January 6, 2003
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ABSTRACT |
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Exosomes are 60-100-nm membrane vesicles
that are secreted into the extracellular milieu as a consequence of
multivesicular body fusion with the plasma membrane. Here we determined
the protein and lipid compositions of highly purified human B
cell-derived exosomes. Mass spectrometric analysis indicated the
abundant presence of major histocompatibility complex (MHC)
class I and class II, heat shock cognate 70, heat shock protein
90, integrin Maturing endosomes accumulate vesicles in their lumen, resulting
in their transformation into multivesicular bodies
(MVB)1 (1). These vesicles
are formed by inward budding of the endosomal limiting membrane and
contain a selected cargo. Proteins that are sorted to the internal
vesicles of MVB potentially may have three distinct fates. The first
possibility is exemplified by ligand-activated epidermal growth factor
receptor, which is ultimately transferred to lysosomes for degradation
(2). A second possibility is that proteins may be stored temporarily in
MVB, as observed for MHC class II in immature dendritic cells (3). MHC
class II-carrying MVB in dendritic cells have also been termed MHC
class II compartments (MIIC), in accordance with similar structures in
B cells (4). MIIC play a crucial role in peptide loading of MHC class
II. In pathogen-stimulated dendritic cells, the internal vesicles of
MVB fuse back with their limiting membrane, thereby allowing subsequent
transfer of peptide-loaded MHC class II to the plasma membrane (3). The
third potential fate of vesicles within MVB is their release into the
extracellular environment as a consequence of fusion of the
MVB-limiting membrane with the plasma membrane. These secreted
MVB-derived vesicles have been called exosomes, which, depending on
their source, may serve a multitude of functions (5-7).
Exosomes are released by a great number of cell types, including
reticulocytes (5), cytotoxic T cells (8), B lymphocytes (9, 10),
dendritic cells (11-13), mast cells (14), platelets (15), and
intestinal epithelial cells (16). The biological functions of exosomes
are generally unclear. Increasing evidence, however, suggests that
exosomes from hematopoietic cells may serve as intercellular
communication vehicles that assist immune responses (6, 7). For
example, B cell-derived exosomes that carry peptide-loaded MHC class II
were demonstrated to stimulate CD4+ T cells (17) and to
specifically bind follicular dendritic cells (18) in vitro.
Furthermore, exosomes derived from cultured dendritic cells that were
loaded in vitro with tumor-derived peptides on MHC class I
stimulated cytotoxic T lymphocytes both in vitro and
in vivo (11).
Functions of exosomes should be reflected by their protein composition.
Given that exosomes are formed as the internal vesicles of MVB,
exosomes can be expected to also contain factors required for MVB
formation and protein sorting therein. Immunoelectron microscopic
studies, Western blot analyses, and peptide mass mapping of exosomes
derived from dendritic cells (12, 13), B lymphocytes (9, 10),
intestinal epithelial cells (16), and other cell types revealed the
presence of common, as well as cell type-specific, proteins. For
example, MHC class II is especially enriched in exosomes derived from B
lymphocytes, dendritic cells, mast cells, and intestinal epithelial
cells. Ubiquitous proteins in exosomes include cytoplasmic proteins,
such as tubulin, actin, and actin-binding proteins, the heat shock
proteins hsc70 (also named hsc71 or hsp73) and hsp90, and trimeric G
proteins, as well as membrane proteins, such as members of the
tetraspanin family (CD9, CD63, CD81, CD82). Sorting of a number of
membrane proteins into the MVB pathway involves ubiquitination of their
cytoplasmic domain (19) and binding of these acquired ubiquitin
moieties to Tsg101 (20). Indeed, Tsg101 (12), as well as c-Cbl (21), a
ubiquitin ligase required for ubiquitination of activated epidermal
growth factor receptor (2), have been detected in isolated exosomes.
Alternatively, membrane proteins may rely on ubiquitinated adaptors for
their sorting in MVB (20, 22). Importantly, sorting of at least some
proteins into the MVB pathway occurs independently of the ubiquitin
system (23). The molecular factors and mechanism(s) behind such
alternative sorting processes in MVB are unknown, and the analysis of
exosomes may help their discovery.
In an approach to understand more about the formation and function of
exosomes we developed a protocol that yielded highly purified exosomes
from human B cells and studied their molecular content and biochemical
properties. Based on exosome characteristics, we propose a model in
which the incorporation of proteins into tetraspanin networks and
detergent-resistant membranes (DRM) at the limiting membrane of MVB may
be conditional for their sorting into the internal vesicles of MVB.
Antibodies, SDS-PAGE, and Western Blotting--
Rabbit anti-MHC
class II (24) was kindly provided by Dr. H. L. Ploegh (Harvard
Medical School), rabbit anti-MHC class II Cell Culture and Exosome Isolation--
RN cells
(HLA-DR15+) were cultured as described (9). We observed
that fetal calf serum contains exosomes (data not shown). To exclude
bovine exosomes, cells were cultured in medium supplemented with fetal
calf serum that had been ultracentrifuged for 60 min at 141,000 × gmax. RN-derived exosomes were isolated
routinely from 800 ml of culture medium containing ~108
RN cells. As a first isolation step, exosomes were collected from the
medium by differential centrifugation, as described (9). In short,
cells were removed by centrifugation for 10 min at 200 × g. Supernatants were collected and centrifuged sequentially twice for 10 min at 500 × gmax, once for
15 min at 2,000 × gmax, once for 30 min at
10,000 × gmax, and once for 60 min at
70,000 × gmax using a SW27 rotor (Beckman
Instruments, Inc., Fullerton, CA). Exosomes were pelleted at the final
centrifugation step and were resuspended in PBS and re-pelleted at
70,000 × gmax. The final pellet routinely
contained ~100 µg of protein. Re-pelleted exosomes were resuspended
in 5 ml of 2.6 M sucrose, 20 mM Tris-HCl, pH
7.2, and floated into an overlaid linear sucrose gradient (2.0-0.25 M sucrose, 20 mM Tris-HCl, pH 7.2) in a SW41
tube for 16 h at 270,000 × gmax.
Gradient fractions of 1 ml were collected from the bottom of the tube
and analyzed for the presence of MHC class II and tetraspanins by
Western blotting. When indicated, gradient fractions were diluted with
3 ml of PBS each and centrifuged for 60 min at 350,000 × gmax, and the pellets were analyzed by SDS-PAGE and Coomassie Blue staining. As a final purification step, 750-µl samples of pooled exosome-containing gradient fractions were added to
200 µl of Dynabeads M-450 (~8 × 107 beads) coated
with monoclonal mouse anti-human MHC class II (Dynal Biotech, Oslo,
Norway). As a negative control, Dynabeads M-450 coated with goat
anti-mouse IgG (Dynal) were used. The Dynabeads that were added to the
exosomes suspensions were first extensively washed with and resuspended
in PBS supplemented with 3 mg/ml bovine serum albumin. For adsorption,
samples were rotated end-over-end for 16 h at 4 °C. The beads
were collected and washed once with PBS with the aid of a magnet
(Dynal). Non-adsorbed membranes were diluted with PBS and collected by
centrifugation for 30 min at 200,000 × gmax in a SW50 tube.
Mass Spectrometric Protein Analyses--
Proteins from
Dynabead-associated exosomes were segregated by SDS-PAGE, stained with
Coomassie Blue, excised from the gels, and analyzed by mass
spectrometry by Protana (Denmark). In-gel tryptic digestion of proteins
was performed as described (25). Approximately 2% of the tryptic
digest was analyzed on a Bruker Reflex MALDI-TOF mass spectrometer
(Bruker, Bremen, Germany), and the obtained peptide maps were queried
against a non-redundant sequence data base. Search criteria was as
follows: mass accuracy, 50 ppm; tryptic peptides, allowed missed
cleavage sites, 1. Samples not unambiguously identified by peptide mass
fingerprints were purified and concentrated using home built
Poros R2 (Applied Biosystems) microcolumns before sequence analysis.
The tryptic peptides were sequenced on a QSTAR quadrupole-TOF mass
spectrometer (Sciex) equipped with a nanoelectrospray source
(ProtanaEngineering). Prior to analysis, the mass spectrometer was
calibrated to a mass accuracy of 20 ppm and a resolution of 9500. The
data were processed with PPSS2 (Protana's Proteomics Software Suite),
and the peptide sequence tags obtained were queried against a
non-redundant sequence data base (26). Search criteria were as follows:
MS mass accuracy, 1.1 Da; MS/MS accuracy, 0.1 Da; tryptic peptides,
allowed missed cleavage sites, 1. For verification of a retrieved
peptide sequences theoretical patterns were compared with the obtained
collision-induced dissociation mass spectra.
Lipid Analysis--
PBS-washed RN cells, Dynabead-associated
exosomes, and ultracentrifuged non-adsorbed membranes were suspended in
a total volume of 3 ml of chloroform/methanol (1:1) (v/v), and lipids
were extracted overnight at 40 °C. The suspensions were then
centrifuged for 10 min at 2000 × g, and the clear
supernatants were dried in a stream of nitrogen. The residue was
dissolved by first adding 60 µl of chloroform followed by 0.96 ml of
methanol and 0.94 ml of water. Each resulting solution was freed of
salts and sucrose by reverse phase chromatography. For this procedure
small pieces of silanized glass fiber wadding were introduced
into glass Pasteur pipettes, and a suspension (1.5 ml; 1:3 (v/v) in
methanol) of LiChroprep RP18 (40-63 µm; Merck) was added. These
columns were subsequently washed three times with 1 ml of
chloroform/methanol (1:1) (v/v), 3 × 1 ml of methanol, and 3 × 1 ml of water. Then the sample solution was applied, and the column
was washed with 3 × 1 ml of water. Bound lipids were eluted with
3 × 1 ml of methanol and 3 × 1 ml of chloroform/methanol
(1:1) (v/v). The eluate was dried under a stream of nitrogen, dissolved
in 0.10 ml of chloroform/methanol (1:1) (v/v), and analyzed by
thin-layer chromatography (TLC). TLC was performed in a horizontal
development chamber (Camag, Muttenz, Switzerland) using 10 × 10-cm silica gel-coated thin-layer plates (Merck) and a mixture of
chloroform/methanol/water (65:25:4, by volume) as the mobile phase.
Lipids were visualized by fine spraying the developed plates with
copper sulfate (10%) (w/v) in phosphoric acid (8%) (w/v) followed by
charring for 8 min at 180 °C. For quantitation the charred plates
were scanned, and the data were analyzed by phosphorimaging using the
TINA software, version 2.0 (Raytest) and compared with known amounts of
reference lipids (cholesterol, dioleoyl phosphatidyl ethanolamine,
bovine heart cardiolipin, dioleoyl phosphatidyl choline, and
sphingomyelin containing stearic or nervonic acid). For the distinction
between cholesterol and its esters thin-layer plates were developed in n-hexane/diethyl ether/acetic acid (60:40:1, by volume). For
an unambiguous identification extracted lipids from B lymphocytes, which were grown in quantity in the presence of
[1-14C]sodium acetate (Amersham Biosciences), were
separated into uncharged and acidic lipids prior to preparative TLC.
Lipids were traced by their radioactivity, scraped, and extracted with
chloroform/methanol (1:1) (v/v). The lipid extracts were then subjected
to mass spectrometry. The separation according to charge was by
DEAE-Sephadex A 25 chromatography in chloroform/methanol/water (3:7:1,
by volume). Acidic lipids were eluted with chloroform/methanol/1
M ammonium acetate (3:7:1, by volume) and freed of salts,
short chain fatty acids, and other less hydrophobic material by reverse
phase chromatography on LiChroprep RP18 as described above.
For electrospray-MS, mass spectra were recorded in the negative or
positive ion mode for acidic or uncharged lipids, respectively, on a
Q-TOF 2 mass spectrometer (Micromass, Manchester, United Kingdom)
equipped with a nanospray source. Lipids were dissolved in
chloroform/methanol (1:1) (v/v). Solutions were injected into the mass
spectrometer by glass capillaries (long type; Protana, Odense, Denmark)
using a capillary voltage of 1000 V and a cone voltage of 50 V at
70 °C. Instrument calibration was done with a mixture of sodium
iodide and cesium iodide in 50% aqueous acetonitrile with 0.1% formic
acid. For MS/MS experiments argon was used as collision gas, and
fragmentation was observed at energy values from 20-50 eV.
For MALDI-TOF-MS, measurements were done on a TOFSpec E
(Micromass, Manchester, United Kingdom) in positive or negative ion mode with an accelerating voltage of 20 kV. For lipid samples matrix
solutions of 2,5-dihydroxybenzoic acid were used in a concentration of
10 mg/ml in methanol. Spectra were calibrated externally by using
suitable reference substances.
Density Gradient Electrophoresis and Cell Surface
Biotinylation--
RN cells were collected and washed with PBS by
centrifugation at 4 °C for 10 min at 200 × g. The
cell surface was biotinylated for 15 min at 0 °C using 5 mg/ml
sulfo-NHS-biotin (Pierce). Excess sulfo-NHS-biotin was quenched with 10 mM NH4Cl in PBS for 20 min at 0 °C after
which the cells were collected and washed twice with homogenization
buffer (10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, 0.25 M sucrose, pH 7.4) by
centrifugation. Cells were homogenized using an EMBL-cell
cracker (ball 8.011 applying 10 strokes), and nuclei were removed by
centrifugation at 900 × gmax for 3 min.
The post-nuclear supernatant was treated with trypsin (25 µg/mg
protein) for 15 min at 37 °C after which trypsin inhibitor (100 µg/mg protein; Sigma) and protease inhibitor mix (Roche Molecular
Biochemicals) were added. Density gradient electrophoresis was
performed as described (27) for 30 min at a constant current of 10 mA.
Fractions of 500 µl were collected from the anodic side and analyzed
by Western blotting for MHC class II
To compare plasma membrane-associated MHC class II with
exosomes-associated MHC class II, intact cells and isolated floated exosomes were biotinylated at 0 °C as described above. After
detergent extraction and ultracentrifugation, pellets were suspended in 0.5 ml of PBS containing 1% Triton X-100, 10 mg/ml bovine serum albumin, 2 mM EDTA, 0.2 mM phenylmethylsulfonyl
fluoride, and 30 µl of Neutravidin-Sepharose beads (Pierce) for
16 h. The beads were washed with PBS and analyzed for bound MHC
class II by Western blotting.
Solubility Assay and Floatation--
Gradient fractions (3 ml in
total) containing floated exosomes were diluted with 26 ml of PBS.
Aliquots of 3.6 ml were mixed with 0.4 ml of PBS containing 10% CHAPS,
10% Triton X-100, 10 mM EDTA, 10 mM
MgCl2, and/or 100 mM methyl-
To demonstrate association of low density lipid material with MHC class
II after detergent solubilization we performed floatation assays.
Floated exosomes from sucrose density gradients were pelleted as above
and suspended in 500 µl of 2.5 M sucrose, 0.5 mM CaCl2, 0.5 mM MgCl2,
1 mM phenylmethylsulfonyl fluoride, 20 mM
Hepes/NaOH, pH 7.2, in the presence or absence of 1% CHAPS by 10 passages through a 23-gauge needle mounted on a syringe. The suspension was overlaid with a 2-0.4 M sucrose gradient in
20 mM Hepes/NaOH, pH 7.2, and centrifuged for 4 h at
200,000 × gmax. Fractions were collected
from the bottom of the tube and analyzed by SDS-PAGE followed by
Coomassie Blue staining or Western blotting.
Immunoelectron Microscopy--
Perfringolysin O modified with
subtilisin Carlsberg and subsequent biotinylation was kindly obtained
from Dr. Ohno-Iwashita (29). Cells were prepared for cholesterol
labeling as detailed elsewhere (30). Perfringolysin O-labeled sections
were fixed in 1% glutaraldehyde and labeled with rabbit polyclonal
anti-DR (24) or anti CD63 followed by protein A coupled to 15 nm of gold. After another fixation with 1% glutaraldehyde, sections were
incubated with anti-biotin antibodies (4 µg/ml) followed by
incubation with protein A coupled to 10 nm of gold. Sections were then
examined and photographed at 80 kV with a JEOL 1200 EX electron
microscope (Tokyo, Japan).
Purification of Secreted Human Exosomes--
Exosomes secreted by
the human B cell line RN were purified in three sequential steps. In
the first step we performed differential centrifugation to collect
membranes from the culture medium that sedimented between
300,000 and 4,200,000 gmax × min (10, 12). In
the second purification step pelleted exosomes were washed and floated
into sucrose density gradients to remove non-membranous serum protein
(complexes). Exosomes, as identified by the presence of MHC class II,
floated up to a density of ~1.15 g/ml (Fig.
1B), consistent with previous
observations (9, 10). The gradient fractions were diluted with PBS and
ultracentrifuged, and the pellets were analyzed by SDS-PAGE and
Coomassie Blue staining (Fig. 1A). The sample buffer used
for SDS-PAGE was supplemented with urea (see "Materials and
Methods") as we found that this greatly enhanced the separation of
exosome-associated proteins by SDS-PAGE. A number of proteins, possibly
originating from fetal calf serum in the culture medium, remained in
the bottom fractions (indicated by the arrows) whereas the
majority of proteins co-distributed with MHC class II in the gradient.
In addition to MHC class II, these proteins include CD86 and the
tetraspanins CD37, CD53, CD63, CD81, and CD82 (data not shown) as
identified previously (10) by Western blotting. As a third purification
step, gradient fractions containing MHC class II (fraction 6-8) were
pooled, and exosomes were immunoadsorbed onto anti-MHC class II-coupled
magnetic beads. Membranes that did not associate with the beads were
collected from the bead supernatant by ultracentrifugation.
Bead-associated and non-associated proteins were analyzed by SDS-PAGE
and Coomassie Blue staining (Fig. 2). The
anti-MHC class II antibody-conjugated beads recovered nearly all
proteins whereas beads coated with negative control antibodies did not
collect any of these proteins. This indicates that all detected
proteins were linked physically to MHC class II-carrying
exosomes.
Protein Composition of Exosomes--
To analyze the identity of
exosome-associated proteins, discernable Coomassie Blue-stained bands
in Fig. 2, lane 1 were excised from the gel and analyzed by
mass spectrometry. Identified proteins are indicated in Fig. 2 and
listed in Table I. Only proteins with a minimum of two matching
peptides are shown. HLA-encoded proteins,
including MHC class I heavy chain and several MHC class II subtypes,
are dominantly present in exosomes. Other membrane proteins that were
identified include Na+/K+-ATPase, the receptor
tyrosine phosphatase CD45, integrin Lipid Composition of B Lymphocytes and Exosomes--
Lipids
extracted from [1-14C]sodium acetate-labeled lymphocytes
were separated according to charge and
relative mobility by TLC, stained, and quantified (see Fig. 3,
lane 2 and Table II). The
nature of these lipids was identified in parallel experiments in which
the position of 14C-labeled lipids was determined on film
rather than by staining. These were then extracted from the TLC
plates and analyzed by electrospray-time-of-flight-mass spectrometry
and MALDI-TOF-MS (Tables III and IV).
Among the acidic lipids were ganglioside GM3 with palmitoyl or
nervonoyl residues. The majority of acidic lipids were, however,
composed of phosphatidylinositol (PI), phosphatidylserine (PS),
phosphatidic acid (PA), bismonoacyl glycerophosphate (BMP), and
cardiolipin (CL) with varying fatty acyl moieties. Their fatty acid
composition was deduced from tandem mass spectra collected in the
negative ion mode. The amount of BMP exceeded that of CL (see Fig.
3A). The uncharged lipids were comprised of sphingomyelin (SM), cholesterol (Chol), phosphatidyl choline,
phosphatidylethanolamine (PE), and ether lipids with an ethanolamine
phosphoryl head group. Like GM3, sphingomyelin contained either
palmitoyl or nervonoyl residues, resulting in two distinct bands by
TLC. SM containing C16:0 was not separated from ganglioside GM3 by TLC
(lower band of the double band in Fig.
3A). Dynabead-bound exosomes and membranes from
exosome-containing sucrose gradient fractions that did not bind to
anti-MHC class II-coated Dynabeads were analyzed for their lipid
content and compared with total cell membranes (see Fig. 3 and
Table II). Exosomes were enriched in
cholesterol (42 versus 20 mol
% in total cell membranes) and in sphingomyelin and ganglioside GM3
(23 versus 13 mol % in total cell membranes) on the expense of the presence of phosphatidyl ethanolamine and its respective ether
lipids, as well as phosphatidyl choline, phosphatidyl inositol, phosphatidyl serine, and phosphatidic acid. As expected, cardiolipin, a
lipid that is predominantly found in mitochondria, was absent from
exosomes. BMP, also referred to as lysobisphosphatidic acid (LBPA),
could also not be detected in exosomes.
Electron Microscopic Detection of Cholesterol on Exosomes--
To
determine the morphological distribution of cholesterol, cryosections
of RN cells were labeled for cholesterol with 10 nm of colloidal gold
using Perfringolysin O and examined by electron microscopy (30). The
sections were double-labeled for either MHC class II (15 nm of gold) or
CD63 (15 nm of gold). MHC class II was detected on the plasma membrane,
in MIIC, and on exosomes within MIIC-plasma membrane fusion profiles.
Consistent with previous observations (10), CD63 was found
predominantly on the internal membranes of MIIC and exosomes rather
than on the plasma membrane. Fig. 4 shows
fusion profiles of MIIC with the plasma membrane. Consistent with the
lipid analyses, cholesterol was predominantly present on secreted
exosomes and much less abundant on the MIIC-limiting membrane and
plasma membrane. We conclude, based on the morphological and
biochemical characterization of exosomes, that exosomes are relatively
enriched in cholesterol.
Exosomes Are Detergent-resistant--
The enrichment in
sphingomyelin, GM3, and cholesterol is a characteristic of so-called
DRM or raft domains (31). Such domains are unusually resistant to
solubilization by non-ionic detergents (32). To investigate whether
exosomes display DRM-like properties we determined their solubility in
the presence of 1% Triton X-100 or 1% CHAPS (Fig.
5). Sucrose gradient fractions containing
exosomes (as in Fig. 1) were pooled and washed with PBS. Aliquots were incubated as indicated for 30 min at 0 or 37 °C in the presence or
absence of 1% CHAPS, 1% Triton X-100, 1 mM EDTA, 1 mM MgCl2, and/or 10 mM
methyl- The Solubility of MHC Class II Depends on Its
Location--
Exosomes are formed as the internal vesicles of
MVB/MIIC. Because the majority of MHC class II in MIIC of RN cells
localizes to these internal vesicles (9), we hypothesized that MHC
class II in MIIC, like exosomes, should be resistant to solubilization in CHAPS. To test this idea, we isolated MIIC from surface-biotinylated cells by density gradient electrophoresis (Fig.
7), a technique that segregates MIIC from
other cellular membranes (33). Each fraction was tested for the
presence of total protein,
To compare exosomes with MIIC for the solubility of MHC class II,
samples of pooled MIIC-containing fractions from density electrophoresis gradients and pooled exosome-containing sucrose gradient fractions were diluted with excess PBS, containing either CHAPS or Triton X-100 or lacking detergent. After 30 min at 0 or
37 °C, DRM were pelleted by ultracentrifugation and analyzed for the
presence of MHC class II by Western blotting (Fig.
8). Exosomes and MIIC were similar with
respect to the solubility of MHC class II (Fig. 8A),
consistent with the notion that the majority of MHC class II in MIIC
localizes to internal vesicles and that these vesicles are released as
exosomes upon exocytic fusion of MIIC with the plasma membrane.
To compare the detergent solubility of MHC class II at the plasma
membrane with that of exosomes, intact cells and isolated exosomes were
first biotinylated. This procedure allowed selective labeling of the
exoplasmic domain of plasma membrane or exosome-associated MHC class
II. After the addition of CHAPS, insoluble MHC class II was pelleted by
centrifugation and solubilized in Triton X-100-containing PBS.
Biotinylated MHC class II was collected using Neutravidin-conjugated Sepharose beads and analyzed by Western blotting. The solubilization of
biotinylated MHC class II from exosomes in CHAPS was inefficient (Fig.
8B), possibly even more so than total MHC class II from non-biotinylated exosomes (Fig. 8A). In contrast to
exosome-related and MIIC-derived MHC class II, however, plasma
membrane-derived MHC class II was entirely solubilized by CHAPS (Fig.
8B). These data indicate that MHC class II is associated
with DRM in exosomes and MIIC but not at the plasma membrane. Possibly,
the incorporation of MHC class II in DRM at the MIIC-limiting membrane
plays a role in its sorting into the MVB internal vesicles.
We developed a method to purify B cell derived exosomes to
homogeneity, thus allowing determination of their protein and lipid composition. In a previous study we already demonstrated the presence of MHC class I and class II on B cell-derived exosomes (9, 10); here we
show that they are among the most prominent proteins. MHC class I and
class II have also been detected on exosomes from dendritic cells (11),
intestinal epithelial cells (16), and T cells (21). MHC class I was
also found to associate with tumor-derived exosomes (34), and MHC class
II was found to associate with mast cell-derived exosomes (14).
Previously, we also demonstrated by immunoelectron microscopy and
Western blotting that the tetraspanins CD63, CD37, CD53, CD81, and CD82
are heavily enriched on B cell-derived exosomes and on the internal
vesicles of MVB (10). One of these tetraspanins, CD63, is in fact also
known as lysosome-associated membrane protein 3 (Lamp 3). Tetraspanins
have also been demonstrated on exosomes derived from dendritic cells
(11, 12), intestinal epithelial cells (16), T cells (21), and platelets
(15). In the current study we failed to detect tetraspanins by the mass spectrometric analyses. Despite their relative enrichment, the only
tetraspanin detected previously (12) in exosomes by mass spectrometric
analyses is CD9, most likely because tetraspanins cannot be recovered
as discrete bands from acrylamide gels (10, 13). Tetraspanins comprise
a large group of ubiquitously expressed 25-50-kDa proteins that
contain a number of conserved residues (35). Tetraspanins associate
with each other, as well as with many Ig superfamily proteins,
proteoglycans, integrins, growth factor receptors, and signaling
enzymes, to form large transmembrane protein networks. Such networks
are involved in a variety of processes at the plasma membrane such as
cell adhesion, cell motility, and signaling (36). Many of the
interactions within these networks are relatively stable in the
presence of detergents. Moreover, detergent solubility assays
demonstrated the association of detergent-resistant lipids with
tetraspanin networks providing them with raft/DRM-like properties
(37).
It has been well established that at least fractions of MHC class II
and MHC class I localize to membrane microdomains, together with
tetraspanins and integrins, as measured by co-immunoprecipitation from
detergent lysates (37-40), flow cytometric energy transfer methods
(41), and competition assays (42). In mild detergents tetraspanins
remain associated with microdomains resembling lipid rafts/DRM whereas
at relatively harsh conditions they can be solubilized as protein webs
that remain stable independently of lipid microdomains (37). The
association of MHC class II with lipid rafts (43), as well as with
tetraspan microdomains distinct from lipid rafts (44), has been
proposed to facilitate antigen presentation. Although several of the
above mentioned studies indicated the presence of such microdomains at
the plasma membrane, their subcellular distribution has not been
investigated systematically. MHC class II may associate with distinct
sets of tertraspanins depending on its subcellular location (45). For
example, CD82 associates with MHC class II in MIIC (46). Here we
demonstrate that MHC class II is in detergent-resistant DRM/protein
webs at MVB rather than at the plasma membrane.
We found that exosomes are enriched in cholesterol,
sphingomyelin, and GM3. These lipids are characteristically
enriched in rafts/DRM. These features, together with the presence
of tetraspanins and the stable association of lipids with
CHAPS-solubilized exosomal protein webs, indicate that exosomes contain
protein/lipid complexes that can be described as webs or DRM/rafts.
These webs or DRM/rafts may contribute to protein sorting in MVB but
may also play a role in the generation of membrane buds and even in
membrane fission (31, 47). We did not observe detectable amounts of BMP
by TLC on exosomes. This is seemingly inconsistent with other studies in which BMP was detected either immunocytochemically on exosomes (18)
or biochemically in a subcellular fraction containing the internal
membranes of MVB (48). Possibly, the amount and distribution of BMP is
cell type-dependent. Furthermore, we observed by
immunoelectron microscopy that in RN
cells BMP is enriched on multilaminar lysosomes rather than on
MVB.2 Certain viruses, such as human cytomegalovirus
(49) and HIV in macrophages (50), assemble at MVB in a process
resembling the formation of MVB internal vesicles. T cells, in contrast
to macrophages, assemble HIV predominantly at the plasma membrane in a
process that requires elements of the same molecular machinery involved
in MVB biogenesis (51). Interestingly, the membrane of HIV, type 1 is,
like exosomes, enriched in cholesterol and sphingomyelin (52), and
lipid rafts have been implicated in HIV, type 1 assembly and release
(53).
At the plasma membrane, glycosylphosphatidylinositol-anchored proteins
have the tendency to be incorporated into DRM/rafts. The fact that
glycosylphosphatidylinositol-anchored proteins are also enriched in
reticulocytes exosomes (54, 55) is consistent with the idea that
exosomes derive from DRM at the MVB-limiting membrane. G The presence of hsc70 in B cell-derived exosomes is consistent with its
detection in exosomes from dendritic cells (12), tumors (34), and
maturing reticulocytes (59, 60). Maturing reticulocytes dispose of
their transferrin receptors by incorporating them into exosomes, and it
has been proposed that a direct interaction between hsc70 and the
transferrin receptor cytoplasmic domain is associated with its
targeting to exosomes (59, 60). In addition to such chaperone
functions, hsc70 is also acting in other processes involving protein
folding and unfolding. For example, hsc70 has been demonstrated to
regulate the disassembly of clathrin coats (61). Clathrin is most often
associated with outward budding of membranes into the cytoplasm.
However, clathrin has also been detected in non-curved lattices on MVB
(62), (22) including MIIC (63). Clathrin in these coats has been
demonstrated to associate with Hrs, an adaptor protein that binds
directly to clathrin and ubiquitinated membrane proteins and is
involved in the sorting of such proteins into the internal vesicles of
MVB (22) (see also below). This process may result in the incorporation of clathrin, which we also detected in exosomes, and hsc70 into MVB
internal vesicles. hsc70 forms complexes with other chaperones, including hsp90 (64), another heat shock protein that we detected in
exosomes. Consistent with their presence in exosomes, these molecular
chaperone complexes have been demonstrated to function in the
translocation of cytoplasmic protein substrates into a subset of
lysosome-like organelles (64). Although this process has been
interpreted to reflect protein translocation across the lysosomal outer
membrane, targeting to the internal vesicles of MVB cannot be excluded.
In addition to actin we detected moesin, an actin-binding
protein of the ERM family in exosomes. Moesin has been demonstrated to
play a role in de novo actin assembly on phagosomal
membranes (65). ERM proteins may play a role in budding processes as
they are incorporated into rhabdoviruses (66) and HIV (67, 68).
As indicated above, ubiquitination of membrane proteins serves as a
signal for their sorting in MVB. Sequential association of sorting
complexes, termed ESCRT 1-3, to their ubiquitin moiety is thought to
select membrane proteins for sorting into the MVB pathway (19, 69, 70).
All components that are required to recruit proteins into the MVB
pathway, including ubiquitin, ESCRT complexes, and the clathrin coat,
are released from assembled cargo prior to the actual packaging into
inwardly budding vesicles at the MVB-limiting membrane. Dissociation of
the ESCRT complexes seems to be regulated by the AAA-ATPase SKD1/Vps4,
and interference with this process results in aberrant sorting in MVB
(20, 71). It should be noted, however, that not all proteins require
ubiquitination for their sorting into the MVB pathway (23). Such
proteins may partition into the inwardly budding vesicles because of
intrinsic properties and preference to partition into raft-like
microdomains or associate with tetraspanins. Endocytosed proteins that
normally recycle, like the transferrin receptor and
acetylcholinesterase, are, when aggregated by antibodies, mistargeted
into the MVB pathway and subsequently secreted in association with
exosomes (72). The hypothesis that protein clustering is an important
determinant for entry into the MVB pathway is also supported by the
observation that interference with transferrin receptor binding to
hsc70 increases its aggregation and association with exosomes (60).
Similarly, MHC class II (73) and associated invariant chain (74) have been shown to bind directly to hsc70, and this interaction may be
important for the trafficking of MHC class II in MVB.
The transmembrane protein tyrosine phosphatase CD45 modulates the
signal that is transduced via the B cell antigen receptor by regulating
the phosphorylation state of Src family kinases and is required for
normal B cell development, tolerance induction, and responsiveness to
antigen (75). Interestingly, CD45 was found to be absent on T
cell-derived exosomes (21). The relevance of the specific association
of CD45 with B cell-derived exosomes is unclear.
Na+/K+-ATPase is generally present at the
plasma membrane. However, its cell surface expression can be regulated
by endocytosis, and it plays a regulatory role in the acidification of
endosomes and lysosomes (76).
The presence of a 4, CD45, moesin, tubulin (
and
), actin,
Gi
2, and a multitude of other proteins. An
4-integrin may direct B cell-derived exosomes to follicular dendritic cells, which were described previously as potential target cells. Clathrin, heat shock cognate 70, and heat shock
protein 90 may be involved in protein sorting at multivesicular bodies.
Exosomes were also enriched in cholesterol, sphingomyelin, and
ganglioside GM3, lipids that are typically enriched in
detergent-resistant membranes. Most exosome-associated proteins,
including MHC class II and tetraspanins, were insoluble in
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
(CHAPS)-containing buffers. Multivesicular body-linked MHC class II was
also resistant to CHAPS whereas plasma membrane-associated MHC class II
was solubilized readily. Together, these data suggest that recruitment
of membrane proteins from the limiting membranes into the internal
vesicles of multivesicular bodies may involve their incorporation into
tetraspanin-containing detergent-resistant membrane domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chain was provided by Dr.
N. Barois (INSERM-CNRS de Marseille-Luminy), and the mouse monoclonal
antibodies anti-CD81 (clone M38) and anti-CD82 (clone C33) were
provided by Dr. O. Yoshie (Kinki University School of Medicine, Osaka,
Japan). Mouse monoclonal anti-MHC class II (CR3/43) was from DAKO
(Glastrup, Denmark), mouse monoclonal anti-CD86 (BU63) was from Ancell
(Lauflingen, Switzerland), mouse monoclonal anti-CD63 (435; CLB1/2) was
from CLB (Amsterdam, The Netherlands), and polyclonal rabbit
anti-biotin was from Sanver Tech (Boechout, Belgium). For the analysis
of exosomes and cell fractions by SDS-PAGE and Western blotting,
samples were incubated for 15 min at 65 °C in urea-containing sample
buffer (5% SDS, 9 M urea, 10 mM EDTA, 2.5%
-mercaptoethanol, 120 mM Tris-HCl, pH 6.8). Proteins
were separated on 12.5, 10, or 7.5% polyacrylamide gels (SDS-PAGE).
For Western blotting, proteins were transferred from polyacrylamide
gels to Immobilon-P membrane (Millipore, Bedford, MA). The membranes
were blocked and probed with antibodies in PBS containing 5% (w/v)
non-fat dry milk (Protivar; Nutricia, Zoetermeer, The Netherlands) and
0.1% (w/v) Tween 20. Primary antibodies were probed with horseradish
peroxidase-conjugated secondary antibodies (DAKO, Glostrup, Denmark)
and detected by enhanced chemiluminescence (Roche Molecular Biochemicals).
-chain. Plasma membrane
proteins were detected by probing Western blots with streptavidin-peroxidase (Sigma) and enhanced chemiluminescence.
-Hexosaminidase was determined as described (28), and total protein
was assayed using the Bio-Rad protein assay (Bio-Rad).
-cyclodextrin
(Sigma) as indicated. The samples were then centrifuged in SW60 tubes for 1 h at 350,000 × gmax. Pellets
were analyzed by SDS-PAGE followed by Coomassie Blue staining or
Western blotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Isolation of exosomes on sucrose density
gradients. Exosomes were collected from RN culture medium by
differential centrifugation. Membranes that pelleted at 420,000 gmax × min were floated up into a sucrose
density gradient. Gradient fractions (from bottom to
top, indicated 1-11) were diluted with PBS, and
membranes were collected by ultracentrifugation and analyzed by 12.5%
SDS-PAGE and Coomassie Blue staining (A) or Western blotting
for MHC class II- (B). Exosomes peaked in fractions 6-8.
Arrows in A at the left indicate
example proteins that did not co-migrate with exosomes;
numbers at the right indicate molecular weight
markers.
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Fig. 2.
Immunopurification of exosomes.
Exosomes, isolated by differential centrifugation and floatation into
sucrose gradients (as in Fig. 1), were immunoadsorbed to Dynal beads
coated with anti-MHC class II or control IgG. The beads were collected
and analyzed by 7.5% (lanes 1-4) or 12.5% (lane
5) SDS-PAGE and Coomassie Blue-stained. Essentially all proteins
were immunoadsorbed to anti-MHC class II-coated beads (lanes
1 and 5), and little protein remained in the
supernatant (lane 3). In contrast, essentially all protein
remained in the supernatant (lane 4) of control IgG-coated
beads (lane 2), indicating the specificity of the procedure.
Coomassie Blue-stained proteins were excised from lanes 1 and 5 and analyzed by nanoelectrospray tandem mass
spectrometry. Identified proteins are indicated in the figure, as well
as in Table I. mIgHC is immunoglobulin-derived from the
mouse anti-MHC class II antibody from the Dynal beads.
4, and the receptor-associated
inhibitory signaling molecule Gi
2, which is
linked to the cytoplasmic face of membranes by a palmitoyl anchor.
Other identified proteins can be grouped in heat shock proteins
(hsp90
and hsc70), cytoskeletal proteins (
and
tubulin and
actin), a member of the ERM
(ezrin-radixin-moesin) family of
cytoskeleton-associated proteins (moesin) and a set of enzymes involved
in glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate
kinase,
-enolase, and fructose-bisphosphate aldolase A). Clathrin
heavy chain-1 and elongation factor 1A were detected, as well. Murine
immunoglobulin heavy chain originated from the anti-MHC class antibody
that was conjugated to the Dynal beads. Tetraspanins were not detected
by mass spectrometry, possibly because of low abundance and/or their
poor resolution characteristics by SDS-PAGE (10).
Proteins from Fig. 2 as identified by mass spectrometry
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Fig. 3.
Analysis of lipids from RN cells and
exosomes. Lipids from total cellular membranes and Dynal
bead-associated exosomes were extracted, subjected to thin-layer
chromatography, and stained. Lane 1, reference lipids, their
nature is indicated on the left side of the figure.
Chol, cholesterol; DOPE, dioleoyl phosphatidyl
ethanolamine; BHCL, cardiolipin from bovine heart;
DOPC, dioleoyl phosphatidyl choline; SM,
sphingomyelin from bovine brain containing stearic or nervonic acid,
resulting in a double band by TLC. Lane 2, lipids from total
RN membranes. Their nature is indicated at the right side of
the figure and was determined in a parallel experiment in which
metabolically 14C-labeled lipids were extracted from cells,
separated by TLC, and analyzed by mass spectrometry. Spot X
is a non-identified compound, spot Y is not identified but
has the mobility of monohexosylceramides, and spot Z at
the front could not be associated with any known lipid and is most
likely related to compound(s) from plastics or other paraphernalia that
were extracted by the solvents. Lane 3, lipids from Dynal
bead-associated exosomes (isolated as in Fig. 2, lane 1).
Lane 4, lipids from non-bound material (as in Fig. 2,
lane 3). The quantified relative amounts of lipids are
depicted in Table II.
Relative lipid compositions as determined from Fig. 3
Mass spectrometric analyses of prominent RN-derived acidic lipids
Mass spectrometric analyses of prominent RN-derived uncharged
lipids
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Fig. 4.
Cholesterol, MHC class II, and CD63
co-localize on exosomes. Cryosections of RN cells were
double-labeled for cholesterol (10 nm of gold) and MHC class II (15 nm
of gold) (A) or CD63 (15 nm of gold) (B).
A shows a fusion profile between a MVB and the plasma
membrane (PM) containing released exosomes (E).
Arrows indicate the site of fusion. The plasma membrane of
an opposing cell is indicated by PM*. Note the presence of
MHC class II on the plasma membrane and on exosomes. Cholesterol is
predominantly present on exosomes. B indicates a similar
structure with exosomes (E) relatively enriched in both
cholesterol and CD63 as compared with the plasma membrane
(PM).
-cyclodextrin and then centrifuged. Pellets were analyzed for
total protein content by SDS-PAGE and Coomassie Blue staining (Fig.
5A) and for the presence of MHC class II by Western blotting
(Fig. 5B). Many exosomal proteins, but not all, were
resistant to solubilization by CHAPS, independently of divalent cations
or temperature. The solubility of some proteins slightly increased when
cholesterol was chelated with methyl-
-cyclodextrin at 37 °C,
suggesting that cholesterol is important for exosomal DRM. The
solubility of exosomes was higher in Triton X-100 compared with CHAPS
but remained incomplete. The same observations were made for
exosome-associated MHC class II. MHC class II was solubilized to a
significant degree only in the concomitant presence of CHAPS and
methyl-
-cyclodextrin at 37 °C or in the presence of Triton X-100.
DRM are characterized by the stable association of both proteins and
lipids and, consequently, have a relative low buoyant density in
sucrose density gradients. To test whether DRM are associated with
exosomes, we performed floatation experiments for CHAPS-treated
exosomes (Fig. 6). Pelleted exosomes were
resuspended in 2.5 M sucrose containing 1% CHAPS and
overlaid with a sucrose density gradient. After ultracentrifugation,
gradient fractions were analyzed for the presence of MHC class II and
the tetraspanins CD81 and CD63. About one-third of each of these
markers floated up into the gradient, indicating their association with
DRM.
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Fig. 5.
Detergent insolubility of exosomes.
Samples of isolated exosomes were suspended in PBS containing no
detergent (contr; lane 1), CHAPS (lanes
2-7), or Triton X-100 (TX100; lane 8). When
indicated, the samples also contained EDTA (lane 3),
MgCl2 (Mg2+; lane 4), or
methyl- -cyclodextrin (M
cD; lanes 6 and
7). The samples were incubated for 30 min at either 0 °C
(lanes 1-4, 6, and 8) or 37 °C
(lanes 5 and 7) and ultracentrifuged at 4 °C
to sediment DRM. Pellets were analyzed either by SDS-PAGE and stained
with Coomassie Blue (A) or by Western blotting for MHC class
II
(B). Closed arrows indicate example
proteins that were entirely solubilized by detergents, open
arrows indicate partially solubilized proteins, and arrow
heads indicate non-solubilized proteins. Molecular weight markers
are on the right. The data are representative of three
independent experiments.
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Fig. 6.
Detergent-resistant exosomal proteins have a
low buoyant density. Isolated exosomes were incubated in the
absence (control) or presence of CHAPS and layered at the
bottom of a sucrose density gradient. After ultracentrifugation,
gradient fractions were tested for the presence of MHC class II
-chain, CD81, and CD63 by Western blotting. Bottom fractions are at
the left. About one-third of all three markers floated up
into the gradient, indicating their association with DRM/rafts. The
data are representative of three independent experiments.
-hexosaminidase (a marker for MVB and
lysosomes), and biotinylated proteins (positioning the plasma
membranes) and Western blotted for MHC class II. As expected, most MHC
class II was found in plasma membrane-containing fractions (fractions
16-21), but a significant amount was associated with MIIC, as
indicated by its co-migration with
-hexosaminidase (fractions
5-10).
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Fig. 7.
Isolation of MIIC by density gradient
electrophoresis. Intact RN cells were biotinylated to label plasma
membrane proteins. The cells were then homogenized, and post-nuclear
supernatants were fractionated by density gradient electrophoresis.
Anodal fractions are at the left. Fractions were analyzed
for the presence of total protein (A, closed
circles), -hexosaminidase (A, open
circles), MHC class II
-chain (B), and biotinylated
plasma membrane proteins (C). MIIC localized to fraction
5-10, and plasma membrane localized to fraction 14-21. The data are
representative of three independent experiments.
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Fig. 8.
The solubility of MHC class II depends on its
location. In A, isolated exosomes and MIIC were
incubated in the absence or presence of CHAPS or Triton X-100
(TX100) at 0 or 37 °C as indicated. The samples were then
ultracentrifuged, and the pellets were analyzed by Western blotting for
MHC class II . MHC class II from MIIC and exosomes has similar
solubilization characteristics. In B, biotinylated intact
cells and biotinylated exosomes were incubated in the presence or
absence of detergents as above. Biotinylated proteins were extracted
with Neutravidin beads from pelleted detergent-resistant DRM that were
resuspended in Triton X-100 and analyzed for the presence of MHC class
II
-chain by Western blotting. Biotinylated MHC class II was almost
entirely extracted by detergents from the plasma membrane but not from
exosomes or MIIC, indicating that the incorporation of MHC class II in
DRM depends on its subcellular location. The data are representative of
three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunits of
heterotrimeric G proteins undergo palmitoylation and/or myristoylation
on their amino-terminal ends and as a consequence are also targeted to
lipid rafts (56). Given our current finding that exosomes contain
Gi
2, it is possible that
Gi
2 is targeted to the internal vesicles of
MVB because of its incorporation into lipid rafts/DRM at the
MVB-limiting membrane. The presence of Gi
2
in exosomes has also been reported for dendritic cell-derived exosomes
(12). The association of Gi
2 with exosomes
may be related either to general MVB functions or specifically to
sorting of G protein-coupled receptors at MVB (57, 58).
4-integrin on B cell-derived exosomes is
intriguing. Exosomes released by maturing reticulocyte also contain
4
1-integrin (77). B cell selection involves their homing to
follicular dendritic cells in the germinal center in a process that is
dependent on the interaction of
4
1 with VCAM-1 (78). B
cell (RN)-derived exosomes bind in vitro to follicular
dendritic cells and not to other cell types, suggesting that follicular dendritic cells are physiological targets for B cell-derived exosomes (18). As for B cells, binding of exosomes to follicular dendritic cells
may require integrin
4. In the germinal center B cells recognize
native antigens that are held in immune complexes at the surface of
follicular dendritic cells by a set of different complement receptors.
Binding of B cells to these antigens is an essential selection step for
their differentiation into memory B cells. A prerequisite for the
stimulation of T helper cells by follicular dendritic cells is their
interaction with MHC class II. However, follicular dendritic cells do
not synthesize MHC class II molecules themselves but rather passively
acquire peptide-loaded MHC class II (79), possibly by binding B
cell-derived exosomes through the interaction of
4
1 with
VCAM-1.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. H. L. Ploegh and Dr. N. Barois for polyclonal anti-DR, Dr. Ohno-Iwashita for the biotinylated Perfringolysin O, and Dr. O. Yoshi for monoclonal antibodies against CD81 and CD82. The expert technical assistance of Petra Michel is gratefully acknowledged.
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FOOTNOTES |
---|
* This work was supported by the European Union (QLRT-2001-00093) and by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 284.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 31-30-2507577 or 31-30-2506551; Fax: 31-30-2541797; E-mail: W.Stoorvogel@Lab.AZU.NL.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M207550200
2 Möbius, W., Van Donselaar, E., Ohno-Iwashita, Y., Shimada, Y., Heijnen, H. F. G., Slot, J. W., and Geuze, H. J. (2003) Traffic 4, in press.
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ABBREVIATIONS |
---|
The abbreviations used are: MVB, multivesicular bodies; DRM, detergent-resistant membranes; MHC, major histocompatibility complex; MIIC, MHC class II containing compartments; hsc, heat shock cognate; hsp, heat shock protein; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; MS/MS, tandem MS; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PI, phosphatidyl inositol; PS, phosphatidylserine; PA, phosphatidic acid; BMP, bismonoacyl glycerophosphate; CL, cardiolipin; SM, sphingomyelin; PE, phosphatidylethanolamine; HIV, human immunodeficiency virus.
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