JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maekawa, S.
Right arrow Articles by Sokawa, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maekawa, S.
Right arrow Articles by Sokawa, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 274, Issue 30, 21369-21374, July 23, 1999


Cholesterol-dependent Localization of NAP-22 on a Neuronal Membrane Microdomain (Raft)*

Shohei MaekawaDagger §, Chihiro Satoparallel , Ken Kitajimaparallel , Nobuo FunatsuDagger , Haruko KumanogohDagger , and Yoshihiro SokawaDagger

From the Dagger  Department of Biotechnology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, 606-8585, § Division of Bioscience, Graduate School of Science and Technology, Kobe-University, Kobe 657-8501, and parallel  Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A membrane microdomain called raft has been under extensive study since the assembly of various signal-transducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl-beta -cyclodextrin was found to be effective to solubilize NAP-22, a neuron-enriched Ca2+-dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dose-dependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The largest pool and concentration of cholesterol in the body exists in the brain. Being a constituent of cell membrane, including myelin, cholesterol is important for the function of this organ, and an inborn defect in cholesterol metabolism is associated with serious neurological and mental dysfunctions (1). Alterations in cholesterol metabolism occur with age and have been implicated in the pathogenesis of Alzheimer's disease (2-7). Brain cholesterol is efficiently protected from exchange with circulating lipoproteins by the blood-brain barrier. In accordance with this, most recent studies have favored the view that the majority of brain cholesterol is synthesized locally, although at a low rate (8, 9). The half-life of cholesterol in the brain has been estimated to be 4-6 months in rats using in vivo and in vitro techniques (10, 11).

Lateral heterogeneities in the classical fluid-mosaic model of cell membranes are now envisaged as caveolae-like microdomains or "rafts" (also referred to as "Triton-insoluble low density fraction," "detergent-insoluble, glycolipid-enriched complexes," or "detergent-resistant membrane domains," and so on), that are enriched in cholesterol, glycosphingolipids, specific membrane proteins, and glycosylphosphatidylinositol (GPI)1-anchored proteins (12-16). Since many signal transducing molecules (e.g. trimeric G proteins, protein-tyrosine kinases, cytoskeletal proteins, calmodulin-binding proteins) are proven to be enriched in the nonionic detergent-insoluble fraction of low density and whose fraction is considered to correspond to the caveolae or to the raft region, this region is recognized to be important not only for the transport of membrane components but also for the compartmentation of these signal-transducing molecules (12-22). Elucidating the molecular interaction in this region, hence, will facilitate understanding the molecular mechanisms of signal transduction in cells and the intracellular sorting mechanisms of this region. In case of the brain, the expression of caveolin, a caveolae marker protein, is relatively low. The brain-derived Triton-insoluble fraction of low density is therefore considered to correspond to the raft region (17, 19, 23). Since the brain is a very complicated system organized for the effective signal transduction as well as a rich source of cholesterol, characterization of this raft region in neuronal cells will be efficient in elucidating the molecular mechanisms of signal transduction as well as the role of cholesterol in this process. In addition, it is suggested that rafts participate in the formation of cell polarity in neuronal cells (12). Furthermore, recent studies have revealed that changes in the membrane cholesterol contents have effects not only on the processing of the amyloid precursor protein and amyloid beta -peptide but also on the Ca2+ signaling (24-28). Elucidating the assembly mechanism of the rafts is, therefore, of primary importance.

In previous studies, we have shown the characterization of NAP-22, a novel calmodulin-binding membrane protein expressed predominantly in the brain (29-31). NAP-22 (also known as CAP-23 and BAPS1) has physicochemical characteristics that are very similar to those of GAP-43 (neuromodulin, F1, B50, p57, and p46) and myristoylated alanine rich C-kinase substrate (MARCKS, p80, and p87) in heat stability, solubilization in 2.5% perchloric acid solution, low hydrophobicity, and anomalous behavior on SDS-PAGE. Namely, the apparent molecular mass of NAP-22 obtained by SDS-PAGE changes depending on the concentration of acrylamide used (58 kDa in 7.5% gel and 45 kDa in 12.5% gel), and this value is much larger than the molecular mass calculated from its amino acid sequence (22 kDa). The myristoylation of NAP-22 was independently confirmed by using a vaculovirus expression system and by the analysis using electrospray mass spectrometry. Also, the membrane localization of NAP-22 was partly ascribed to the myristoylation in its N terminus (30, 32, 33). Further biochemical studies showed its strong Ca2+-dependent association with calmodulin (31). Tissue fractionation studies showed that NAP-22 accumulates in a Triton-insoluble fraction during brain development and is recovered, after applying a sucrose density centrifugation, in a low density region, called "raft" (18). SDS-PAGE analysis of this fraction showed that NAP-22 is one of the major proteins of brain raft, and further studies showed the localization of other proteins such as GAP-43 (neuromodulin), several GPI-anchored proteins, Src family tyrosine kinases, and trimeric G proteins (18). Since the enrichment of the latter three protein groups in rafts prepared from various cells is well recognized, neuronal raft is characterized with the enrichment of two calmodulin-binding proteins, NAP-22 and GAP-43 (12-19).

To elucidate the assembly mechanism of the raft region, we first tried the biochemical fractionation of the raft. Extraction of whole lipids with chloroform and methanol resulted in the solubilization of GAP-43 and NAP-22, suggesting the localization of these proteins through lipid anchors. Butanol extraction was also effective in eluting out these proteins. Since the detergent insolubility of this fraction is attributed to the enrichment of cholesterol and sphingomyelin (12-17), extraction of cholesterol with methyl-beta -cyclodextrin was attempted. This treatment resulted in the selective solubilization of NAP-22. Further in vitro studies showed that NAP-22 binds liposomes in a cholesterol-dependent manner and that this binding is inhibited by calmodulin.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of Brain Raft-- All steps were carried out at 4 °C. Whole brain was isolated from 2-week-old rats and cooled in ice-cold phosphate-buffered saline and frozen at -80 °C until use. For the preparation of raft, frozen whole brain was thawed, minced with scissors, and homogenized in TME solution (10 mM Tris-HCl, 1 mM MgCl2, 1 mM EGTA, pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, and 2% Triton X-100. An aliquot of 2.4 M sucrose solution was added to this homogenate, and the final sucrose concentration was adjusted to 0.8 M. The sample was then placed on the bottom of the centrifuge tube and overlaid with 0.7 M sucrose in TME solution and with TME solution successively. After centrifugation at 70,000 × g for 6 h (Hitachi SW27-2), a clear white band concentrated on the interface of TME solution and 0.7 M sucrose solution was collected and diluted with TME solution containing 1% Triton X-100. After centrifugation at 100,000 × g for 60 min, the pellet fraction was suspended in TME solution and was frozen as brain raft fraction.

Butanol Extraction and NAP-22 Preparation from Brain Raft Fraction-- Brain raft fraction (obtained from 60-g brain) was mixed with 1 volume of butanol, shaken vigorously, and incubated for 20 min at 25 °C. After centrifugation for 20 min at 20,000 × g and 25 °C to separate the butanol phase (upper) and the water phase (lower), the water phase was recovered and dialyzed against TE solution (10 mM Tris-HCl, 0.2 mM EGTA, pH 7.5). After mixing with Triton X-100 (final concentration 1%), the sample was applied to a DEAE-Sepharose column (1.6 × 10 cm), that was equilibrated with TE solution. The column was washed with TE solution containing 0.1% Triton X-100, and bound proteins were eluted with a linear gradient of NaCl (0-500 mM) in TE solution. After SDS-PAGE, fractions containing NAP-22 was recovered and fractionated further under 40% saturation of ammonium sulfate. Precipitated proteins were removed with centrifugation at 20,000 × g for 30 min. The resulting supernatant was applied to a phenyl-Sepharose column (1.4 × 6.5 cm). Proteins were eluted with 2 M NaCl solution in TME, followed with 1.5 M NaCl solution in TME. Fractions containing NAP-22 were collected and dialyzed against 10 mM Tris-HCl, pH 7.5. The sample was then applied to a hydroxyapatite column (1.4 × 6.5 cm), and proteins were eluted with a linear gradient of potassium phosphate buffer (20-350 mM, pH 7.0). Eluted NAP-22 was dialyzed against TE solution and stored at -80 °C until use.

Lipid Isolation and Analysis-- For lipid analysis, brain raft fraction was further purified by NaCl wash and recentrifugation. Namely, the raft fraction was suspended in TME solution containing 1 M NaCl and centrifuged at 100,000 × g for 60 min. The pellet was suspended in TME solution containing 1% Triton X-100 and 0.8 M sucrose, and the discontinuous sucrose gradient centrifugation was repeated. The membrane fraction that floated through the 0.7 M sucrose solution was collected, diluted, and centrifuged. After washing twice with TME solution to remove remaining Triton X-100, the sample was used for the lipid analysis. The isolation procedures for total lipids and for neutral and acidic lipids were described previously (13, 34). Briefly, total lipids were extracted with chloroform/methanol (2:1 and 1:1, v/v) and chloroform/methanol/water (30:60:8, v/v/v). The combined supernatants were applied to a DEAE-Sephadex A-25 column (CH3COO- form, 1-ml bed volume, equilibrated with 1 column volume of chloroform/methanol/water (30:60:8, v/v/v)). The neutral lipid fraction was eluted with 1 column volume of chloroform/methanol/water (30:60:8) and 2 column volumes of methanol. Acidic lipid fraction was eluted with 2 column volumes of 0.3 M ammonium acetate in methanol. The acidic fraction was desalted by a Sephadex G-50 column (0.5 × 51 cm, water). Neutral and acidic lipid fractions were analyzed by HPTLC as described previously (35). In short, HPTLC was prewashed with chloroform/methanol/water (60:35:8, v/v/v). Samples were loaded on the silica gel HPTLC plate, developed in solvent A (chloroform/methanol/CH3COOH/HCOOH/water, 35:15:6:2:1, v/v/v/v/v) until the solvent front ascended to about 4.5 cm from the bottom for neutral lipid analysis or about 6 cm for acidic lipid analysis. Following this development, excess solvent on the plates was evaporated in a fume hood for 15 min and then in a vacuum desiccator for 15 min. The plates were then developed in solvent B (hexane/diisopropylether/acetic acid, 65:35:2, v/v/v) until the solvent front ascended to the top. Excess solvent was evaporated as described above. The plates were then dipped into a 3% cupric acetate (w/v), 8% phosphoric acid (v/v) solution and heated at 180 °C for 15 min. Each of the bands was quantitated by an ATTO Densitograph lane and spot analyzer (ATTO, Japan, version 5.0) as referenced to authentic lipid samples. For glycolipid analysis, acidic lipid fractions and authentic gangliosides were loaded on HPTLC, developed in solvent C (chloroform/methanol/0.25% CaCl2, 55:45:10, v/v/v), and visualized with the resorcinol reagent (36). Each band was purified by a preparative TLC and was analyzed by GLC (Shimadzu GC-14A, Japan) as described previously (37).

Cholesterol Extraction with Methyl-beta -cyclodextrin (MCD)-- MCD was employed to remove cholesterol from membrane fractions (38-40). After dilution with a solution containing 20 mM Tris-HCl, 5 mM EDTA, pH 7.5, brain raft was collected through centrifugation and suspended in a solution of 20 mM Tris-HCl, 5 mM MgCl2, pH 7.5, which contained various amounts of MCD. After incubation at 37 °C or on ice, fractions were centrifuged at 100,000 × g for 30 min. The supernatants and pellets were separated and processed for SDS-PAGE and a cholesterol assay. In this study, cholesterol was assayed spectrophotometrically using a diagnostic kit (Cholesterol C-Test Wako catalog no. 274-46401) (40).

Liposome Binding-- Based on the lipid contents in the brain raft fraction described below, phosphatidylcholine (PC)-based liposomes were used. Lipids solubilized in chloroform/methanol were dried under a stream of nitrogen gas. Liposomes were obtained through sonication in ME solution (10 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2,, 0.2 mM EGTA, 1 mM dithiothreitol, pH 7.5) in the presence of various amounts of the proteins. In some cases, sonication was done in MC solution (ME solution containing 0.4 mM CaCl2). After incubation at 37 °C, the samples were centrifuged at 100,000 × g for 20 min. The supernatants and pellets were separated and analyzed with SDS-PAGE. In some experiments, the pellet fraction was suspended in ME solution, loaded on a discontinuous Ficoll gradient (2, 6, and 10% Ficoll in ME solution, top to bottom), and recentrifuged at 170,000 × g for 5 h at 35 °C. After centrifugation, concentrated materials at the interfaces were collected for analysis with SDS-PAGE and Western blotting.

Others-- DEAE- and phenyl-Sepharose were obtained from Amersham Pharmacia Biotech. Hydroxyapatite (ceramic hydroxyapatite type I) was purchased from Bio-Rad. For liposome construction, dioleoylphosphatidylcholine, phosphatidylserine (bovine brain), and phosphatidylinositol (porcine liver) obtained from Sigma; phosphatidylethanolamine (PE, chicken egg) obtained from Doosan Serdary Research Laboratory; and sphingolipid (egg) obtained from Avanti Polar Lipids Inc. were used. For lipid analysis, triacylglycerol, cholesterol (egg), cholesterol assay kit (Cholesterol Test C, Wako), glucoceramide, and galactosylceramide were purchased from Wako (Japan). Gangliosides were gifts from Dr. Masao Iwamori (Kinki University, Japan). Silica gel HPTLC plates were purchased from Baker. Protein determination, calmodulin preparation, and SDS-PAGE were performed as described previously (29). Densitometric scan of the Coomassie Brilliant Blue-stained gels was done to measure the amount of NAP-22 using a scanner and the NIH Image program. Known amounts of NAP-22 were elelctrophoresed in the same gel for the standard to quantify the protein.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of NAP-22 Using Butanol Extraction and the Lipid Content of Brain Raft-- Since many of the lipid-anchored proteins are known to be hydrophilic proteins, removal of lipids from raft is anticipated to solubilize these proteins if these proteins are anchored in raft simply through the lipid insertion into the lipid bilayer. Extraction of lipids with various alcohols, such as methanol, ethanol, butanol, and pentanol, was tried, and butanol extraction was found to be effective to solubilize several proteins including NAP-22. Butanol extraction separates the components of raft into three parts: the butanol phase, the water phase, and the precipitate. Protein components in these fractions analyzed with SDS-PAGE are shown in Fig. 1. Over 90% of the proteins were recovered in the precipitate fraction. NAP-22, GAP-43, and Thy-1 were identified as major proteins recovered in the water phase from their mobilities in SDS-PAGE and reactivities with specific antibodies. Since over 80% of NAP-22 in the raft was solubilized with butanol, this extraction method was used to purify the protein. NAP-22 was separated from other proteins through several column procedures and used in this experiment. Its purity is shown in Fig. 1 (lane 4) and also confirmed with Western blotting using a monoclonal antibody (data not shown). Table I shows the lipid composition of raft and the partition of lipids after butanol treatment. The lipid content of brain raft is characterized by the enrichment of PC and cholesterol. A lesser amount of SM existed in this fraction compared with other cell-derived rafts (13, 41). After butanol extraction, about 90% of lipids were recovered in the butanol phase. Some amount of gangliosides and a small amount of other lipids were recovered in the water phase, and no lipid was detected in the precipitate fraction. Solubilization of NAP-22 and other proteins in the water phase suggested that these proteins localize to raft through lipid binding rather than through protein binding.


View larger version (53K):
[in this window]
[in a new window]
  		Fig. 1.   Solubilization of NAP-22 and GAP-43 after butanol extraction. Brain raft fraction (lane 1) was extracted with butanol, and proteins recovered in the water phase (lane 2) and in the precipitate (lane 3) were analyzed with SDS-PAGE using a 12% acrylamide gel. Sample volumes were adjusted, and the same volume of each samples was analyzed to represent protein recovery after this fractionation. In the butanol phase, no protein was recovered. NAP-22 was further purified using several column procedures and was used for liposome binding (lane 4, 0.3 µg of protein). The bands of NAP-22, GAP-43, and Thy-1 are shown.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Lipid composition of raft and butanol-treated fractions

Solubilization of NAP-22 with MCD Extraction-- As described in a previous report, NAP-22 is one of the main components in the brain raft (18). Since low buoyant density of rafts from several cells and tissues is ascribed to the enrichment of SM and cholesterol, cholesterol extraction from raft was attempted using beta -cyclodextrin. beta -Cyclodextrin and its methylated form (MCD) are recognized to be effective at removing cholesterol from cells and isolated membranes (38-40). The addition of MCD to the raft fraction resulted in a dose-dependent, specific solubilization of a protein (Fig. 2A). The mobility of this protein coincided with that of NAP-22 under different acrylamide concentrations and exhibited the same retardation behavior in gels of decreasing acrylamide concentration. Also, a monoclonal antibody against NAP-22 reacted with this protein (Fig. 2B). For these reasons, this protein was identified as NAP-22. Interestingly, the extent of NAP-22 solubilization was correlated fairly well with the extraction of cholesterol (Fig. 2C). Consequently, it is calculated that extraction of about 460 cholesterol molecules resulted in the solubilization of one NAP-22 molecule. Analysis of the lipid contents of the MCD-solubilized fraction showed that except for some solubilization of PE, little or no solubilization of other lipids by MCD occurred, consistent with the results reported by others (data not shown; Refs. 38-40). This result strongly suggests that NAP-22 localizes in raft through the interaction with cholesterol.


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 2.   Specific solubilization of NAP-22 with MCD. A, protein solubilization with MCD treatment. After incubation of brain raft with MCD (0-50 mM) for 60 min on ice, the samples were centrifuged at 100,000 × g for 30 min. Supernatants (sup) and pellets (ppt) were recovered. Pellets were suspended in the original volume to represent the solubilization effect after analysis of the same volume with SDS-PAGE. MCD concentrations used were 0 mM (lane 1), 5 mM (lane 2), 10 mM (lane 3), 15 mM (lane 4), 30 mM (lane 5), and 50 mM (lane 6). In lanes 7 and 8, known amounts of purified NAP-22 (0.15 and 0.3 µg) were applied to measure the solubilized amounts of NAP-22 by the densitometric scan. B, identification of the major solubilized protein as NAP-22. a, SDS-PAGE analysis of the solubilized protein. Using 8 and 12% acrylamide gels, 0 and 15 mM MCD extract (lanes 1 and 2), purified NAP-22 (lane 3), and marker proteins (lane M; 1, phosphorylase (97 kDa); 2, BSA (68 kDa); 3, ovalbumin (43 kDa)) were electrophoresed. Note the mobility shift of NAP-22 under different concentrations of acrylamide. Only appropriate regions of the Coomassie Brilliant Blue-stained gels are shown. b, Western blotting of the extracts (lane 1, 0 mM extract; lane 2, 15 mM extract) and purified NAP-22 (lane 3) were electrophoresed and immunoblotted using an anti-NAP-22 monoclonal antibody (29). C, solubilization of NAP-22 and cholesterol with MCD. The amounts of NAP-22 (open circle ) and cholesterol () in the supernatants were plotted for used MCD concentrations.

Liposome Binding of NAP-22-- To confirm the molecular interaction between NAP-22 and cholesterol, liposome binding of NAP-22 was studied with the centrifugation assay. Based on the result of the lipid analysis of brain raft, PC-based liposomes were constructed in the presence of NAP-22 and were pelleted by centrifugation. Binding of NAP-22 to the cholesterol-containing liposomes was observed, and this binding was cholesterol-specific, since basically little or no binding of NAP-22 to other liposomes containing PE, SM, or phosphatidylserine was observed (Fig. 3A). Since aggregated proteins also come to the pellet fraction by the sedimentation assay, density-dependent separation of the liposomes was attempted. Pelleted material was suspended and loaded on the top of a discontinuous Ficoll gradient to be centrifuged. A clear white band was observed on the interface between 0 and 2% Ficoll solution. NAP-22 was also concentrated in this region (Fig. 3B). This result clearly shows the liposome association of NAP-22. Further studies revealed that the binding of NAP-22 to the liposomes is dependent on the cholesterol dose (Fig. 3C). Increasing amounts of NAP-22 to the constant amount of the cholesterol-containing liposomes proved a dose-dependent binding of the protein, and a Scatchard analysis showed a binding ratio of approximately 760 cholesterol molecules/1 molecule of NAP-22 (Fig. 3D).


View larger version (41K):
[in this window]
[in a new window]
  		Fig. 3.   Liposome binding of NAP-22. A, binding of NAP-22 to various liposomes. After mixing of NAP-22 (0.91 µM) with liposomes constructed from various lipids, these samples were centrifuged at 100,000 × g for 20 min at 35 °C. The supernatants (sup) and pellets (ppt) were separated and analyzed with SDS-PAGE. Lanes 1-5 contained PC (0.4 mg/ml) and additional lipids (0.4 mg/ml each). Lane 1, PC; lane 2, PE; lane 3, cholesterol; lane 4, SM; lane 5, phosphatidylserine. B, liposome sedimentation through a Ficoll gradient. A liposome fraction of PC, cholesterol, and NAP-22 was constructed and centrifuged as described for A. The pellet fraction was suspended in ME solution (lane 1) and loaded on a Ficoll gradient. After centrifugation, the top fraction (lane 2) and interface fractions (0-2% (lane 3), 2-6% (lane 4), and 6-10% (lane 5)) and the pellet fraction (lane 6) were recovered to be analyzed by Western blotting using an anti-NAP-22 antibody. C, cholesterol-dependent liposome binding of NAP-22. Liposomes containing constant amounts of PC (dioleoylphosphatidylcholine, 0.4 mg/ml) and PE (0.2 mg/ml) and various amounts of cholesterol (0-0.6 mg/ml) were mixed with 0.45 µM NAP-22. After incubation and centrifugation as described in Fig. 2, the amounts of bound NAP-22 were measured. D, binding of NAP-22 to the cholesterol-containing liposome. A liposome fraction containing dioleoylphosphatidylcholine (0.4 mg/ml), PE (0.2 mg/ml), and cholesterol (0.4 mg/ml) was mixed with various amounts of NAP-22 and incubated. After centrifugation, the supernatants (s) and pellets (p) were analyzed with SDS-PAGE as described for A. NAP-22 used was as follows: 0.13 µM (lane 1), 0.27 µM (lane 2), 0.45 µM (lane 3), 0.67 µM (lane 4), 0.89 µM (lane 5), 1.11 µM (lane 6), and 1.34 µM (lane 7).

Since NAP-22 binds calmodulin in a Ca2+-dependent manner, the effect of calmodulin on the binding was studied. The presence of calmodulin did inhibit the binding in a stoichiometric manner (Fig. 4A, lanes 2-6). The addition of calmodulin after the formation of NAP-22-containing liposomes was also effective (lanes 7 and 8). In these experiments, calmodulin in the micromolar concentration range effectively dissociated NAP-22 from the liposomes (Fig. 4B). This result coincides well with the high affinity binding of NAP-22 to calmodulin reported previously (31). Using brain raft fraction, this solubilization effect of calmodulin on NAP-22 was studied. Calmodulin did solubilize NAP-22, although the effective amount of calmodulin was much larger than in the case of the liposomes (Fig. 5).


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of calmodulin on the NAP-22 binding to the cholesterol-containing liposome. A, liposome binding of NAP-22 analyzed with SDS-PAGE. Liposome binding was done as described in the legend to Fig. 3D, in the presence of NAP-22 (0.9 µM) and various amounts of calmodulin in the MC solution. In lane 1, cholesterol was omitted to test the cholesterol effect on the binding of NAP-22 in MC solution shown in lane 2. Calmodulin used was as follows: 0 µM (lanes 1 and 2), 0.7 µM (lane 3), 1.4 µM (lanes 4 and 7), 2.9 µM (lane 5), and 4.3 µM (lanes 6 and 8). In lanes 7 and 8, calmodulin was added after the liposome construction (3 min before the start of centrifugation). B, a dose-dependent inhibitory effect of calmodulin on the binding of NAP-22 to cholesterol-containing liposomes.


View larger version (41K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of exogenous calmodulin on the association of NAP-22 with brain raft. A, brain raft fraction (1.7 mg of protein/ml) in MC solution was incubated with various amounts of calmodulin (lane 1, 0 µM; lane 2, 8.4 µM; lane 3, 16.8 µM; lane 4, 23.7 µM) for 20 min at 37 °C. After centrifugation for 20 min, the supernatants and the pellets were collected and analyzed with SDS-PAGE. In lane N, purified NAP-22 was applied as a standard. B, the amounts of NAP-22 recovered in the supernatants were plotted versus the concentration of calmodulin added.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Membrane microdomains, called rafts and caveolae, are considered to play important roles not only in the signal transduction through the cell membrane but also in the transport of lipid components within the cell. In the case of caveolae, a 22-kDa phosphoprotein caveolin plays a vital role in the formation of the caveola structure (42-45). Caveolin is multiply palmitoylated and binds cholesterol (46). In the case of brain raft, caveolin was not detected, at least as a major component (17-19, 23). The assembly mechanism of the raft in neurons was, hence, unclear. In this study, we showed the co-solubilization of NAP-22 and cholesterol from brain raft fraction by MCD treatment, along with the binding of NAP-22 to the cholesterol-containing liposomes. Considering that NAP-22 is one of the main components in the brain raft, as described in the previous study, and no other major protein component was eluted out after the elimination of membrane cholesterol using MCD, NAP-22 is expected to play an important role in the formation of raft. The fact that a clear dose-dependent elution of NAP-22 through the elimination of cholesterol is observed in a relatively low concentration range of MCD suggests that some part of NAP-22 localizes in raft mainly through the interaction with cholesterol. Longer incubation or higher concentration of MCD causes further elimination of cholesterol, but the elution of NAP-22 does not correlate well with the elution of cholesterol (Fig. 2). This less extractable pool of NAP-22 may reside in raft, at least in part, through the interaction with other proteins.

Interestingly, the association of NAP-22 with cholesterol containing liposomes was inhibited with calmodulin. This result implies that the raft structure is, at least in part, under Ca2+ regulation. The presence of GAP-43 in raft is especially noteworthy concerning this point, because this protein binds calmodulin in the absence of Ca2+ ions, and increasing the concentration of Ca2+ ions weakens the binding (47-49). Considering that the binding of calmodulin to NAP-22 is much stronger than its binding to GAP-43 (31), the transfer of calmodulin from GAP-43 to NAP-22 could occur concomitantly with the increasing Ca2+ concentration, which could cause the dissociation of NAP-22 from raft.

According to our results, the extraction of about 460 cholesterol molecules in the brain raft resulted in the dissociation of one molecule of NAP-22. In the synaptosome membrane, over 85% of cholesterol exists in the inner leaflet of the membrane (50). Assuming that this partition ratio of cholesterol is true to the raft membrane and the cholesterol is evenly extracted from both leaflets of membrane by MCD, our results indicate that one molecule of NAP-22 covers 390 molecules of cholesterol in the inner leaflet of raft. In the case of liposome binding experiments, the maximum binding ratio of NAP-22 to cholesterol was 1:760. Since even distribution of cholesterol in both leaflets is anticipated in case of the artificial liposomes, one molecule of NAP-22 was calculated to cover 380 cholesterol molecules in the liposomes. The values obtained using isolated raft and using artificial liposomes are, thus, correlated fairly well. Since NAP-22 is a very hydrophilic protein having little or no hydrophobic region in the molecule except the N-terminal myristic acid, it is thought that the association of this protein to the cholesterol-containing liposomes takes place through the interaction with the hydroxyl group of cholesterol. Further studies using the artificial protein expression system will help assign the cholesterol interaction region within the protein.

The analysis of the lipid components in brain raft showed the enrichment of PC and cholesterol. The presence of lipid components in a low density fraction of the brain was also reported by Moss and White (51). In their experiment, embryonic chick brain was extracted with 3% Nonidet P-40, and membranes concentrated on the interface of 10 and 30% sucrose solution was collected for analysis. A high PC content was also reported by them. An enrichment of PC is, hence, observed in these brain derived rafts, although the significance of this is not yet cleared. In contrast to the fairly equal amount of PC, the cholesterol content of our fraction was much higher than their results. The difference has not been cleared at present, because the detergents used, the origin and the age of brains, and the extraction procedures are different. Our study revealed that the main lipid components of 2-week-old rat brain raft were PC and cholesterol. The molar ratio of SM in brain raft was rather low compared with other cell or tissue-derived raft (13, 51). This result was noteworthy because brain tissue contains myelin, a rich source of SM. Since the bands of myelin basic proteins, marker proteins of the myelin membrane, were detected in the SDS-PAGE pattern of the raft fraction, some fraction of myelin membrane was determined to have recovered in the brain raft fraction obtained from 2-week-old rat brain (data not shown).

As in the case of other cell-derived raft, neuronal raft also contains many signal transduction molecules such as receptor type and nonreceptor type tyrosine kinases, trimeric G proteins, and GPI-anchored cell adhesion molecules (17-19). Furthermore, localization in raft is reported for the amyloid precursor protein, beta -amyloid protein, several gangliosides, and prion protein (24-28, 52-54). Dissociation of NAP-22 from neuronal raft by calmodulin implies the presence of the exchange mechanism of raft components by the Ca2+ signal. Specific localization of NAP-22 in the synaptic region assists this hypothesis. Since cellular cholesterol levels will influence signal transduction pathways, the regulation of raft construction will be important (55-57). Another possible role of NAP-22 is its participation in the sorting of cholesterol-containing vesicles within the cell. Brain cholesterol is efficiently protected from exchange with circulating lipoproteins by the blood-brain barrier. Recent studies have favored the view that the majority of brain cholesterol is synthesized locally (10, 11). In neuron, cholesterol is synthesized in the cell body, neither in the axon nor in the presynaptic region; therefore, some mechanism is needed to deliver cholesterol through the axon. Raft may participate in the sorting of cholesterol in neuron as suggested in the case of epithelial cells (58-60). In this context, the targeted sorting mechanism in the neuron is interesting. Several studies showed the axonal sorting of some GPI-anchored proteins in cortical neurons, although dendritic sorting of some GPI-anchored proteins is reported in the granule cell of the cerebellum (61-63). According to the recent studies by Jereb and Banker (64) using viral vectors in cultured hippocampal neurons, the sorting information present in apical proteins is not sufficient to target these proteins to axons, although the dendritic sorting depends on the protein sequence. Considering the importance of raft to the sorting of proteins and lipids, further studies on raft will be helpful in understanding the molecular background of cell polarity (65).

    ACKNOWLEDGEMENTS

We are grateful to Dr. Masao Iwamori (Kinki University, Japan) for providing us with gangliosides. We also thank H. Teramura for technical expertise.

    FOOTNOTES

* This study was supported by Grants-in-Aid for Scientific Research on Priority Areas 07279222 and for Scientific Research (B) 11490022 from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 85-75-724-7789; Fax: 85-75-724-7760; E-mail: smaekawa@ipc.kit.ac.jp.

    ABBREVIATIONS

The abbreviations used are: GPI, glycosylphosphatidylinositol; MCD, methyl-beta -cyclodextrin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Tint, G. S., Irons, M., Elias, E. R., Batta, A. K., Frieden, R., and Chen, T. S. (1994) N. Engl. J. Med. 330, 107-113[Abstract/Free Full Text]
2. Roth, G. S., Joseph, J. A., and Mason, R. P. (1995) Trends Neurosci. 5, 203-206
3. Igbavboa, U., Avdulov, N. A., Schroeder, F., and Wood, W. G. (1996) J. Neurochem. 66, 1717-1725[Medline] [Order article via Infotrieve]
4. Lutjohann, D., Breuer, O., Ahlborg, G., Nennesmo, I., Siden, A., Diczfalusy, U., and Bjorkhem, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9799-9804[Abstract/Free Full Text]
5. Zhang, Y., Appelkvist, E. L., Kristensson, K., and Dallner, G. (1996) Neurobiol. Aging 17, 869-875[CrossRef][Medline] [Order article via Infotrieve]
6. Mason, R. P., Shoemaker, W. J., Shajenko, L., Chambers, T. E., and Herbette, L. G. (1992) Neurobiol. Aging 13, 413-419[CrossRef][Medline] [Order article via Infotrieve]
7. Jarvik, G. P., Wijsman, E. M., Kukull, W. A., Schllenberg, G. D., Yu, C., and Larson, E. B. (1995) Neurology 45, 1092-1096[Abstract]
8. Dietschy, J. M., Turley, S. D., and Spady, D. K. (1993) J. Lipid Res. 34, 1637-1659[Medline] [Order article via Infotrieve]
9. Snopes, G. J., and Suter, U. (1997) Subcell. Biochem. 28, 173-204[Medline] [Order article via Infotrieve]
10. Serougne-Gautheron, C., and Chevallier, F. (1973) Biochim. Biophys. Acta 316, 244-250[Medline] [Order article via Infotrieve]
11. Andersson, M., Elmberger, P. G., Edlund, C., Kristensson, K., and Dallner, G. (1990) FEBS Lett. 269, 15-18[CrossRef][Medline] [Order article via Infotrieve]
12. Simons, K., and Ikonen, E. (1997) Nature 387, 596-572
13. Brown, D. A., and Rose, J. K. (1992) Cell 68, 533-544[CrossRef][Medline] [Order article via Infotrieve]
14. Brown, D. A., and London, E. (1998) Annu. Rev. Cell Dev. Biol. 14, 111-136[CrossRef][Medline] [Order article via Infotrieve]
15. Lisanti, M. P., Scherer, P. E., Tang, Z., and Sargiacomo, M. (1994) Trends Cell Biol. 4, 231-235 [CrossRef][Medline] [Order article via Infotrieve]
16. Anderson, R. G. W. (1998) Annu. Rev. Biochem. 67, 199-225[CrossRef][Medline] [Order article via Infotrieve]
17. Olive, S., Dubois, C., Schachner, M., and Rougon, G. (1995) J. Neurochem. 65, 2307-2317[Medline] [Order article via Infotrieve]
18. Maekawa, S., Kumanogoh, H., Funatsu, N., Takei, N., Inoue, K., Endo, Y., Hamada, K., and Sokawa, Y. (1997) Biochim. Biophys. Acta 1323, 1-5[Medline] [Order article via Infotrieve]
19. Wu, C., Butz, S., Ying, Y., and Anderson, R. G. W. (1997) J. Biol. Chem. 272, 3554-3559[Abstract/Free Full Text]
20. Schroeder, R. J., Ahmed, S. N., Zhu, Y., London, E., and Brown, D. A. (1998) J. Biol. Chem. 273, 1150-1157[Abstract/Free Full Text]
21. Hanada, K., Nishijima, M., Akamatsu, Y., and Pagano, R. E. (1995) J. Biol. Chem. 270, 6254-6260[Abstract/Free Full Text]
22. Gorodinsky, A., and Harris, D. A. (1995) J. Cell Biol. 129, 619-627[Abstract/Free Full Text]
23. Cameron, P. L., Refflin, J. W., Bollag, R., Rasmussen, H., and Cameron, R. (1997) J. Neurosci. 17, 9520-9535[Abstract/Free Full Text]
24. Hartmann, H., Eckert, A., and Mueller, W. E. (1994) Biochem. Biophys. Res. Commun. 200, 1185-1192[CrossRef][Medline] [Order article via Infotrieve]
25. Liang, J-S., Fine, R. E., Abraham, C. R., and Sipe, J. D. (1996) Biochem. Biophys. Res. Commun. 219, 962-967[CrossRef][Medline] [Order article via Infotrieve]
26. Bodovitz, S., and Klein, W. L. (1996) J. Biol. Chem. 271, 4436-4440[Abstract/Free Full Text]
27. Howland, D. S., Trusko, S. P., Savage, M. J., Reaume, A. G., Lang, D. M., Hirsch, J. D., Maeda, N., Siman, R., Greenberg, B. D., Scott, R. W., and Flood, D. G. (1998) J. Biol. Chem. 273, 16576-16582[Abstract/Free Full Text]
28. Simons, M., Keller, P., De Strooper, B., Beyreuther, K., Dotti, C. G., and Simons, K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6460-6464[Abstract/Free Full Text]
29. Maekawa, S., Maekawa, M., Hattori, S., and Nakamura, S. (1993) J. Biol. Chem. 268, 13703-13709[Abstract/Free Full Text]
30. Maekawa, S., Matuura, Y., and Nakamura, S. (1994) Biochim. Biophys. Acta 1218, 119-122[Medline] [Order article via Infotrieve]
31. Maekawa, S., Murofushi, H., and Nakamura, S. (1994) J. Biol. Chem. 269, 19462-19465[Abstract/Free Full Text]
32. Caroni, P., Aigner, L., and Schneider, C. (1997) J. Cell Biol. 136, 679-692[Abstract/Free Full Text]
33. Mosevitsky, M. I., Capony, J. P., Skladchikova, G. Y., Novitskaya, V. A., Plekhanov, A. Y., and Zakharov, V. V. (1997) Biochimie 79, 373-384[Medline] [Order article via Infotrieve]
34. Ariga, T., Macala, L. J., Saito, M., Margolis, R. K., Greene, L. A., Margolis, R. U., and Yu, R. K. (1988) Biochemistry 27, 52-58[CrossRef][Medline] [Order article via Infotrieve]
35. Macala, L. J., Yu, R. K., and Ando, S. (1983) J. Lipid Res. 24, 1243-1250[Abstract]
36. Svennerhorn, L. (1957) Biochim. Biophys. Acta 24, 604-611[Medline] [Order article via Infotrieve]
37. Song, Y., Kitajima, K., Inoue, S., and Inoue, Y. (1991) J. Biol. Chem. 266, 21929-21935[Abstract/Free Full Text]
38. Kilsdonk, E. P. C., Yancey, P. G., Stoudt, G. W., Bangerter, F. W., Johnson, W. J., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 17250-17256[Abstract/Free Full Text]
39. Neufeld, E. B., Cooney, A. M., Pitha, J., Dawidowicz, E. A., Dwyer, N. K., Pentchev, P. G., and Blanchette-Mackie, E. J. (1996) J. Biol. Chem. 271, 21604-21613[Abstract/Free Full Text]
40. Gimpl, G., Burger, K., and Fahrenholtz, F. (1997) Biochemistry 36, 10959-10974[CrossRef][Medline] [Order article via Infotrieve]
41. Mescher, M. F., and Apgar, J. R. (1986) Adv. Exp. Med. 184, 387-400
42. Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y-S., Glenney, J. R., and Anderson, R. G. W. (1992) Cell 68, 673-682[CrossRef][Medline] [Order article via Infotrieve]
43. Lipardi, C., Mora, R., Colomer, V., Paladino, S., Nitsch, L., Rodriguez-Boulan, E., and Zurzolo, C. (1998) J. Cell Biol. 140, 617-626[Abstract/Free Full Text]
44. Vogel, U., Sandvig, K., and van Deurs, B. (1998) J. Cell Sci. 111, 825-832[Abstract]
45. Okamoto, T., Schlegel, A., Scherer, P. E., and Lisanti, M. P. (1998) J. Biol. Chem. 273, 5419-5422[Free Full Text]
46. Murata, M., Peranen, J., Schreiner, R., Wieland, F., Kurzchalia, T. V., and Simons, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10339-10343[Abstract/Free Full Text]
47. Liu, Y., and Storm, D. R. (1990) Trends Pharmacol. Sci. 11, 107-111[CrossRef][Medline] [Order article via Infotrieve]
48. Gerendasy, D. D., Herron, S. R., Jennings, P. A., and Sutcliffe, J. G. (1995) J. Biol. Chem. 270, 6741-6750[Abstract/Free Full Text]
49. Gamby, C., Waage, M. C., Allen, R. G., and Baizer, L. (1996) J. Biol. Chem. 271, 26698-26705[Abstract/Free Full Text]
50. Igbavboa, U., Avdulov, N. A., Chochina, S. V., and Wood, W. G. (1997) J. Neurochem. 69, 1661-1667[Medline] [Order article via Infotrieve]
51. Moss, D. J., and White, C. A. (1992) Eur. J. Cell Biol. 57, 59-65[Medline] [Order article via Infotrieve]
52. Bouillot, C., Prochiantz, A., Rougon, G., and Allinquant, B. (1996) J. Biol. Chem. 271, 7640-7644[Abstract/Free Full Text]
53. Lee, S-J., Liyanage, U., Bickel, P., Xia, W., Lansbury, P. T., Jr., and Kosik, K. S. (1998) Nat. Med. 4, 730-734[CrossRef][Medline] [Order article via Infotrieve]
54. Vey, M., Pilkuhn, S., Wille, H., Nixon, R., DeArmond, S. J., Smart, E. J., Anderson, R. G. W., Taraboulos, A., and Prusiner, S. B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14945-14949[Abstract/Free Full Text]
55. Pike, L. J., and Miller, J. M. (1998) J. Biol. Chem. 273, 22298-22304[Abstract/Free Full Text]
56. Furuchi, T., and Anderson, R. G. W. (1998) J. Biol. Chem. 273, 21099-21104[Abstract/Free Full Text]
57. Rao, A. M., Igbavboa, U., Semotuk, M., Schroeder, F., and Wood, W. G. (1993) Biochem. Int. 23, 45-52
58. Posse de Chaves, E., Rusinol, A. E., Vance, D. E., Campenot, R. B., and Vance, J. E. (1997) J. Biol. Chem. 272, 30766-30773[Abstract/Free Full Text]
59. Fielding, C. J., and Fielding, P. E. (1997) J. Lipid Res. 38, 1503-1521[Abstract]
60. Fiedler, K., Kobayashi, T., Kurzchalia, V., and Simons, K. (1993) Biochemistry 32, 6365-6373[CrossRef][Medline] [Order article via Infotrieve]
61. Craig, A. M., and Banker, G. (1994) Annu. Rev. Neurosci. 17, 267-310[CrossRef][Medline] [Order article via Infotrieve]
62. Powell, S. K., Rivas, R., Rodriguez-Boulan, E., and Hatten, M. E. (1997) J. Neurobiol. 32, 223-236[CrossRef][Medline] [Order article via Infotrieve]
63. Ledesma, M. D., Simons, K., and Dotti, C. G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3966-3971[Abstract/Free Full Text]
64. Jereb, M., and Banker, G. (1998) Neuron 20, 855-867[CrossRef][Medline] [Order article via Infotrieve]
65. Weimbs, T., Low, S. H., Chapin, S. J., and Mostov, K. E. (1997) Trends Cell Biol. 7, 393-399 [Medline] [Order article via Infotrieve]

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 EndocrinologyHome page
S. S. Gouraud, K. Heesom, S. T. Yao, J. Qiu, J. F. R. Paton, and D. Murphy
Dehydration-Induced Proteome Changes in the Rat Hypothalamo-Neurohypophyseal System
Endocrinology, July 1, 2007; 148(7): 3041 - 3052.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
U. Igbavboa, J. M. Pidcock, L. N. A. Johnson, T. M. Malo, A. E. Studniski, S. Yu, G. Y. Sun, and W. G. Wood
Cholesterol Distribution in the Golgi Complex of DITNC1 Astrocytes Is Differentially Altered by Fresh and Aged Amyloid beta -Peptide-(1-42)
J. Biol. Chem., May 2, 2003; 278(19): 17150 - 17157.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
S. Grimmer, B. van Deurs, and K. Sandvig
Membrane ruffling and macropinocytosis in A431 cells require cholesterol
J. Cell Sci., July 15, 2002; 115(14): 2953 - 2962.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
T. Laux, K. Fukami, M. Thelen, T. Golub, D. Frey, and P. Caroni
GAP43, MARCKS, and CAP23 Modulate PI(4,5)P2 at Plasmalemmal Rafts, and Regulate Cell Cortex Actin Dynamics through a Common Mechanism
J. Cell Biol., June 26, 2000; 149(7): 1455 - 1472.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Seno, M. Kishimoto, M. Abe, Y. Higuchi, M. Mieda, Y. Owada, W. Yoshiyama, H. Liu, and F. Hayashi
Light- and Guanosine 5'-3-O-(Thio)triphosphate-sensitive Localization of a G Protein and Its Effector on Detergent-resistant Membrane Rafts in Rod Photoreceptor Outer Segments
J. Biol. Chem., June 8, 2001; 276(24): 20813 - 20816.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow C