THE JOURNAL OF BIOLOGICAL CHEMISTRY
18240 18244, 1997
1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of Human Plasma Membrane Phospholipid
Scramblase
A PROTEIN MEDIATING TRANSBILAYER MOVEMENT OF PLASMA MEMBRANE PHOSPHOLIPIDS*
(Received for publication, April 25, 1997, and in revised form, May 19, 1997)
Quansheng Zhou, Ji Zhao, James G. Stout, Robert A. Luhm, Therese Wiedmer, and Peter J. Sims
From the Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201-2178
The rapid movement of phospholipids (PL) between
plasma membrane leaflets in response to increased in-
tracellular Ca21 is thought to play a key role in expres-
sion of platelet procoagulant activity and in clearance of
injured or apoptotic cells. We recently reported isola-
tion of a ;37-kDa protein in erythrocyte membrane that
mediates Ca21-dependent movement of PL between
membrane leaflets, similar to that observed upon eleva-
tion of Ca21 in the cytosol (Basse, F., Stout, J. G., Sims, P.
J., and Wiedmer, T. (1996) J. Biol. Chem. 271, 17205
17210). Based on internal peptide sequence obtained
from this protein, a 1,445-base pair cDNA was cloned
from a K-562 cDNA library. The deduced PL scram-
blase protein is a proline-rich, type II plasma mem-
brane protein with a single transmembrane segment
near the C terminus. Antibody against the deduced C-
terminal peptide was found to precipitate the ;37-kDa
red blood cell protein and absorb PL scramblase activ-
ity, confirming the identity of the cloned cDNA to eryth-
rocyte PL scramblase. Ca21-dependent PL scramblase
activity was also demonstrated in recombinant protein
expressed from plasmid containing the cDNA. Quantita-
tive immunoblotting revealed an approximately 10-fold
higher abundance of PL scramblase in platelet (;104
molecules/cell) than in erythrocyte (;103 molecules/
cell), consistent with apparent increased PL scramblase
activity of the platelet plasma membrane. PL scram-
blase mRNA was found in a variety of hematologic and
nonhematologic cells and tissues, suggesting that this
protein functions in all cells.
The plasma membrane phospholipids (PL)1 are normally
asymmetrically distributed, with phosphatidylcholine (PC) and
sphingomyelin located primarily in the outer leaflet, and the
aminophospholipids, phosphatidylserine (PS) and phosphati-
* This work was supported in part by Grant R01 HL36946 from the
NHLBI, National Institutes of Health (to P. J. S. and T. W.) and a
Grant-In-Aid from the American Heart Association (to T. W.). The costs
of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertise-
ment in accordance with 18 U.S.C. Section 1734 solely to indicate this
fact.
Recipient of Research Fellowship Award, American Heart Associa-
tion, Wisconsin Affiliate.
To whom correspondence should be addressed: Blood Research
Inst., Blood Center of Southeastern Wisconsin, P.O. Box 2178, Milwau-
kee, WI 53201-2178. Tel.: 414-937-3850; Fax: 414-937-6284; E-mail:
peter_s@smtpgate.bcsew.edu.
1
The abbreviations used are: PL, phospholipid(s); EST, expressed
sequence tag; MBP, maltose binding protein; NBD-PC, 1-oleoyl-2-[6(7-
nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phospho-
choline; OG, N-octyl-b-D-glucopyranoside; PC, phosphatidylcholine; PS,
phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; bp, base
pair(s); PCR, polymerase chain reaction; MOPS, 4-morpholinepropane-
sulfonic acid.
18240
Vol. 272, No. 29, Issue of July 18, pp.
Printed in U.S.A.
dylethanolamine restricted to the cytoplasmic leaflet (1, 2). An
increase in intracellular Ca21 due to either cell activation, cell
injury, or apoptosis causes a rapid bidirectional movement of
the plasma membrane PL between leaflets, resulting in expo-
sure of PS and phosphatidylethanolamine at the cell surface (1,
35). This exposure of the plasma membrane aminophospho-
lipids has been shown to promote assembly and activation of
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several key enzymes of the coagulation and complement sys-
tems, as well as to accelerate the clearance of injured or apo-
ptotic cells by the reticuloendothelial system, suggesting that
Ca21-induced remodeling of plasma membrane PL is central to
both vascular hemostatic and cellular clearance mechanisms
(1, 6 9).
We recently reported isolation of a ;37-kDa integral mem-
brane protein from human erythrocytes that when reconsti-
tuted into liposomes mediated a Ca21-dependent, bidirectional
scrambling of PL between membrane leaflets mimicking the
action of Ca21 at the endofacial surface of the erythrocyte
membrane (10, 11). Evidence for protein(s) in platelet that
mediates a similar PL scramblase function when incorpo-
rated into liposomes has also been reported (12). Here we
report the cDNA cloning and deduced structure of the PL
scramblase from human erythrocyte and show evidence that
this same protein is expressed in human platelet and various
other cell lines and tissues where plasma membrane PL scram-
blase activity has been observed.
EXPERIMENTAL PROCEDURES
MaterialsEgg yolk PC, brain PS, and 1-oleoyl-2-[6(7-nitrobenz-2-
oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine (NBD-
PC) were obtained from Avanti Polar Lipids. Expressed sequence tag
(EST) clone with GenBanky accession number gb AA143025 was ob-
tained through American Type Culture Collection (ATCC 962235). All
restriction enzymes and amylose resin were from New England Bio-
Labs, Inc. KlenTaq polymerase was from CLONTECH Laboratories,
wheat germ agglutinin Sepharose was from Sigma, isopropyl-b-D-thio-
galactopyranoside was from Eastman Kodak, factor Xa was from He-
matologic Technologies, and Bio-Beads SM-2 were from Bio-Rad. N-Oc-
tyl-b-D-glucopyranoside (OG) and Glu-Gly-Arg chloromethyl ketone
were from Calbiochem. Sodium dithionite (Na2S2O4, Sigma) was freshly
dissolved in 1 M Tris, pH 10, at a concentration of 1 M.
PL Scramblase Isolation and Amino Acid SequencingPL scram-
blase was purified as described previously (10, 11), with the following
modifications. The active fraction eluting from Mono S was concen-
trated and exchanged into 150 mM NaCl, 20 mM Tris, 0.1 mM EGTA,
0.1% Nonidet P-40, pH 7.4, and absorbed against 5 ml of wheat germ
agglutinin-Sepharose to remove trace contaminating glycophorins. The
breakthrough material was concentrated and exchanged into 20 mM
Tris, 0.1 mM EGTA, 0.02% Nonidet P-40, pH 7.4, and subjected to
SDS-PAGE under reducing conditions in a 10% NuPAGE gel (Novex,
San Diego, CA). The band at ;37 kDa was visualized with 0.1% Bril-
liant Blue R-250 and excised for amino acid analysis and sequencing
(University of Michigan Protein and Carbohydrate Structure Facility).
450 pmol of this protein was subjected to in situ cleavage with 10 mg/ml
CNBr in 70% formic acid, the cleaved peptides were extracted into 60%
acetonitrile, 10% trifluoroacetic acid, dried in a speed vacuum, and
This paper is available on line at http://www.jbc.org
 cDNA Encoding the Plasma Membrane Phospholipid Scramblase
resolved by SDS-PAGE and electroblotted onto sequencing grade poly-
vinylidene difluoride. Peptides were observed by staining with Coomas-
sie Blue, excised, and subjected to microsequencing using Edman chem-
istry on a model 494 Applied Biosystems sequencer run with standard
cycles, yielding the sequence PAPQPPLNCPPGLEYLSQIDQILIHQ-
QIELLE. This same sequence was partially confirmed in a second
preparation of PL scramblase purified from erythrocyte ghosts and
subjected to internal peptide sequencing as above. A BLAST homology
search revealed 100% identity to the translation product of EST clone
gb AA143025 with no significant sequence homology to any protein in
available data bases.
Isolation of PL Scramblase cDNA by Plaque HybridizationThe
568-bp insert of EST clone gb AA143025 was labeled with [a-32P]dCTP
by Random Primed DNA Labeling Kit (Boehringer Mannheim) and
used to screen a cDNA library derived from human erythroleukemic cell
line K-562 in lgt11 (CLONTECH). Escherichia coli strain Y1090r was
transformed by K-562 cDNA library (4.86 3 105 pfu) and poured on 27
agarose plates (15-cm diameter, 18,000 plaque-forming unit/plate).
Plaques were lifted onto Hybond-N Nylon membranes (Amersham
Corp.). After UV-cross-linking and prehybridization in a solution com-
posed of 5 3 Denhardt, 5 3 SSC, 1% SDS, and 200 mg/ml herring sperm
DNA for 3 h at 68 C, the membranes were hybridized in the same
solution containing 5 ng/ml 32P-labeled probe for 16 h at 68 C. The
membranes were washed once with 1 3 SSC, 0.1% SDS, then three
times with 0.2 3 SSC, 0.1% SDS for 20 min at 65 C, and exposed to
x-ray film. Secondary plaque lifts and hybridization were carried out on
32 strongly positive plaques at a density of 50 100 plaques/plate.
Single positive and well isolated plaques were picked and amplified.
The length of cDNA insert was examined by PCR with lgt11 reverse
and forward primers. Six clones with cDNA inserts of .1.4 kilobase
pairs were selected for DNA sequencing.
DNA SequencingDNA was sequenced on an Applied Biosystems
DNA Sequencer model 373 Stretch using PRISM Ready Reaction
DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer) and com-
binations of vector and insert sequence primers.
Cloning of PL Scramblase into pMAL-C2 Expression VectorTo ex-
press PL scramblase as a fusion protein with maltose binding protein
(MBP), cDNA encoding PL scramblase was cloned into pMAL-C2 (New
England BioLabs). PCR was performed on a full-length clone using the
primers 59-TCAGAATTCGGATCCATGGACAAACAAAACTCACAGAT-
G-39 with an EcoRI site before the ATG start codon and 59-GCTTGCC-
TGCAGGTCGACCTACCACACTCCTGATTTTTGTTCC-39 with a SalI
site after the stop codon. KlenTaq polymerase (CLONTECH) was used
to ensure high fidelity amplification. The PCR product was digested
with EcoRI and SalI and isolated by electrophoresis on 1% low melting
agarose gel and purification with Wizard kit (Promega). The amplified
cDNA was cloned into pMAL-C2 vector digested with EcoRI and SalI,
immediately 39 of MBP. This construct was amplified in E. coli strain
TB1, and the sequence of the cDNA insert of plasmids from single
colonies was confirmed.
Expression and Purification of PL Scramblase-MBP Fusion Protein
10 ml of E. coli TB1 transformed with scramblase cDNA-pMAL-C2 were
used to inoculate 1 liter of rich LB containing 2 mg/ml glucose, 100
mg/ml ampicillin, and the bacteria were allowed to grow for about 4 h at
37 C. When A600 reached ;0.5, isopropyl-b-D-thiogalactopyranoside
was added to a final concentration of 0.3 mM. After 2 h of incubation at
37 C, the cells were centrifuged at 4000 3 g for 20 min. The cell pellet
was suspended in 50 ml of 20 mM Tris, 200 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (column buffer), and
subjected to a freeze/thaw cycle. After sonication (3 3 30 s on ice) and
centrifugation at 43,000 3 g for 1 h, the supernatant was applied to 10
ml of amylose resin. The column was washed with 20 volumes of column
buffer, and the scramblase-MBP fusion protein eluted with the same
buffer containing 10 mM maltose. Digestion of MBP-PL scramblase
protein with factor Xa was routinely performed at 1/100 (w/w) ratio of
enzyme and monitored by SDS-PAGE. In addition to MBP, the product
of this digest is the PL scramblase translation product containing the
N-terminal extension Ile-Ser-Glu-Phe-Gly-Phe (codons 26 to 21).
Reconstitution of PL Scramblase or Scramblase Fusion Protein into
ProteoliposomesReconstitution into proteoliposomes was performed
essentially as described previously (10, 11). Briefly, a mixture of PC and
PS (9:1 molar ratio) was dried under a stream of nitrogen and resus-
pended in 100 mM Tris, 100 mM KCl, 0.1 mM EGTA, pH 7.4 (Tris buffer).
Protein samples to be reconstituted were added to the liposomes at a
final lipid concentration of 4 mg/ml in the presence of 60 mM OG and
dialyzed overnight at 4 C against 200 volumes of Tris buffer containing
1 g/liter Bio-Beads SM-2. To liberate PL scramblase from MBP, the
proteoliposomes were incubated for 3 h at room temperature in the
18241
presence of 1/40 (w/w) factor Xa. The digestion was terminated by the
addition of 100 mM Glu-Gly-Arg chloromethyl ketone. Completeness of
the digest was monitored by SDS-PAGE. Following dialysis, the pro-
teoliposomes were labeled in the outer leaflet by the addition of 0.25 mol
% fluorescent NBD-PC (in dimethyl sulfoxide; final solvent concentra-
tion, 0.25%).
PL Scramblase ActivityPL Scramblase activity was measured as
described previously (10, 11). Routinely, proteoliposomes labeled with
NBD-PC were incubated for 2 h at 37 C in Tris buffer in the presence
or the absence of 2 mM CaCl2. Proteoliposomes were diluted 25-fold in
Tris buffer containing 4 mM EGTA and transferred to a stirred fluores-
cence cuvette at 23 C. Initial fluorescence was recorded (SLM Aminco
8000 spectrofluorimeter; excitation, 470 nm; emission, 532 nm), 20 mM
dithionite was added, and the fluorescence was continuously monitored
for a total of 120 s. The difference in nonquenchable fluorescence ob-
served in the presence versus the absence of CaCl2 was attributed to
Ca21-induced change in NBD-PC located in the outer leaflet (10, 11, 13).
Ionized [Ca21] was calculated using FreeCal version 4.0 software (gen-
erously provided by Dr. Lawrence F. Brass, University of Pennsylvania,
Philadelphia, PA).
Antibody against PL Scramblase C-terminal PeptideThe peptide
CESTGSQEQKSGVW, corresponding to amino acids 306 318 of the
predicted open reading frame of PL scramblase with an added N-
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terminal cysteine, was synthesized and conjugated to keyhole limpet
hemocyanin (Protein Core Facility, Blood Research Institute). Anti-
serum to this protein was raised in rabbit (Cocalico Biologicals, Inc.),
and the IgG fraction was isolated on protein A-Sepharose-CL4B (Sig-
ma). Peptide-specific antibody was isolated by affinity chromatography
on UltraLink Iodoacetyl beads (Pierce) to which peptide CES-
TGSQEQKSGVW was conjugated. This affinity-purified antibody (anti-
306 318) was used for immunoprecipitation and Western blotting of
PL scramblase (see Results and Discussion).
Immunoprecipitation of PL ScramblasePL scramblase purified
from human erythrocytes was 125I-labeled with Iodogen (Pierce), free
iodide removed by gel filtration, and the protein was incubated (4 C,
overnight) with either anti-306 318,or an identical quantity of preim-
mune rabbit IgG (1 mg/ml in 150 mM NaCl, 10 mM MOPS, 50 mM OG,
pH 7.4) or no IgG as control. The IgG was precipitated with protein
A-Sepharose and washed exhaustively, and protein bands were re-
solved by 8 25% SDS-PAGE (Phast System, Pharmacia Biotech Inc.)
under reducing conditions. Radioactive bands were visualized by auto-
radiography. To determine whether antibody to this peptide specifically
removed the functional activity associated with the purified erythrocyte
PL scramblase protein, the supernatant fractions remaining after im-
munoprecipitation were reconstituted in liposomes for activity meas-
urements, performed as described above. For these experiments, unla-
beled erythrocyte PL scramblase substituted for the 125I-labeled
protein.
Western Blot Analysis2 3 108 washed platelets, 2 3 108 erythrocyte
ghost membranes, 0.9 pmol of purified recombinant PL scramblase
(obtained by factor Xa digest of the PL scramblase-MBP fusion protein),
and 0.3 pmol of PL scramblase purified from human erythrocyte were
each denatured by boiling in 40 ml of sample buffer containing 10%
SDS, 4%b-mercaptoethanol, and 1 mM EDTA, and protein bands were
resolved by SDS-PAGE. After transfer to nitrocellulose, the blocked
membrane was incubated with 1 mg/ml of anti-306 318, and the blot
was developed with horseradish preoxidase-conjugated goat anti-rabbit
IgG (Sigma) using Chemiluminescence Reagent (DuPont).
Protein ConcentrationsProtein concentrations were estimated
based upon optical density at 280 nm, using extinction coefficients (M21
cm21) of 39,000 (PL scramblase), 64,500 (MBP), and 105,000 (PL scram-
blase-MBP fusion). PL scramblase contained in human platelet and
erythrocyte membranes was estimated by quantitative immunoblotting
of the detergent extracts, with reference to known quantities of purified
MBP-PL scramblase fusion protein.
Northern Blot AnalysisHuman multiple tissue Northern blot and
human cancer cell line multiple tissue Northern blot membranes were
obtained from CLONTECH. The blots were prehybridized with Ex-
pressHyb (CLONTECH) at 68 C for 30 min and hybridized with Ex-
pressHyb containing 5 ng/ml 32P-labeled PL scramblase cDNA probe at
68 C for 1 h, then washed, and exposed to x-ray film. After develop-
ment, the blots were stripped and hybridized with 32P-labeled b-actin
cDNA probe using identical conditions.
RESULTS AND DISCUSSION
Cloning of PL Scramblase cDNAPL scramblase was puri-
fied from human erythrocyte membranes and cleaved with
18242 cDNA Encoding the Plasma Membrane Phospholipid Scramblase
FIG. 1. cDNA and deduced amino acid sequence of PL scram-
blase. The deduced amino acid sequence of the predicted open reading
frame is shown under the nucleotide sequence (GenBank accession
number AF008445). The 32 residues of peptide sequence that were
obtained from cyanogen bromide digest of purified erythrocyte PL
scramblase are indicated by single underline. Also indicated are the
residues comprising a predicted inside-to-outside transmembrane do-
main (Ala291Gly309; double underline) and a protein kinase C phospho-
rylation site (Thr161; asterisk). See Experimental Procedures for
details.
cyanogen bromide, and Edman degradation was performed on
a 12-kDa peptide fragment to obtain 32 residues of peptide
sequence (Fig. 1, underlined sequence). This peptide sequence,
plus the anticipated methionine residue N-terminal to the pre-
dicted site of cyanogen bromide cleavage, was identified in the
translation product of a 568-bp EST clone deposited in Gen-
Bank by the I.M.A.G.E. Consortium (clone identification num-
ber 505141). No other significant matches to this sequence
were identified in any protein data base. The EST clone was
used to screen a human K-562 leukemic cell cDNA library. Of
32 positive clones identified by plaque hybridization, six clones
were sequenced yielding 1445 bp of cDNA (Fig. 1). The open
reading frame encodes a protein without a signal sequence that
contains 318 residues with a calculated mass of 35.1 kDa, a
theoretical pI of 4.8, and a single predicted transmembrane
helix near the C terminus (residues Ala291Gly309), in good
agreement with the physical properties observed for the ;37-
kDa protein band we tentatively identified as PL scramblase in
human erythrocyte membrane (10). Whereas the deduced pro-
tein sequence is notable for its high proline (12%) content,
homology searching failed to reveal significant concensus to
identifiable protein family or domain structures, with the ex-
ception of a single potential protein kinase C phosphorylation
site (Thr161). The possibility that PL scramblase function is
mediated by a phosphoprotein has previously been suggested
based on an observed decrease in PL scrambling activity in
erythrocytes depleted of ATP (14).
Analysis of the cDNA-derived protein sequence (Tmpred pro-
gram, ISREC server, University of Lausanne, Epalinges, Swit-
zerland) revealed a strongly preferred (p , 0.01) inside-to-
FIG. 2. Immunoprecipitation of erythrocyte PL scramblase. PL
scramblase purified from human erythrocytes was precipitated with
either anti-306 318 IgG (bar 1) or preimmune rabbit IgG (bar 2), and
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protein remaining in the supernatant was reconstituted into liposomes
for measurement of residual PL scramblase activity. Data normalized
to PL scramblase activity were measured for identical controls omitting
antibody (100%; bar 3). Error bars denote the means 6 SD (n 5 4).
Inset, erythrocyte PL scramblase was labeled with 125I and precipitated
with anti-306 318 antibody, and the resulting pellet was analyzed by
SDS-PAGE (lane 1). Matched controls incubated with either preim-
mune rabbit IgG (lane 2) or no IgG (lane 3) served as control. See
Experimental Procedures for details. Data of single experiment are
shown, representative of two so performed.
outside orientation of the predicted 19-residue transmembrane
helix, consistent with a type II plasma membrane protein. Most
of the polypeptide (residues 1290) thereby extends from the
cytoplasmic membrane leaflet, leaving a short exoplasmic tail
(residues 310 318). The predicted orientation of this protein is
consistent with the anticipated topology of PL scramblase in
the erythrocyte membrane, where lipid-mobilizing function is
responsive to [Ca21] only at the endofacial surface of the
membrane (3, 5, 10, 11, 15, 16).
To confirm that the cDNA we cloned from the K-562 cDNA
library actually encodes the same protein purified as PL scram-
blase from human erythrocyte membrane, we raised a rabbit
antibody against the deduced C terminus predicted from the
open reading frame of the cloned cDNA (codons 306 318). As
shown in Fig. 2, this antibody precipitated the ;37-kDa red cell
protein we tentatively identified as PL scramblase and also
absorbed the functional activity detected in this isolated eryth-
rocyte membrane protein fraction. As also evident from Fig. 2
(inset), we often observed the partial proteolysis of 37-kDa PL
scramblase to a polypeptide of ;30 kDa. The apparent suscep-
tibility of this protein to proteolytic degradation may account
for the reported rapid loss of activity observed in earlier at-
tempts to purify PL scramblase from platelet (12).
Expression and Membrane Reconstitution of Recombinant PL
ScramblaseRecombinant PL scramblase was expressed in E.
coli as fusion protein with MBP, purified by amylose affinity
chromatography, and incorporated into PC/PS liposomes for
assay of PL scramblase activity. When incorporated into lipo-
somes, the recombinant protein mediated a Ca21-dependent
transbilayer movement of NBD-PC mimicking the activity of
PL scramblase isolated from erythrocyte. PL scramblase activ-
ity was observed both for the chimeric MBP-PL scramblase
fusion protein (not shown) and for recombinant PL scramblase
liberated from MBP through proteolytic digestion with factor
Xa (Fig. 3). By contrast, no such activity was observed for
control protein consisting of the pMAL-C2 translation product
MBP lacking the PL scramblase cDNA insert. The specific PL
 cDNA Encoding the Plasma Membrane Phospholipid Scramblase
FIG. 3. Activity assay of recombinant PL scramblase. Purified
PL scramblase-MBP fusion protein (0 43 3 10211 mol; abscissa) was
reconstituted into liposomes (1 mmol of total PL), and MBP was proteo-
lytically removed by incubation with factor Xa in presence of 0.1 mM
EGTA. After digest to release MBP, the proteoliposomes were recovered
for determination of PL scramblase activity, measured in the absence
(E) or the presence () of 2 mM CaCl2 as described under Experimental
Procedures. Data are corrected for nonspecific transbilayer migration
of NBD-PC probe in identically matched control liposomes containing
either MBP or no added protein (,2% NBD-PC sequestered; not
shown). Error bars denote the means 6 SD (n 5 3). Data of single
experiment are shown, representative of two so performed. Similar
results were also obtained for proteoliposomes containing intact PL
scramblase-MBP fusion protein, omitting the factor Xa digest (not
shown).
mobilizing activity of recombinant PL scramblase expressed
and purified from E. coli was approximately 50% of that ob-
served for the endogenous protein purified from the erythrocyte
membrane, which is likely due to incomplete folding of the
recombinant protein. Half-maximal [Ca21] required for activa-
tion was approximately 100 200 mM for recombinant protein
purified from E. coli versus ;40 mM for the erythrocyte-derived
protein, raising the possibility that altered folding or an un-
known post-translational modification in mammalian cells af-
fects the putative Ca21 binding site (10, 11). In addition to
activation by Ca21, the transbilayer migration of PL in eryth-
rocytes is accelerated upon acidification of the inside leaflet to
pH , 6.0 (in absence of Ca21), a response that is also observed
in proteoliposomes containing PL scramblase purified from
erythrocyte membranes (11). A similar acid-dependent activa-
tion of PL mobilizing function was also exhibited by proteoli-
posomes incorporating recombinant PL scramblase purified
from E. coli (not shown).
Platelet PL ScramblaseIn addition to the presumed role of
PL scramblase in PS exposure following cell injury and upon
repeated sickling of SS hemoglobin red cells, the capacity of
activated platelets to rapidly mobilize aminophospholipids
across the plasma membrane is thought to play a central role in
the initiation of thrombin generation required for plasma clot-
ting (17). Whereas incubation with Ca21 ionophore causes a
marked acceleration in transbilayer movement of plasma mem-
brane PL in both platelets and erythrocytes, the apparent rate
of transbilayer PL migration in platelet exceeds that in eryth-
rocyte by approximately 10-fold, implying either a higher abun-
dance of PL scramblase or the action of another component in
platelet with enhanced PL scrambling function (18, 19). Zwaal
18243
FIG. 4. Immunoblotting of PL scramblase in human erythro-
cytes and platelets. 2 3 108 platelets (lane 1) and ghost membranes
from 2 3 108 erythrocytes (lane 2) were separated by SDS-PAGE,
transferred to nitrocellulose, and Western blotted with anti-306 318
antibody as described under Experimental Procedures. Lane 3 con-
tains 0.9 pmol of factor Xa cleaved recombinant PL scramblase, and
lane 4 contains 0.3 pmol of PL scramblase purified from erythrocytes.
Data of single experiment are shown, representative of three so
performed.
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FIG. 5. Expression of PL scramblase in multiple human tissues
and cancer cell lines. Northern blotting with PL scramblase cDNA is
shown for equal amounts of poly(A) RNA obtained from the human
tissues indicated (A) and from the following human cancer cell lines (B):
promyelocytic leukemia HL-60, epithelial cancer HeLa S3, chronic my-
elogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitts
lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma
A549, and melanoma G361. Lower panels show results for b-actin. Blots
were developed as detailed under Experimental Procedures. Data of
single experiment are shown. kb, kilobases.
and associates recently reported evidence for the existence of
protein(s) in platelet with functional properties similar to that
of PL scramblase we isolated from erythrocyte (10 12). To
determine whether the protein we now identify in the erythro-
cyte membrane is also found in platelets, we probed platelets
with antibody against PL scramblase residues 306 318. As
shown in Fig. 4, this antibody blotted a single protein in plate-
let with similar mobility to the ;37-kDa PL scramblase in
erythrocyte. Based on quantitative immunoblotting with anti-
306 318, we estimate approximately 104 molecules/cell in
platelet versus 103 molecules/cell in erythrocyte, consistent
with the increased PL scramblase activity and procoagulant
function observed for human platelets versus erythrocytes.
Tissue DistributionIn addition to platelet and red blood
cell, PL scramblase activity has been observed in many other
cells, and this Ca21-induced response is thought to be central to
the rapid movement of PS and phosphatidylethanolamine from
inner plasma membrane leaflet to the surface of perturbed
endothelium and a variety of injured and apoptotic cells (17).
The resulting exposure of PS at the cell surface is thought to
play a key role in removal of such cells by the reticuloendo-
thelial system, in addition to activation of both the plasma
complement and coagulation systems (8, 9, 17). Whereas the
molecular mechanism(s) in each circumstance remains unre-
18244 cDNA Encoding the Plasma Membrane Phospholipid Scramblase
solved, evidence for a specific platelet membrane protein func-
tioning to accelerate migration of PL between membrane leaf-
lets at increased cytosolic [Ca21] has been reported (12),
similar to the proposed role of PL scramblase in red blood cells
(10, 11). It was thus of interest to determine whether mRNA for
this protein is expressed in nucleated cells where PL scram-
blase-like activity has been observed. As shown by Fig. 5,
Northern blotting with PL scramblase cDNA revealed tran-
scripts of ;1.6 and ;2.6 kilobases in all tissues and cell lines
tested. Some tissue-to-tissue and cell line variability in the
relative abundance of these two transcripts is apparent, the
significance of which remains to be determined. Also notable
was markedly reduced expression in HL-60 and the lymphoma
lines Raji and MOLT-4, whereas abundant message was de-
tected in spleen, thymus, and peripheral leukocytes. In addi-
tion to the transformed cell lines shown, mRNA for PL scram-
blase was also confirmed in human umbilical vein endothelial
cells (not shown). Whereas these data imply that the same
protein identified as mediating accelerated transbilayer flip-
flop of the erythrocyte membrane PL also plays a similar role in
the plasma membrane of platelets, leukocytes, and other cells,
actual confirmation for this role of PL scramblase awaits anal-
ysis of a cell line that is selectively deficient in this protein. In
Scott syndrome, a bleeding disorder related to an inherited
deficiency of plasma membrane PL scramblase function, eryth-
rocytes and other cells deficient in PL scramblase activity were
found to contain normal amounts of the PL scramblase protein
(11).2 Furthermore, despite the apparent deficiency in Scott
syndrome cells of endogenous PL scramblase function, when
PL scramblase protein from these cells was purified and recon-
stituted in proteoliposomes containing exogenous PL, it exhib-
2
Q. Zhou, J. Zhao, J. G. Stout, P. J. Sims, and T. Wiedmer,
unpublished data.
ited normal Ca21-dependent PL mobilizing activity (11). This
suggests that in addition to the known regulation by intracel-
lular [Ca21], the activity of PL scramblase in the plasma mem-
brane is regulated by other as yet unidentified membrane or
cytoplasmic component(s).
AcknowledgmentsThe excellent technical assistance of Mary J.
Blonski and Timothy T. OBryan is gratefully acknowledged. The as-
sistance of Dr. Philip C. Andrews in protein sequencing and Trudy
Holyst in peptide sequencing is also gratefully acknowledged.
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MEMBRANES AND BIOENERGETICS:
Molecular Cloning of Human Plasma
Membrane Phospholipid Scramblase: A
PROTEIN MEDIATING
TRANSBILAYER MOVEMENT OF
PLASMA MEMBRANE
PHOSPHOLIPIDS

Quansheng Zhou, Ji Zhao, James G. Stout,

Robert A. Luhm, Therese Wiedmer and Peter

Downloaded from http://www.jbc.org/ at University of Toronto on March 7, 2015

J. Sims
J. Biol. Chem. 1997, 272:18240-18244.
doi: 10.1074/jbc.272.29.18240

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