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Brief report
Stomatin, flotillin-1, and flotillin-2 are major integral proteins of erythrocyte
lipid rafts
Ulrich Salzer and Rainer Prohaska
Lipid rafts are sphingolipid- and cholesterolrich membrane microdomains that are
insoluble in nonionic detergents, have a
low buoyant density, and preferentially
contain lipid-modified proteins, like glycosyl phosphatidylinositol (GPI)-anchored
proteins. The lipid rafts were isolated
from human erythrocytes and major protein components were identified. Apart
from the GPI-anchored proteins, the most
abundant integral proteins were found to
be the distantly related membrane proteins stomatin (band 7.2b), flotillin-1, and
flotillin-2. Flotillins, already described as
lipid raft components in neurons and
caveolae-associated proteins in A498 kidney cells, have not been recognized as
red cell components yet. In addition, it
was shown that the major cytoskeletal
proteins, spectrin, actin, band 4.1, and
band 4.2, are partly associated with the
lipid rafts. Stomatin and the flotillins are
present as independently organized highorder oligomers, suggesting that these
complexes act as separate scaffolding
components at the cytoplasmic face of
erythrocyte lipid rafts. (Blood. 2001;97:
1141-1143)
© 2001 by The American Society of Hematology
Introduction
The concept of lipid rafts as domains of lateral organization of the
plasma membrane1-3 has gained great importance recently, because
it helps to understand diverse membrane processes such as signal
transduction in hematopoietic cells2-4 and sorting of glycosyl
phosphatidylinositol (GPI)-anchored proteins.2 In erythrocytes,
these membrane microdomains have not been investigated in
detail. Fluorescence microscopy revealed that in the red cell
membrane there are domains of unequal enrichment of different
phospholipids,5 and GPI-anchored proteins were shown to resist
membrane extraction by Triton X-100 at 4°C.6 The recent finding
that lipid rafts of epithelial cells are enriched in stomatin7 raised the
question of whether stomatin is similarly organized in the red cell
membrane, where it is a major integral protein.8-12
The aim of this study was, therefore, to isolate the lipid rafts of
erythrocytes and identify their major protein components. We show
that stomatin, flotillin-1, and flotillin-2 are highly abundant integral
proteins in these rafts.
Study design
Cells
Whole blood was obtained from healthy donors by venipuncture and
collected into heparinized tubes. Erythrocytes were pelleted (200g, 10
minutes) and subsequently washed 5 times with 150 mM NaCl and 10 mM
Tris-Cl, pH 7.5 (TBS). Blood samples from 2 patients with overhydrated
hereditary stomatocytosis (OHSt) were kindly provided by Dr Arnulf
Pekrun, University of Göttingen; the red cells were stored at ⫺80°C.
cleavage and mixed peptide sequencing14 (ABI model 476A) was performed, or Western blotting13 was performed using monoclonal antibodies
against ␣-spectrin, band 3, and ␤-actin (Sigma, St Louis, MO), flotillin-1
and ESA/flotillin-2 (Transduction Laboratories, San Diego, CA), glycophorin-C and stomatin,8 with subsequent detection by horseradish peroxidase–goat–antimouse immunoglobulin G (Promega, Madison, WI) and the
Supersignal chemiluminescence kit (Pierce, Rockford, IL). Additionally,
proteins were identified by mass spectrometry (Bruker Reflex III MALDITOF-MS) of their tryptic peptides.
Preparation of lipid rafts
Method A. Erythrocytes were lysed in 9 vol ice-cold 0.5% Triton X-100 in
TBS, incubated for 20 minutes on ice, and centrifuged (10 minutes,
15 000g, 4°C). The pellet was resuspended in cold 60% sucrose and 0.5%
Triton X-100 in TBS, with a final sucrose concentration of 40%. A total of
500 ␮L of this suspension was placed in centrifuge tubes (Beckman
13 ⫻ 51 mm), overlayed with 1.5 mL 30% sucrose in TBS, and 1 mL 10%
sucrose in TBS, and centrifuged in a precooled SW50.1 rotor (Beckman) for
17 hours, 230 000g, 4°C. Fractions (150 ␮L) were collected from the top;
lipid raft fractions were pooled, diluted with an equal volume TBS, pelleted
(10 minutes, 15 000g, 4°C), and stored at 4°C for subsequent analyses.
Method B. Erythrocytes were similarly lysed in 4 vol ice-cold 1%
Triton X-100 in TBS and, after 20 minutes on ice, mixed with an equal
volume 80% sucrose in 0.2 M Na2CO3, overlayed with 2 mL 30% and 1 mL
10% sucrose in TBS, and centrifuged as above. The diluted lipid raft
fraction was pelleted at 100 000g for 1 hour (Beckman TLA-100.1).
Extraction of lipid rafts with sodium carbonate
Protein samples were analyzed by gel electrophoresis/silver staining, as
previously described.13 For the identification of protein bands, CNBr
Lipid rafts isolated by method A were resuspended in 200 ␮L ice-cold 0.1
M Na2CO3, incubated for 10 minutes on ice, and pelleted by ultracentrifugation (Beckman TLA-100.1, 200 000g, 15 minutes, 4°C). The pellet was
resuspended in 200 ␮L 0.1 M Na2CO3, and aliquots of the supernatant and
suspended pellet were analyzed by gel electrophoresis/silver staining,
immunoblotting, and for acetylcholinesterase (AChE) activity.15
From the Institute of Medical Biochemistry, University of Vienna, Vienna
Biocenter, Vienna, Austria.
Vienna, Vienna Biocenter, Dr Bohr-Gasse 9/3, A-1030 Vienna, Austria; e-mail:
[email protected].
Submitted July 31, 2000; accepted October 10, 2000.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Identification of proteins
Supported by grant P12907 from
wissenschaftlichen Forschung (FWF).
the
Fonds
zur
Förderung
der
Reprints: Rainer Prohaska, Institute of Medical Biochemistry, University of
BLOOD, 15 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 4
© 2001 by The American Society of Hematology
1141
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1142
SALZER and PROHASKA
BLOOD, 15 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 4
immunoblotting. For gradient calibration, molecular weight standards were
used: albumin (66 kd), ␤-amylase (200 kd), and apoferritin (440 kd). AChE
(150 kd) was used as internal membrane protein marker.
Results and discussion
Figure 1. Identification and characterization of human erythrocyte lipid raftassociated proteins. (A) A total of 150 ␮L of packed red cells was extracted with
0.5% Triton X-100 on ice and centrifuged. The lipid rafts were prepared from the
detergent-insoluble pellet by discontinuous density gradient centrifugation as described in method A. Twenty 150 ␮L fractions were collected from the top and pooled
according to their contents. Aliquots of these pools were analyzed by 11% polyacrylamide gel electrophoresis/silver staining (top panel), Western blotting, and for AChE
activity, as indicated. Lane 1, pellet resuspended in 300 ␮L TBS; lane 2, fractions 17
to 20 (high density); lane 3, fractions 9 to 16 (medium density); lane 4, fractions 7 to 8
(lipid rafts). Fractions 1 to 6 (low density, not shown) did not contain protein. (B) Lipid
rafts prepared by method A were extracted with Na2CO3 and pelleted. Aliquots of the
total sample before extraction (T), the supernatant (S), and resuspended pellet (P)
were analyzed by silver staining/immunoblotting and for AChE activity. (C) Lipid rafts
containing only integral proteins were prepared in one step from 100 ␮L of packed red
cells as described in method B. The raft proteins were analyzed by gel electrophoresis/
silver staining, Western blotting, and for AChE activity, as indicated. (D) Analysis of
oligomeric complexes. Lipid rafts prepared by method A were solubilized in 0.5%
Triton X-100 at 37°C, and 200 ␮L of the extract was subjected to sucrose density
(5%-30%) centrifugation. Eighteen fractions were collected and analyzed by immunoblotting, as indicated. Molecular masses of marker proteins are in kilodaltons.
To isolate erythrocyte lipid rafts, we incubated red cells with Triton
X-100 on ice, followed by centrifugation to concentrate the
detergent-insoluble material and to separate it from the soluble
membrane proteins band 3 and glycophorin and from hemoglobin,
which disturbs immunoblot analyses. Step gradient ultracentrifugation of this pellet yielded a whitish band floating in the low-density
region of the gradient (method A). This material, which we further
refer to as lipid rafts, contained over 70% of the GPI-anchored
protein AChE and virtually all of stomatin (Figure 1A). Stomatin’s
unusually low solubility in Triton X-100 8,10,11 can be explained
now by its association with lipid rafts rather than binding to the
cytoskeleton. Variable amounts of the cytoskeletal proteins actin,
spectrin, and proteins 4.1 and 4.2 were also present in the floating
fractions. The cytoskeleton-interacting membrane proteins glycophorin C (Figure 1A) and band 3 (not shown) were absent from the
rafts. Lipid raft-associated cytoskeletal components have already
been described in other cells,16,17 and actin was identified in
raft-related caveolae.18 For red cells, the interacting proteins or
lipids remain to be determined.
The prominent 45-kd band (Figure 1A-C) was analyzed by
peptide sequencing, mass spectrometry, and Western blotting and
found to contain the raft proteins flotillin-1 and flotillin-2.19-23
Flotillins form hetero-oligomeric complexes with caveolins in
A498 kidney cells,21 whereas in neurons they cocluster with
activated GPI-anchored adhesion molecules in noncaveolar
micropatches.22,23
To distinguish between the integral and peripheral raftassociated proteins, we performed alkaline extraction using 0.1 M
Na2CO3 (Figure 1B). Stomatin, flotillins, and AChE proved to be
integral components of the lipid rafts, whereas the cytoskeletal
proteins were solubilized. An alternative one-step approach to
purify lipid rafts devoid of peripheral proteins (method B) yielded
essentially the same results (Figure 1C). These data also indicate
that potentially different lipid raft populations (small rafts) were
not lost during the first pelleting step of detergent-insoluble
complexes (method A).
Because stomatin forms homo-oligomers in epithelial cells,13
we addressed the question of the oligomeric state of stomatin and
the flotillins in red cell lipid rafts. After solubilization, these
proteins showed similar high-migration velocities in a linear
sucrose gradient (Figure 1D), indicating that they are organized in
high-order oligomeric complexes. However, immunoprecipitation
experiments failed to coprecipitate stomatin and flotillins (not
Analysis of oligomeric complexes
Proteins of isolated lipid rafts were dissolved in 200 ␮L 0.5% Triton X-100
in TBS, by incubation at 37°C for 20 minutes. After centrifugation (10
minutes, 15 000g) the supernatant was placed on top of a linear 5%-to-30%
sucrose gradient (12 mL) in 0.5% Triton X-100 in TBS, and centrifuged for
17 hours at 180 000g in a Beckman SW40 rotor at 4°C. Eighteen fractions
(0.68 mL) were collected from the top. Aliquots were analyzed by
Figure 2. Identification of stomatin, flotillin-1, and flotillin-2 in normal and OHSt
erythrocyte membranes. Erythrocytes from a healthy donor (N) and 2 OHSt
patients (1,2) were hypotonically lysed, and the prepared ghosts were analyzed by
Western blotting, as indicated.
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BLOOD, 15 FEBRUARY 2001 䡠 VOLUME 97, NUMBER 4
shown), suggesting that these proteins form independent oligomeric aggregates. These complexes probably act as different
scaffolding components at the cytoplasmic face of red cell lipid
rafts. It remains to be determined whether they function as docking
sites for the cytoskeleton or signaling components.
Stomatin is missing in erythrocytes from OHSt patients,10,11 but
the cause of this disease is still unknown.24 In the light of our
findings, it is conceiveable that OHSt erythrocytes have a defect in
the assembly and/or maintenance of lipid rafts leading to the loss of
stomatin and possibly other lipid raft proteins; however, flotillin-1
and flotillin-2 are present in OHSt erythrocytes (Figure 2). Future
RED CELL LIPID RAFTS
1143
studies on OHSt will have to consider possible alterations of red
cell lipid rafts.
In conclusion, the present study shows that the distantly related
membrane proteins25 stomatin, flotillin-1, and flotillin-2 are the
most abundant integral proteins of red cell lipid rafts, where they
are independently organized in high-order oligomeric complexes.
Acknowledgments
We thank Diethelm Gauster for peptide sequencing and Edina
Csaszar for mass spectrometric analyses.
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2001 97: 1141-1143
Stomatin, flotillin-1, and flotillin-2 are major integral proteins of
erythrocyte lipid rafts
Ulrich Salzer and Rainer Prohaska
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