Sphingomyelin chain length influences the distribution ofGPI-anchored proteins in rafts in supported lipid bilayers

ASHLEY E. GARNER1, D. ALASTAIR SMITH2, & NIGEL M. HOOPER1

1Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, and Leeds Institute of

Genetics, Health and Therapeutics, University of Leeds, and 2Institute of Molecular Biophysics and the Astbury Centre for

Structural Molecular Biology, University of Leeds, Leeds, UK

(Received 4 May 2006 and in revised form 7 November 2006)

AbstractGlycosyl-phosphatidylinositol (GPI)-anchored proteins are enriched in cholesterol- and sphingolipid-rich lipid rafts withinthe membrane. Rafts are known to have roles in cellular organization and function, but little is understood about the factorscontrolling the distribution of proteins in rafts. We have used atomic force microscopy to directly visualize proteins insupported lipid bilayers composed of equimolar sphingomyelin, dioleoyl-sn -glycero-3-phosphocholine and cholesterol. Thetransmembrane anchored angiotensin converting enzyme (TM-ACE) was excluded from the liquid ordered raft domains.Replacement of the transmembrane and cytoplasmic domains of TM-ACE with a GPI anchor (GPI-ACE) promoted theassociation of the protein with rafts in the bilayers formed with brain sphingomyelin (mainly C18:0). Association with therafts did not occur if the shorter chain egg sphingomyelin (mainly C16:0) was used. The distribution of GPI-anchoredproteins in supported lipid bilayers was investigated further using membrane dipeptidase (MDP) whose GPI anchorcontains distearoyl phosphatidylinositol. MDP was also excluded from rafts when egg sphingomyelin was used butassociated with raft domains formed using brain sphingomyelin. The effect of sphingomyelin chain length on thedistribution of GPI-anchored proteins in rafts was verified using synthetic palmitoyl or stearoyl sphingomyelin. Both GPI-ACE and MDP only associated with the longer chain stearoyl sphingomyelin rafts. These data obtained using supportedlipid bilayers provide the first direct evidence that the nature of the membrane-anchoring domain influences the associationof a protein with lipid rafts and that acyl chain length hydrophobic mismatch influences the distribution of GPI-anchoredproteins in rafts.

Keywords: Atomic force microscopy, lipid rafts, sphingomyelin, GPI-anchored protein, cholesterol

Abbreviations: ACE, angiotensin converting enzyme; AFM, atomic force microscopy; CHO, Chinese hamster

ovary; DOPC, dioleoyl-sn-glycero-3-phosphocholine; DRM, detergent resistant membrane; GPI, glycosyl-

phosphatidylinositol; GPI-ACE, GPI anchored form of angiotensin converting enzyme; ld, liquid disordered; lo,

liquid ordered; MDP, membrane dipeptidase; octyl glucoside, n-octyl b-D-glucopyranoside; TBS, Tris-buffered

saline; TM-ACE, transmembrane form of angiotensin converting enzyme.

Introduction

The existence of distinct domains in biological

membranes has prompted considerable interest in

recent years [1�3]. One such domain, enriched in

sphingolipids and cholesterol, the so-called lipid raft,

serves to cluster specific proteins and lipids and has

been implicated in a variety of cellular functions

including protein sorting, membrane trafficking and

signal transduction [4,5]. The specific lipid composi-

tion of rafts renders them resistant to solubilization

by certain detergents and this has been exploited to

isolate detergent resistant membrane (DRM) frac-

tions from cells [6�8]. Studies on model membrane

systems have shown that lipid bilayers composed of

sphingolipids, cholesterol and unsaturated phospho-

lipids, phase separate to form liquid-ordered (lo)

domains in a sea of liquid-disordered (ld) lipids

[9,10]. These lo domains, like rafts, are enriched in

sphingolipids and cholesterol, and are resistant

to detergent solubilization. Such model membrane

Correspondence: Nigel M. Hooper, Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological

Sciences, and Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, UK. Tel: �/44 113 343 3163.

Fax. �/44 113 343 3167. E-mail: n.m.hooper@leeds.ac.uk

Molecular Membrane Biology, May�June 2007; 24(3): 233�242

ISSN 0968-7688 print/ISSN 1464-5203 online # 2007 Informa UK Ltd

DOI: 10.1080/09687860601127770

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systems provide a means of investigating the biophys-

ical properties of rafts and can present valuable

insights into lipid-lipid and lipid-protein interactions

[5,11].

One of the major classes of proteins associated

with lipid rafts are the glycosyl-phosphatidylinositol

(GPI)-anchored proteins [6,12]. The distribution of

GPI-anchored proteins in rafts is attributable pre-

dominantly to their lipid chains, which are typically

saturated, and therefore preferentially pack with the

more ordered, saturated chains of raft lipids [13,14].

Several studies have shown that a GPI anchor will

target proteins to rafts in both model membranes

[15�17] and cells [18�20]. However, these investi-

gations have relied upon detergent solubilization to

determine the distribution of raft and non-raft

proteins, a method which subsequent reports have

suggested is susceptible to artefacts and may cause

protein redistribution [21�24].

In the present study the use of detergents was

avoided by directly visualizing supported lipid bi-

layers using atomic force microscopy (AFM), a

technique that has been successfully utilized to

image lipid bilayers, lo raft domains and membrane

proteins in recent years [25�27]. Although it has

been reported that the GPI-anchored placental

alkaline phosphatase partitions into lo domains in

model membranes [28�30], no study has directly

compared the distribution of a transmembrane

polypeptide anchored protein with that of a GPI-

anchored protein in model membranes. Here we

present data on the distribution of the transmem-

brane protein, angiotensin converting enzyme (TM-

ACE) within supported lipid bilayers composed of

equimolar sphingomyelin, dioleoyl-sn-glycero-3-

phosphocholine (DOPC) and cholesterol, and com-

pare this distribution with that obtained using a

GPI-anchored form of ACE (GPI-ACE) and the

GPI-anchored membrane dipeptidase (MDP). In

addition we study the effect of sphingomyelin chain

length on the raft localisation of GPI-ACE and

MDP using natural sphingomyelin mixtures from

egg and brain which are comprised of mainly C16:0

and C18:0 chains, respectively. Synthetic palmitoyl

and stearoyl sphingomyelin were used to verify that

the acyl chain length was responsible for the

distribution of GPI-anchored proteins in egg and

brain sphingomyelin rafts.

Materials and methods

Purification of TM-ACE, GPI-ACE and MDP

TM-ACE was purified from porcine kidney cortex,

following solubilization of the membranes with

Triton X-100, by lisinopril-Sepharose affinity chro-

matography as described previously [31]. The pro-

tocol was adapted to replace the Triton X-100 with 2

mM n-octyl -D-glucopyranoside (octyl glucoside) in

the final isolation buffer. Chinese hamster ovary

(CHO) cells stably expressing GPI-ACE [18,32]

were cultured in Ham’s F12 medium (Cambrex Bio

Science, Berkshire, UK) supplemented with 10%

GIBCO foetal bovine serum (Invitrogen, Paisley,

UK), penicillin (50 units/ml) and streptomycin (50

units/ml) (both from Cambrex Bio Science, Berk-

shire, UK). Confluent cells were washed and

scraped into phosphate buffered saline (20 mM

Na2HPO4, 2 mM NaH2PO4, 150 mM NaCl, pH

7.4). Cells were pelleted at 500 g for 5 min,

sonicated, centrifuged at 100,000 g for 1 h and

then solubilized using octyl glucoside (60 mM).

Following centrifugation at 100,000 g for 1 h, GPI-

ACE was purified from the supernatant fraction by

lisinopril-Sepharose affinity chromatography [31]

with the final isolation buffer containing 2 mM octyl

glucoside. MDP was purified from porcine kidney

cortex, following solubilization from the membranes

with octyl glucoside, by cilastatin-Sepharose affinity

chromatography as described previously [33] with 2

mM octyl glucoside in the final protein isolation

buffer. Enzyme activity was determined using Hip-

puryl-His-Leu as substrate for TM-ACE and GPI-

ACE [31] and Gly-D-Phe as substrate for MDP

[33]. Protein concentration was determined using

the bicinchoninic acid assay with bovine serum

albumin as standard [34]. For SDS polyacrylamide

gel electrophoresis, proteins were separated on 7�17% (w/v) acrylamide gradient gels as described

previously [31] with Precision protein prestained

standards (Bio-Rad, Hertfordshire, UK).

Formation of supported lipid bilayers

Egg sphingomyelin, brain sphingomyelin, N-pal-

mitoyl-D-erythro-sphingosylphosphoryl-choline

(palmitoyl sphingomyelin), and N-stearoyl-D-ery-

thro-sphingosylphosphoryl-choline (stearoyl sphin-

gomyelin) were purchased from Avanti Polar

Lipids (Alabaster, USA). DOPC and cholesterol

were purchased from Sigma-Aldrich (Dorset, UK).

Equimolar mixtures of sphingomyelin, DOPC and

cholesterol were prepared in chloroform:methanol

(3:1 volume ratio) and dried under argon for 2 h.

The dried lipid mixtures were rehydrated in Tris-

buffered saline (TBS; 5 mM Tris/HCl, 100 mM

NaCl, pH 7.6) to a concentration of 2 mg/ml and

the purified protein (1�2 mg per ml hydrated lipid)

was added. For control samples, the final protein

isolation buffer containing 2 mM octyl-glucoside

was used in the absence of purified protein.

Vesicles were formed by a process of vortexing,

234 A. E. Garner et al.

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dialyzing in TBS overnight and then sonicating in

a bath sonicator (Ultrawave Ltd, Cardiff, UK) for

30 min. Supported lipid bilayers were prepared by

transferring 10 ml of sample on to freshly cleaved

mica followed by 80 ml TBS containing 2 mM

CaCl2. After 3 min the bilayer was washed twice

with TBS before imaging by AFM.

Atomic force microscopy

A multimode atomic force microscope with a

Nanoscope IIIa controller (Digital Instruments,

Santa Barbara, USA) and an E-scanner was used

to image samples in TBS with a fluid cell (Digital

Instruments). Images were recorded in tapping

mode using oxide-sharpened, silicon nitride tips

mounted on cantilevers with nominal spring con-

stants of 0.32 Newton/m, oscillating to a frequency

between 7 and 9 KHz. The set point was adjusted

during imaging to minimize the force whilst scan-

ning at a rate of 1�2 Hz. Nanoscope software was

used to flatten the images.

Statistical analysis of protein distribution

A minimum of three separate experiments were

performed for each of the proteins in each of the

lipid mixtures. Multiple scans of 5�10 mm square

were recorded for each experiment and 9 represen-

tative images were used for analysis. The total

number of proteins in raft and non-raft regions was

analyzed using Statistica (Statsoft, UK). Analysis of

the AFM data showed that the number of proteins

which were incorporated into lo rafts was a contin-

uous random variable with a probability density

function which approximated to a normal distribu-

tion. Significance testing was carried out to deter-

mine whether there was a difference between the

mean protein incorporation into lo and a value for Z

determined. A one-tailed test was performed, with

the null hypothesis being rejected if �/3.291B/ZB/

3.291 inferring significance at the 0.5% level.

Results

To investigate the effect of the type of anchor on the

distribution of a protein in the membrane, two forms

of ACE were purified. The endogenous transmem-

brane form of ACE (TM-ACE) was purified from

porcine kidney, while a GPI-anchored form of ACE

(GPI-ACE) was purified from CHO cells that had

been transfected with the cDNA in which the

sequence encoding the transmembrane and cytosolic

domains of human ACE had been replaced with the

sequence encoding a C-terminal GPI anchor attach-

ment signal [18,32]. Both proteins migrated as a

single band of 180 kDa on SDS polyacrylamide gel

electrophoresis (Figure 1). TM-ACE and GPI-ACE

had similar specific activities of 1.98 mmol/min/mg

and 1.91 mmol/min/mg, respectively, with Hippuryl-

His-Leu as substrate, similar to that reported pre-

viously [31].

Supported lipid bilayers composed of equimolar

sphingomyelin, DOPC and cholesterol using either

egg (mainly C16:0; Avanti Polar Lipids) or brain

(mainly C18:0; Avanti Polar Lipids) sphingomyelin

were formed and imaged by AFM (Figure 2a�2d).

Both the lipid mixtures exhibited phase separation

and formed lo ‘raft’ domains which were approx. 0.7

nm higher than the surrounding ld ‘non-raft’ lipids,

as described previously [25,29]. In the bilayers

containing egg sphingomyelin 35.3% of the surface

area was in the higher lo raft domains, while in the

brain sphingomyelin bilayers 34.0% was in the lodomain.

The addition of TM-ACE and GPI-ACE to the

lipid vesicles during preparation enabled these two

proteins to be incorporated into the lipid bilayer and

their distribution between the phases to be investi-

gated. AFM images (n�/9) were analyzed from

repeated experiments for both TM-ACE and GPI-

ACE in lipid mixtures containing either egg or brain

sphingomyelin (Figure 3). Both proteins were visua-

lized as small protruding particles in the AFM

image. The total number of protein molecules in

the lo and ld regions was counted in order to

determine the percentage of each protein associated

with the lo rafts (Table I). Surprisingly, in the

supported lipid bilayers containing egg sphingomye-

lin both the TM-ACE and GPI-ACE were located

Figure 1. Purification of TM-ACE, GPI-ACE and MDP. TM-

ACE, GPI-ACE and MDP were purified as described in the

Experimental section, analysed on a 7�17% polyacrylamide SDS

gel and stained with Coomassie Brilliant Blue. The positions of

the molecular weight markers (kDa) are shown.

Distribution of GPI-anchored proteins in bilayers 235

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almost exclusively in the ld non-raft phase with only

2.4% and 1.5%, respectively, present in the lo rafts

(Figure 3a, 3c). However, in bilayers formed from

the lipid mixture containing brain sphingomyelin,

although the TM-ACE was still found almost

exclusively (96.8%) in the ld phase, a significant

proportion (41.3%) of the GPI-ACE was associated

with the lo raft domains (Figure 3b, 3d). These

results indicate directly that the type of membrane

anchor on ACE determines its distribution between

raft and non-raft domains of the membrane,

although this differential distribution is only seen

when bilayers contain brain sphingomyelin.

In order to determine whether the sphingomyelin

species affected the distribution of another GPI-

anchored protein between rafts and non-raft do-

mains, we investigated the distribution of MDP, a

well-characterized GPI-anchored protein whose

complete anchor structure has been determined

[35]. MDP purified from porcine kidney migrated

as a single band of 45 kDa on SDS polyacrylamide

gel electrophoresis (Figure 1) and had a specific

activity of 42.3 mmol/min/mg with Gly-D-Phe as

substrate, similar to that reported previously [33].

MDP was incorporated into lipid vesicles of equi-

molar sphingomyelin, DOPC and cholesterol using

either egg or brain sphingomyelin (Figure 4). Images

(n�/9) from repeated experiments were analyzed in

order to determine the percentage of the protein in lorafts with each sphingomyelin species (Table I). As

with GPI-ACE, MDP was also essentially excluded

from lo rafts when egg sphingomyelin was used in the

Figure 2. AFM images of supported lipid bilayers containing either egg, brain, palmitoyl or stearoyl sphingomyelin. Supported lipid

bilayers composed of equimolar sphingomyelin, DOPC and cholesterol were imaged in fluid using tapping mode AFM. Surface images of

bilayers containing (a) egg sphingomyelin, (b) brain sphingomyelin, (e) palmitoyl sphingomyelin or (f) stearoyl sphingomyelin. (c), (d), (g)

and (h) cross-sections of images in (a), (b), (e) and (f), respectively, at the lines indicated. The arrows indicate a height difference of �/0.7

nm between the phases in all the lipid bilayers. All images are 5 mm scans with 10 nm height scale. Bar�/1 mm.

236 A. E. Garner et al.

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lipid mixture (Figure 4a). However, when brain

sphingomyelin was used, the majority (92.8%) of

the MDP was localized in the lo raft domains (Figure

4b).

To verify that the association of the GPI-anchored

proteins with brain sphingomyelin rafts was attribu-

table to the length of the sphingomyelin acyl chains,

bilayers were formed from lipid mixtures containing

either synthetic palmitoyl or stearoyl sphingomyelin

(Avanti Polar Lipids) (Figure 2e�2h). Both the lipid

mixtures exhibited phase separation and formed lodomains which were approx. 0.7 nm higher than the

surrounding ld lipids. In the bilayers made from

palmitoyl sphingomyelin 33.9% of the surface area

was in the lo raft domains, while in the stearoyl

sphingomyelin bilayers 36.5% was in the lo domains.

Both GPI-ACE and MDP exhibited a similar

distribution in the synthetic palmitoyl and stearoyl

Figure 3. Distribution of TM-ACE and GPI-ACE in supported lipid bilayers. Supported lipid bilayers of equimolar sphingomyelin, DOPC

and cholesterol containing either TM-ACE or GPI-ACE were imaged in fluid using tapping mode AFM. (a) TM-ACE in bilayers

containing egg sphingomyelin; the protein is almost exclusively located in ld non-raft regions. (b) TM-ACE in bilayers containing brain

sphingomyelin; TM-ACE is excluded from the lo raft domains. (c) GPI-ACE in bilayers containing egg sphingomyelin; the protein is

confined to ld non-raft regions. (d) GPI-ACE in bilayers containing brain sphingomyelin; 38% of the protein is located in lo rafts. All images

are 10 mm scans with 10 nm height scale. Bar�/1 mm.

Table I. Statistical analysis of the distribution of proteins located in lo rafts.

Protein Sphingomyelin Number of proteins counted Protein in lo rafts% (mean9/SE)

TM-ACE Egg 576 2.49/1.4

Brain 454 3.29/2.1

GPI-ACE Egg 507 1.59/1.6

Brain 520 41.39/5.4*

Palmitoyl 393 0.89/1.2

Stearoyl 416 42.29/3.1*

MDP Egg 334 1.29/1.4

Brain 382 92.89/3.0*

Palmitoyl 288 0.99/1.4

Stearoyl 321 93.99/3.5*

Random AFM images (n�/9 for each combination of protein and sphingomyelin) from up to 3 repeated experiments were analysed for the

number of protein molecules of TM-ACE, GPI-ACE or MDP located in lo raft regions of supported lipid bilayers. Either egg, brain,

palmitoyl or stearoyl sphingomyelin was used to form bilayers of equimolar sphingomyelin, DOPC and cholesterol; *Z value of�/3.291

indicating that at the 0.5% level there is a significant difference in the mean protein incorporation into lo.

Distribution of GPI-anchored proteins in bilayers 237

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sphingomyelin rafts as they did in egg and brain

sphingomyelin rafts (Figure 5); 42.2% of GPI-ACE

and 93.9% of MDP were localized to lo raft domains

when stearoyl sphingomyelin was used in the lipid

bilayer mixture (Table I). Conversely, only 0.8% of

GPI-ACE and 0.9% of MDP associated with the

palmitoyl sphingomyelin rafts (Table I).

Discussion

The distribution of membrane proteins in a lipid

bilayer can be influenced by a variety of factors,

including the length of the transmembrane domain,

oligomerization with other proteins, and the nature

of its acylation [36]. By incorporating proteins into

supported lipid bilayers, we have for the first time

Figure 4. Distribution of MDP in supported lipid bilayers. Supported lipid bilayers of equimolar sphingomyelin, DOPC and cholesterol

containing MDP were imaged in fluid using tapping mode AFM. (a) MDP in bilayers containing egg sphingomyelin; the protein is almost

exclusively located in ld non-raft regions. (b) MDP in bilayers containing brain sphingomyelin; the protein is almost exclusively located in loraft domains. All images are 2.5mm scans with 5nm height scale. Bar�/1 mm.

Figure 5. Distribution of GPI-ACE and MDP in supported lipid bilayers containing either palmitoyl or stearoyl sphingomyelin. Supported

lipid bilayers composed of equimolar sphingomyelin, DOPC and cholesterol were imaged in fluid using tapping mode AFM. (a) GPI-ACE

in bilayers containing palmitoyl sphingomyelin; the protein is confined to ld non-raft regions. (b) GPI-ACE in bilayers containing stearoyl

sphingomyelin; 46.3% of the protein is located in lo rafts. (c) MDP in bilayers containing palmitoyl sphingomyelin; the protein is almost

exclusively located in ld non-raft regions. (d) MDP in bilayers containing stearoyl sphingomyelin; the protein is predominantly (91.2%)

located in lo raft domains. Images (a) and (b) are 10 mm scans with 10 nm height scale. (c) and (d) are 2.5 mm scans with 5 nm height scale.

Bar�/1 mm.

238 A. E. Garner et al.

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directly visualized by AFM the effect of (i) a GPI

anchor versus a transmembrane anchor on the

partitioning of a membrane protein in rafts, and

(ii) sphingomyelin chain length on the distribution of

GPI-anchored proteins in rafts. TM-ACE, a trans-

membrane protein which is not located in lipid rafts

in the plasma membrane of cells [37,38], was

essentially excluded from the lo raft domains in the

supported lipid bilayer. However, when the trans-

membrane and cytosolic domains in TM-ACE were

exchanged for a GPI anchor and the resulting GPI-

ACE was incorporated into a bilayer, 41% of the

protein entered the lo rafts when the bilayer con-

tained brain sphingomyelin. This result directly

demonstrates that it is the GPI anchor and not the

ectodomain that is responsible for targeting the

protein to lo rafts in this system. Although one might

have expected the orientation of reconstituted pro-

teins to be randomized, with half the molecules

facing inwards, experimental data show that the

asymmetric orientation of ectoenzymes like ACE

and MDP is maintained upon reconstitution into

artificial lipid bilayers [39,40].

AFM revealed that placental alkaline phosphatase

was located almost exclusively (92%) in lo rafts in

supported lipid bilayers containing brain sphingo-

myelin [29]. Whether our observation that only 41%

of GPI-ACE was associated with lo rafts suggests

that another factor could be limiting the inclusion of

this protein in the raft domains is not clear. Inter-

estingly though, analysis of the transfected CHO

cells that express GPI-ACE showed that upon

treatment with Triton X-100 and separation on a

sucrose gradient, only 52.8% of the GPI-ACE was

located in DRMs [18], suggesting that its partial

localization to rafts in both cells and model mem-

branes may be an intrinsic feature of this protein.

Accurate measurement of protein dimensions by

AFM is extremely challenging. The triangular shape

of the AFM tip can impede accurate width measure-

ments due to the side of the tip making contact with

the particle before the apex � referred to as ‘tip

convolution’. As a result width measurements can be

greatly amplified and, for irregular shaped particles

such as proteins, these effects may be further

exaggerated, especially when the particles are not

directly fixed to the mica surface as in a lipid bilayer.

Height measurements, although more accurate than

width, are also susceptible to inaccuracies due to

lack of control of electrostatic forces between the tip

and the protein [41] as well as the physical forces of

tapping. Therefore, in order to obtain accurate

measurements of protein dimensions by AFM,

conditions have to be specifically tailored to the

individual protein. This was not possible within this

study which required consistent conditions for direct

comparison between different proteins. So it is not

possible for us to obtain accurate measurements for

the dimensions of the proteins in our AFM images

and therefore it is not possible for us to determine if

the protein particles within our images correspond to

individual proteins.

However, we have compared the relative dimen-

sions of the protein particles within the same images,

between replica samples and in the various lipid

mixtures and have concluded: (i) For all 3 proteins

used, in all the replica images, the largest particle

does not exceed 4�/ the smallest particle. Therefore

the largest possible aggregate present contains 4 of

the smallest particles. For all images, this 4�/

aggregate represents less than 8% of the total

number of particles in a single image; (ii) for all 3

proteins used, in all the replica images, the smallest

particle represents at least 60% of the total number

of particles in a single image; (iii) for images where

the protein is distributed between the lo and ld phase,

there is no significant difference between the particle

sizes in each phase. Therefore, inclusion or exclusion

into the lo phase does not appear to be dependent on

particle size or aggregation; and (iv) for all 3 proteins

used, comparing the average particle size in each of

the lipid mixtures shows no significant difference in

particle size when different sphingomyelin species

are used in the bilayer. Therefore, the lipid mixture

does not appear to affect protein size or aggregation.

In conclusion, although we cannot rule out some

degree of protein aggregation, we consider the

observations and conclusions made in our study

are independent of aggregation. It should also be

noted that other AFM studies show a similar

distribution of particle sizes when the protein is

reconstituted in bilayers [29,42].

Although the nature of the lipids which form the

bilayer has been shown to affect the distribution of

some transmembrane proteins [42�44], this has not

been explored before for GPI-anchored proteins.

Exchanging brain sphingomyelin for egg sphingo-

myelin caused GPI-ACE to be excluded from the loraft domains. To further investigate the significance

of the sphingomyelin species on the distribution of

another GPI-anchored protein in lo rafts, we utilized

MDP, an endogenous GPI-anchored protein that,

following Triton X-100 extraction, is found exclu-

sively in DRMs [45]. When incorporated into

supported lipid bilayers containing egg sphingomye-

lin, MDP was excluded from lo rafts but the

exchange of egg sphingomyelin for brain sphingo-

myelin resulted in 93% of MDP being located in the

lo raft domains. The predominant difference be-

tween egg and brain sphingomyelin is the acyl chain

length. Egg sphingomyelin is primarily (84%) com-

posed of palmitoyl (C16:0) acyl chains and brain

Distribution of GPI-anchored proteins in bilayers 239

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sphingomyelin mainly (88%) consists of lipids with

chain lengths of C18 or longer. The effect of

sphingomyelin acyl chain length on the raft associa-

tion of GPI-anchored proteins was confirmed using

synthetic palmitoyl and stearoyl sphingomyelin li-

pids. Both GPI-ACE (42%) and MDP (94%)

localized to the stearoyl sphingomyelin raft domains

but were excluded from lo rafts when palmitoyl

sphingomyelin was used.

Despite brain sphingomyelin containing approx.

20% unsaturated acyl chains the surface area cov-

ered by the lo phase was very similar to that seen

with stearoyl sphingomyelin. We assume that the

unsaturated sphingomyelin molecules (all of which

are monounsaturated) have been incorporated into

the lo phase. Monounsaturated sphingolipids can

form the lo phase as long as there is sufficient

cholesterol. Previous studies have shown that the

phase transition of brain sphingomyelin is eliminated

by the addition of equimolar cholesterol, indicative

of the formation of a single lo phase [46]. In

addition, the unsaturated chains are all C24:1D15

(Avanti) and there is evidence to suggest that double

bonds which occur beyond CD13 are too far down the

acyl chain to interfere with the interaction between

the cholesterol and sphingomyelin [47].

The GPI anchor of MDP consists almost exclu-

sively of distearoyl (C18:0) acyl chains [35]. The

incorporation of MDP into egg or palmitoyl sphin-

gomyelin rafts would require the slightly longer acyl

chains in the GPI anchor of MDP to associate with

the tightly packed, shorter acyl chains of the

sphingomyelin. Such an interaction has been shown

to occur in lipid bilayers where the longer fatty acid

interdigitates into the lower lipid leaflet [48]. The

findings of the present study suggest that such an

interaction is unfavourable in the ordered lo do-

mains, at least in supported lipid bilayers, and that

the saturated acyl chains of the GPI anchor associate

with the unsaturated ld lipids in preference to

interdigitation. In contrast, brain and stearoyl sphin-

gomyelin provide slightly longer acyl chains, of

equivalent length to the acyl chains in the GPI

anchor of MDP, and the MDP GPI anchor is able to

insert into the outer leaflet of the lo raft regions

formed from the sphingomyelin without causing

interdigitation. In model bilayers prepared from

mixtures containing sphingomyelin, unsaturated

phosphatidylcholine and cholesterol, the outer and

inner leaflets in lo domains are coupled [5]. How-

ever, in cell membranes, the inner leaflet has a

different lipid composition to that of the outer leaflet

[49]. Whether the transmembrane asymmetry found

in biological membranes would allow the interdigita-

tion of the long acyl chains in a GPI anchor with the

inner bilayer remains to be seen.

The mismatch of the length of the hydrophobic

portion of the polypeptide chain in transmembrane

proteins with that of the surrounding lipids has been

reported as a method of sorting such proteins into

lipid rafts [50�53]. The results of the present study

suggest that hydrophobic mismatch also influences

the distribution of GPI-anchored proteins in rafts.

The studies which reported the association of

placental alkaline phosphatase, which, like MDP,

consists of a distearoyl GPI anchor [54], with lipid

rafts also used brain sphingomyelin or a synthetic

stearoyl sphingolipid [29,30]. Another study found

that the variant surface glycoprotein from Trypano-

soma brucei which contains C14 acyl and alkyl chains

did not readily reconstitute into model membranes

containing brain sphingomyelin [17]. Together these

studies give further support to our hypothesis that

GPI-anchored proteins are targeted to rafts when the

sphingomyelin species has an equivalent acyl chain

length as the GPI anchor. The analysis of the acyl

chain lengths of the sphingomyelin species in DRMs

extracted from mast cells revealed a similar compo-

sition of both C16:0 and C18:0 chains [55], while

DRMs from rat brain membranes contained pre-

dominantly C18:0 sphingomyelin [56]. What is not

known from these studies is whether some individual

rafts in the membrane consist predominantly of

C16:0 sphingomyelin, while others consist primarily

of C18:0 sphingomyelin, but our data would suggest

that differences in sphingomyelin chain length com-

position may determine the segregation of particular

GPI-anchored proteins into particular rafts.

Acknowledgements

A. E. Garner is in receipt of a studentship from the

Biotechnology and Biological Sciences Research

Council (BBSRC) of Great Britain. The financial

support of the BBSRC and the Medical Research

Council of Great Britain is gratefully acknowledged.

We thank Dr S. Connell for assistance with the

AFM, M. Nimick for assistance with the purification

of ACE and MDP, Dr N. T. Watt for assistance with

the statistical analysis and Dr R. A. Skidgel (Uni-

versity of Illinois at Chicago, USA) for the cDNA

encoding GPI-ACE.

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