AbstractWe have synthesized radioiodinated photoactivatable phosphatidylcholine (125-N3-PC) and phosphatidylserine (125I-N3-PS). After incubation with red blood cells in the dark, the labeled PC could be extracted but not the corresponding PS molecule, indicating that the latter was transported by the aminophospholipid translocase, but not the former. When irradiated immediately after incorporation, N3-PS, but not N3-PC, partially blocked subsequent translocation of spin-labeled aminophospholipids. Analysis of probe distribution by SDSpolyacrylamide gel electrophoresis revealed that 125I-N3-PS labeled seven membrane bound components with molecular masses between 140 and 27 kDa: one (or several) of these components should correspond to the aminophospholipid translocase

Devaux, Philippe F.

Fellmann, Pierre

Hervé, Paulette

Zachowski, Alain

Elsevier - Publisher Connector 

Labeling of human erythrocyte membrane proteins by photoactivatable radioiodinated phosphatidylcholine and phosphatidylserine A search for the aminophospholipid translocase 

Volume 223, number 2, 315-320 FEB 05252 November 1987 
Labeling of human erythrocyte membrane proteins by 
photoactivatable radioiodinated phosphatidylcholine and 
phosphatidylserine 
A search for the aminophospholipid translocase 
Alain Zachowski, Pierre Fellmann, Paulette Her& and Philippe F. Devaux 
Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, F-75005 Paris, France 
Received 14 August 1987 
We have synthesized radioiodinated photoactivatable phosphatidylcholine (‘2SI-NJ-PC) and phosphatidyl- 
serine (i2’I-N3-PS). After incubation with red blood cells in the dark, the labeled PC could be extracted but 
not the corresponding PS molecule, indicating that the latter was transported by the aminophospholipid 
translocase, but not the former. When irradiated immediately after incorporation, Ns-PS, but not N3-PC, 
partially blocked subsequent ranslocation of spin-labeled aminophospholipids. Analysis of probe distribu- 
tion by SDSpolyacrylamide gel electrophoresis revealed that 1251-N3-PS labeled seven membrane bound 
components with molecular masses between 140 and 27 kDa: one (or several) of these components should 
correspond to the aminophospholipid translocase. 
Phospholipid flip-flop; Nitrene; Erythrocyte; Spin-label; ESR 
1. INTRODUCTION 
Phospholipids are asymmetrically distributed 
between the two leaflets of the human erythrocyte 
membrane: phosphatidylcholine and sphingo- 
myelin are the major components of the outer 
layer, while the aminophopholipids, phospha- 
tidylserine and phosphatidylethanolamine, are 
located preferentially on the inner layer [ 1,2]. This 
stable asymmetry seems to be produced and main- 
Correspondence address: A. Zachowski, Institut de 
Biologie Physico-Chimique, 13 rue Pierre et Marie 
Curie, F-75005 Paris, France 
AbbreviationxBSA, bovine serum albumin; ESR, elec- 
tron spin resonance; PS, phosphatidylserine; PC, 
phosphatidylcholine; PE, phosphatidylethanolamine; 
TLC, thin-layer chromatography 
tained by a specific ATP requiring transport pro- 
tein, the existence of which was first postulated in 
our laboratory from ESR experiments [3]. In 
subsequent work, we have shown that PS and PE 
use the same carrier system which can be inhibited 
by protein reagents such as N-ethylmaleimide or 
ions such as calcium and vanadate [4]. Several 
laboratories have now shown that when exogenous 
aminophospholipids are introduced into the outer 
leaflet of the erythrocyte membrane, they are 
rapidly transported to the inner leaflet providing 
the cells contain ATP [5,6]. The question remains 
to identify which protein(s) of the erythrocyte sur- 
face is (are) involved. In a recent study Schroit et 
al. [7] introduced a radioiodinated photoac- 
tivatable phosphatidylserine molecule in human 
erythrocytes. Their probe preferentially labeled 30 
kDa protein which may be responsible for the 
phosphatidylserine translocation. We have syn- 
Published by Elsevier Science Publishers B. V. (Biomedical Division) 
00145793/87/$3.50 0 1987 Federation of European Biochemical Societies 315 
Volume 223, number 2 FEBS LETTERS November 1987 
thesized radioiodinated photoactivatable 
phospholipids slightly different from the ones used 
by Schroit and collaborators. Here we show that 
the phosphatidylserine analogue used in our ex- 
periments is an inhibitor of aminophospholipid 
translocation after irradiation. It labels several 
bands on gel electrophoresis, leaving several possi- 
ble attributions for the aminophospholipid 
translocase. 
addition of 10~1 of 5 mg/ml chloramine T in 0.5 
M phosphate buffer, pH 7.5. After 30 s vortexing, 
the reaction was stopped with 40 ~1 sodium 
metabisulfite (50 mg/ml)Kl (20 mg/ml) in 0.05 M 
phosphate buffer. The ‘251-N3 phospholipid was 
then extracted four times with 250 ~1 chloroform 
and analysed by TLC on silica gel plates and by 
autoradiography. When necessary, the phopho- 
lipid was purified on CM-52 cellulose column ac- 
cording to [lo]. 
2. MATERIALS AND METHODS 2.2 Photoactivation 
2.1. Synthesis 
The synthesis of photoactivatable phospholipids 
(Fig. 1) was adapted from [8] and carried out in the 
dark. 150 pmol of 4-azido-2-hydroxybenzoic ester 
of N-hydroxysuccinimide (Pierce) and 155 prnol of 
fl-alanine ethyl ester hydrochloride (Serva) were 
dissolved in 6 ml chloroform containing 45 ~1 
triethylamine and stirred for 30 min. The eth- 
yl N-(2-hydroxy-4-azidobenzoyl)-3-aminopropa- 
noate formed was purified by TLC and transform- 
ed quantitatively into the corresponding acid by an 
overnight incubation in 1.5 ml dioxane/2.7 ml 
sodium hydroxide solution (2 mol per liter of 
water). After acidification to pH 3, the reaction 
mixture was extracted three times by chloroform. 
The overall yield was better than 70%. The 
esterification of this acid onto lysophosphatidyl- 
choline to yield photoactivatable PC was perform- 
ed according to Samuel et al. [9]. Photoactivatable 
PC was transformed into photoactivatable PS by 
the phospholipase D-catalysed base-exchange r ac- 
tion as described by Comfurius and Zwaal [lo]. 
Iodination of the photoactivatable phospholipids 
was carried out as follows: 5 pg N3-lipid in 20 ~1 
chloroform were mixed with 20 pl Na1251 (2 mCi, 
carrier-free; Amersham); iodination was started by 
Freshly drawn erythrocytes were washed five 
times in 130 mM NaC1, 20 mM phosphate buffer, 
pH 7.4, and treated by 5 mM di-isopropyl- 
fluorophosphate to inhibit phospholipase ac- 
tivities. They were then brought to 50% hematocrit 
and incubated in the dark with the photoac- 
tivatable phospholipids for either 2 or 60 min at 
37°C. Cell suspension was then diluted 20 times 
with ice-cold buffer, transferred into a Petri dish 
and placed on ice 10 cm below a 30 W, 3 12 nm-U’V 
lamp for 10 min. Cells were then washed twice in 
NaCl-phosphate buffer. 
2.3. Aminophospholipid translocase-binding 
assay 
!? 
FH,-0-C-_(CH&,,-CH, 
0- CH-0-$-CH,- CH,--NH-C 
R-0-A-0-;H, o 
6H00 N3 
0 
6 
IL?51 
. 
R= CH,-CH,-N-_(CH,), ‘*%N,-PC 
R&H*-Y-NH; ‘*‘I-N,-PS 
coo- 
Fig.1. Structure of ‘*‘I-N3-PC and r2%-NJ-PS. 
The activity of the phospholipid translocase was 
measured by the spin-label assay [3,4]. Briefly 
spin-labeled PS or PE were introduced at 4°C in 
erythrocytes and their translocation towards the 
inner leaflet monitored by measurement of the 
fraction of spin-labels resistant o externally added 
ascorbate. In order to determine if the photoac- 
tivatable lipids bind to the translocase, two types 
of experiments were run. First, the outside-inside 
distribution of spin-labeled PE was assessed in the 
presence of non-irradiated NYPS or N3-PC. 
Secondly, erythrocytes supplemented with either 
N3-PS or N3-PC (2% of membrane phospholipids) 
were incubated at 37°C (for 2 or 30 min), and ir- 
radiated. Samples were subsequently washed twice 
with 1% BSA in phosphate buffer and twice in 
buffer alone, and spin-labeled PS transmembrane 
reorientation measured. Controls without 
photoactivation were run in parallel. 
2.4. Analysis of photolysis products 
White ghosts were prepared from washed, UV- 
irradiated erythrocytes by repetitive washings in 5 
316 
Volume 223, number 2 FEBS LETTERS November 1987 
mM phosphate buffer and solubilised in elec- 
trophoresis sample buffer containing SDS and fl- 
mercaptoethanol. Electrophoresis was performed 
using Laemmli’s discontinuous system [ 111, with a 
5% acrylamide stacking gel and a 10% acrylamide 
separating gel, at a constant current intensity of 40 
mA per gel. Gels were then stained, dried and 
autoradiographed for two weeks. 
3. RESULTS 
3. I. Inhibition of aminophospholipid 
translocation 
Non-irradiated NYPC in the erythrocyte mem- 
brane did not impair the transmembrane reloca- 
tion of spin-labeled PE, which shows the absence 
of competition between the spin-labeled PE and 
the photoactivatable PC. By contrast, N3-PS in- 
hibited the relocation of spin-labeled PE (not 
shown) and, thus competes with PE for the 
60 120 180 
Time (min.1 
Fig.2. Kinetics of transmembrane relocation at 4°C of 
spin-labeled PS in erythrocytes irradiated in the absence 
of NJ-phospholipids (0), in the presence of N3-PC (Ir), 
in the presence of N3-PS after a 2 min incubation (A) or 
a 30 min incubation and albumin extraction (0). 
Transmembrane relocation was assayed as described in 
[3,41. 
translocase. From the decrease in the initial veloci- 
ty of PE reorientation, we inferred an apparent 
affinity of N3-PS for the aminophospholipid 
translocase comparable to that of spin-labeled PE, 
i.e. 2: 50 FM [4]. Photoactivation of N3-PC em- 
bedded in the red cell membrane did not affect 
reorientation kinetics assayed with spin-labeled PS 
(fig.2). On the other hand, UV irradiation of 
N3-PS, following 2 min preincubation, reduced the 
initial velocity of spin-labeled PS to 72 f 4% of 
the control; after 30 min preincubation the inhibi- 
tion was less pronounced, leaving 87 + 3% of the 
velocity. Control experiments run in the conditions 
of photoactivation but without actual irradiation, 
showed that subsequently to the washing with 
BSA, there was no inhibition of the aminophos- 
pholipid reorientation. Thus, NJ-phospholipids in- 
cubated with cells for a short period of time are 
fully extractable. 
a bc de 
*43 
,30 
420 
*I4 
Fig.3. Autoradiographs obtained after electrophoresis 
of photoaffinity-labeled erythrocyte membranes (see 
section 2). Gels are stained with Coomassie blue (right 
lane) and autoradiographed. Lanes: a and b, two dif- 
ferent concentrations of ?-Nx-PC; c, ‘251-N3-PS 
without irradiation; d and e, “‘I-N3-PS illuminated 
after 2 min or 60 min incubation at 37°C. Arrows in- 
dicate the migration of molecular mass marker proteins. 
317 
Volume 223. number 2 FEBS LETTERS November 1987 
3.2. Membrane labeling 
Fig.3 shows the electrophoretic pattern of 
Coomassie blue-stained membrane proteins and 
the autoradiographic patterns obtained under 
various conditions. In the absence of irradiation, 
no spot can be detected except at the dye front 
(lane c). The pattern observed after photoactiva- 
tion following a 2 min incubation at 37°C is dif- 
ferent for 1251-N3-PC (lanes a and b) and 
1251-N3-PS (lane d): several bands are well in- 
dividualized with the latter probe while the former 
one indicates an efficient coupling of undistinct 
pattern. If cells incubated with less ‘*‘I-N3-PC are 
analysed (lane b), onecan see that the major com- 
ponent labeled comigrates with band 3 protein 
(nomenclature according to Steck [12]). UV-light 
irradiation of cells which had been incubated 60 
Fig.4. Density profiles of autoradiographs: erythrocytes 
incubated with 1251-N3-PS for 2 min (A) or 60 min. (B) 
at 37°C before irradiation, or pretreated with an excess 
of cold N,-PC (C) or cold Ns-PS (D) prior to addition 
of ‘251-N3-PS; erythrocytes incubated with N- 
ethylmaleimide before addition of rz51-NYPS (E). 
min at 37°C in the presence of ‘251-Ns-PS led to a 
pattern (lane e) which closely resembled that ob- 
tained after a short incubation (lane d), with the 
exception of a more intense spot comigrating with 
proteins of M,- 27000. This is more obvious on 
the profiles obtained by autoradiograph scanning 
(fig.4): trace A shows the labelling by ‘251-Ns-PS 
after a 2 min incubation while trace B was obtained 
after a 60 min incubation. If, prior to incubation 
with ‘251-N3-PS, cells were incubated and ir- 
radiated in the presence of an excess of cold N3-PC 
or cold Ns-PS, the autoradiography profiles ob- 
tained are different as shown in fig.4C and D. A 
pre-treatment of erythrocytes by 1 mM N- 
ethylmaleimide as described in [4] abolishes all 
subsequent labeling by ‘*‘I-Ns-PS, except for com- 
ponents migrating at the dye front (trace E. fig.4). 
If, after a 60 min incubation at 37”C, cells were 
washed with BSA prior to UV illumination, no 
labeling by ‘*‘I-Ns-PC remained while labeling by 
1251-N3-PS exhibited a profile resembling that of 
trace B. 
DISCUSSION 
In this paper, we have described the synthesis of 
radioiodinated photoactivatable phospholipids 
(1251-N~-PC and 1251-Ns-PS), which we have used 
for the differential labeling of human erythrocyte 
membrane proteins. Both lipids incorporated 
spontaneously into the outer leaflet of erythrocytes 
from which they could be extracted by washing 
with BSA. However, if N3-PS was allowed to in- 
cubate in red cells, prior to BSA extraction, it re- 
mained in the erythrocytes, suggesting a 
translocation towards the inner leaflet by the 
aminophospholipid translocase. Similar observa- 
tions were made by Schroit et al. [7], using dif- 
ferent photoactivatable phospholipids; these 
authors interpreted their data as being indicative of 
a ‘specific immobilisation’ of the PS derivative in 
the cells’ outer leaflet. That the N3-PS selectively 
interacts with the aminophospholipid translocase 
and not the NJ-PC, is also indicated by the ex- 
periments where the photoactivatable lipids are 
coupled to the proteins by irradiation. Indeed, 
after UV irradiation, N3-PS, and not NYPC, par- 
tially inhibited the activity of the transport enzyme 
as judged from subsequent assays conducted with 
spin-labeled aminophospholipids (fig.2). The in- 
318 
Volume 223, number 2 FEBS LETTERS November 1987 
hibition very likely indicated a covalent binding of 
N3-PS to the aminophospholipid translocase. Note 
that if Nj-PS was incubated with the membranes 
before irradiation, hence was allowed to reach the 
inner leaflet before chemical coupling, then no in- 
hibition took place. This suggests that the affinity 
of phosphatidylserine derivatives to the 
translocase, from the inner layer, is low. 
As can be seen from the autoradiographs (fig.3), 
r2’I-N3-PC and 125 I-Nj-PS label differently the 
proteins of the red blood cell membrane. First, as 
already quoted by Schroit et al. [7], there is a 
higher yield of binding with the PC derivative than 
with the PS derivative. If irradiation is carried out 
under condition of weak binding, the PC 
derivative labels mainly band 3 protein, which 
probably reflects solely the fact that band 3 ac- 
counts for approximately 50% of the total intrinsic 
proteins of erythrocyte membranes [13]. 1251- 
Nj-PS migrates with several bands which are 
resolved in the scans shown in fig.4A. We detect 
seven major membrane components which are 
migrating as the following proteins (according to 
the nomenclature of Steck [12]): unknown (- 140 
kDa), band-3 (95 kDa), PAS-l (80 kDa), PAS-4 
(67 kDa), band 5 or PAS-2 (44 kDa), band 6 (32 
kDa) and PAS-3 or band 7 (27 kDa). An additional 
band seen at 17 kDa corresponds most likely to a 
hemoglobin polypeptide. Incubation at 37°C 
which allows some 1251-Ns-PS to be transported in- 
to the inner membrane leaflet, is accompanied by 
an increased labelling of the 27 kDa peptide. Either 
this polypeptide is easier to label from the inside of 
the cell or we detect two comigrating proteins, one 
of which being localized on the cytoplasmic face. 
Since the inhibitory property of Ns-PS on the 
aminophospholipid translocation is lost when 
membranes are irradiated after incubation at 
37”C, this band does not appear to be a good can- 
didate for the the protein being searched. This 
band would correspond to the major band labeled 
by Schroit et al. 171 with their photoactivatable PS, 
since, according to these authors, it comigrates 
with band 7. 
Binding experiments carried out with prior ir- 
radiation in the presence of cold N3-PC or cold 
N3-PS (fig.4C and D) show that “‘I-N3-PS: (i) 
does not compete for binding with N3-PC; but (ii) 
competes with N3-PS. This latter conclusion 
proves that the iodinated derivative is chemically 
, equivalent to the noniodinated molecule and that 
the number of phosphatidylserine binding sites is 
limited, except perhaps on band 3 protein. Finally 
preincubation with N-ethyl-maleimide (fig.4E) 
clearly confirms that the photoexcited nitrenes 
have a preference for SH groups [14]. Thus the 
distribution of radiolabel among the components 
of red blood cells is probably sensitive to the chain 
length of the fatty acid which bears the nitrene 
residue. It is therefore not surprising that our 
autoradiograph patterns are not identical to the 
ones published recently by Schroit et al. [7]. In fact 
the two investigations are quite complementary. 
Definitive assignment of the aminophospholipid 
translocase will await for a different type of la- 
beling. 
ACKNOWLEDGEMENTS 
We thank Drs Bensaude and Bienvenue for their 
help during the chemical synthesis. Dr P. Douzou 
is acknowledged for allowing us to use a scan den- 
sitometer. This work was supported by the Centre 
National de la Recherche Scientifique (UA 526), 
the Institut National de la Sante et de la Recherche 
Medicale and the Universite Paris VII. 
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Labeling of human erythrocyte membrane proteins by photoactivatable radioiodinated phosphatidylcholine and phosphatidylserine A search for the aminophospholipid translocase

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