Myelin proteolipid protein (PLP), but not DM-20, is an inositol hexakisphosphate-binding protein.

Myelin proteolipid protein (PLP) and its alternatively spliced isoform, DM-20, are the major integral membrane proteins of central nervous system myelin. It is known that PLP and DM-20 are delivered to myelin by a finely regulated vesicular transport system in oligodendrocytes. Evolutionarily, it is believed that ancestral DM-20 acquired a PLP-specific exon to create PLP, after which PLP/DM-20 became a major component of central nervous system myelin. We purified PLP as an inositol 1,3,4,5-tetrakisphosphate-binding protein after solubilization in a non-organic solvent. However, under the isotonic condition, PLP binds inositol hexakisphosphate (InsP6) significantly, not inositol 1,3,4,5-tetrakisphosphate. Most of the InsP6-binding proteins are involved in vesicular transport, suggesting the involvement of PLP in vesicular transport. We separated DM-20 from PLP by CM-52 chromatography and showed that DM-20 has no InsP6 binding activity. These findings indicate that the PLP-specific domain confers the InsP6 binding activity and this interaction may be important for directing PLP transport to central nervous system myelin.

Eucaryotic cells are subdivided into membrane-bounded compartments. These functional organelles contain sets of proteins and other molecules specific to themselves. The intracellular vesicular transport system delivers specific proteins to their destination. A knowledge of this mechanism is essential for understanding how these compartments are created and maintained within eucaryotic cells.
The oligodendrocyte provides both an opportunity and a challenge for studying the machinery of intracellular vesicular transport. Oligodendrocytes are glial cells in the central nervous system which synthesize unique functional component "myelin." Myelin is composed of multilamellar stacks of plasma membrane surrounding individual axons and plays a significant role in supporting fast nerve conduction. To create and maintain this myelin, oligodendrocyte must deliver large amounts of proteins and lipids to this component via vesicular transport (1).
Generally, integral membrane proteins are co-translationally inserted into the rough endoplasmic reticulum membrane and then transported to the plasma membrane via the Golgi apparatus (2). Myelin proteolipid protein (PLP) 1 is the major integral membrane protein of central nervous system myelin. PLP mRNA is associated with polysomes on the rough endoplasmic reticulum (3) and an immunoreactive product has been detected in membranous structures, such as the Golgi apparatus, of oligodendrocytes in vivo (4 -6). As expected for a protein being processed through the vesicular transport pathway, a significant lag exists between translation of PLP on the rough endoplasmic reticulum and its insertion into the myelin membrane (3,7). Mutations within the PLP gene causes severe dysmyelination (8), at least in part caused by an impaired protein transport system. In one of the PLP mutants, the jimpy mouse, for example, the mutated PLP protein accumulates in the rough endoplasmic reticulum and very little PLP is found in myelin (6). In oligodendrocytes of the transgenic mouse overexpressing the wild type PLP gene there is a swelling in the Golgi apparatus and PLP is rarely found in myelin (9,10). Therefore, it is very important to study the regulation of the PLP transport system in order to understand the efficient vesicle transport system of oligodendrocyte.
PLP is a highly conserved protein. In mammals, the amino acid sequences of PLP of bovine, rat, mouse, and human are 99% identical, suggesting that PLP has indispensable functions (11). DM-20 is a less abundant proteolipid of mammalian central nervous system myelin, the mRNA of which is produced by alternative splicing of the PLP-mRNA precursor (12)(13)(14). It is important to ascertain the function conferred upon DM-20 by the addition of this PLP-specific domain. InsP 6 is found at concentrations from 10 to 100 M in many kinds of cells (15,16). Although, the function of InsP 6 has not yet been clarified, several recent findings have suggested a physiological role for InsP 6 . Several proteins involved in intracellular vesicular transport have been identified as InsP 6 -binding proteins. A clathrin assembly protein, AP-2 (17)(18)(19)(20), may be an essential protein in the endocytotic recycling pathway of all cells (21). Binding of InsP 6 inhibits the clathrin assembly me-diated by AP-2 (22) and AP-3, a synapse-specific clathrin assembly protein (23,24). Coatomer, a cytosolic protein complex containing subunits of non-clathrin-coated Golgi intercisternal transport vesicles, also binds InsP 6 (25). These findings indicate that InsP 6 is closely related to vesicular transport.
In this study, we have purified PLP using a non-organic solvent and showed that PLP is an InsP 6 -binding protein, while DM-20 is not. Apparently, DM-20 acquired InsP 6 binding activity by gaining a PLP-specific domain and thereafter became the major central nervous system myelin component PLP with InsP 6 binding activity. Thus, this binding property of PLP may play a crucial role in targeting vesicles containing PLP to central nervous system myelin.  (12 Ci/mmol), and PP-InsP 5 were obtained from DuPont NEN. Ins-1,4,5-P 3 , Ins-1,3,4,5-P 4 , and CHAPS were from Dojindo Laboratories. Ins-1,3,4,5,6-P 5 was from Boehringer Mannheim, while InsP 6 was from Sigma. The hybridoma of anti-PLP monoclonal antibodies (AA3 and AH7-2a) were kindly supplied from Dr. Marjorie B. Lees (26). The hybridomas were cultured in HYBRIDOMA-SFM (Life Technologies, Inc.) medium.

Materials-[
General Methods-Protein concentrations were determined using the Bio-Rad protein assay (the Bradford protein assay) with bovine serum albumin as standard. All the purification steps and protein handling were performed at 4°C or on ice. The pH of all buffers was adjusted at room temperature and was not corrected for cooling to 4°C. 6 Binding-InsP 4 and InsP 6 binding were measured by the slightly modified polyethylene glycol precipitation method as described previously (27). Assay mixture contained 2 mg/ml ␥-globulin, 20 mM HEPES-NaOH at pH 7.2 for the [ 3 H]InsP 4 -binding assay and 0.15 M KCl, 0.2 mg/ml ␥-globulin, 50 mM HEPES-KOH at pH 7.2 for the [ 3 H]InsP 6 binding assay.

Measurement of [ 3 H]InsP 4 and [ 3 H]InsP
Purification of IP4BP2b (PLP)-Preparation of the P2/P3 membrane fraction from young adult male ddY mouse cerebella (18 g) and the solubilization with 1% Triton X-100 were carried out according to the method described previously (27). The supernatant (1% Triton X-100 extract) (ϳ200 ml) was applied to a column of DE-52 (Whatman) ( 2.6 ϫ 11 cm) equilibrated with 1% Triton X-100, 10% glycerol, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 M leupeptin, 10 M pepstatin A, 1 mM 2-mercaptoethanol, and 50 mM Tris-HCl, pH 8.0 (Buffer 1). The column was washed with 70 ml of Buffer 1. DE-52 flow-through fractions were stored at Ϫ70°C. Stored DE-52 flowthrough fractions from two experiments were combined (ϳ400 ml) and incubated with 5 ml of packed heparin-agarose (Sigma) for 1 h at 4°C on a rotator. The heparin-agarose was poured into a column ( 1.0 cm), and nonadherent proteins were collected and discarded. The column was washed with 40 ml of Buffer 2 (10 mM CHAPS, 0.1 mM PMSF, 10 M leupeptin, 10 M pepstatin A, 1 mM 2-mercaptoethanol, and 50 mM EPPS-NaOH, pH 8.0) containing 0.05 M NaCl and 40 ml of Buffer 2 containing 0.25 M NaCl, and the InsP 4 binding activities were eluted with a linear gradient of 0.25-1.0 M NaCl, Buffer 2 (50 ml total) at 0.5 ml/min. Peak fractions of the InsP 4 binding activity were pooled and diluted with 15 volumes of the solution containing 0.1 mM PMSF, 10 M leupeptin, 10 M pepstatin A, 1 mM 2-mercaptoethanol, 50 mM HEPES-NaOH, pH 7.5 (Buffer 3), and incubated with 8 ml of packed cationexchange gel CM-52 (Whatman) for 1 h at 4°C on a rotator. They were poured into a column, and nonadherent proteins were discarded. The column was washed with 40 ml of Buffer 3 containing 10 mM CHAPS, 0.05 M NaCl and the binding activities were eluted with a linear gradient of 0.05-0.5 M NaCl, 10 mM CHAPS, Buffer 3 (50 ml total) at 0.5 ml/min. Peak fractions of the InsP 4 binding activity were pooled, concentrated, and applied to a column of Sephacryl S-300 (Pharmacia) ( 1.0 ϫ 57 cm) equilibrated with 10 mM CHAPS, 0.5 M NaCl, Buffer 3. Peak fractions of InsP 4 binding activity were pooled, concentrated, and stored at Ϫ70°C.
SDS-PAGE and Immunoblot Analysis-SDS-PAGE for protein profile analysis or immunoblotting were carried out by the method of Laemmli (28). The proteins were visualized with Coomassie Brilliant Blue R-250 or by silver staining with Silver Stain II kit (Wako).
After electrophoresis, the proteins were transferred to a nitrocellulose membrane (Hybond-C; Amersham) using semidry system (ATTO). The membrane was soaked in 5% skim milk in PBS containing 0.1% Tween 20 (T-PBS) for 1 h at room temperature to block nonspecific binding and then incubated in the culture supernatant of hybridoma producing monoclonal antibody for 60 min at 37°C. After washing with T-PBS, the membrane was incubated with biotinylated goat anti-rat IgG (Fc region specific) (Jackson Immunoresearch Laboratories) (1:200) in 5% skim milk, T-PBS. Immunoreactive bands were visualized using Vectastain ABC kit (Vector Laboratories) according to the manufacturer's protocol. Finally, the membrane was treated with 0.08% diaminobenzidine, 0.009% hydrogen peroxide, 0.04% NiCl 2 /imidazol (5 g/ml), PBS.
NH 2 -terminal Sequence Analysis of IP4BP2b-The purified IP4BP2b (approximately 14.4 g) was separated by SDS-PAGE on 12% gel. After separation, the proteins were transferred to a polyvinylidene difluoride membrane (0.2 m) (Bio-Rad) with CAPS transfer buffer (CAPS-NaOH, pH 11.0, 10% methanol). These proteins were visualized with Coomassie Brilliant Blue R-250 and protein bands were cut from the blots. The membrane pieces were applied to a gas-phase protein sequencer (Applied Biosystem). The sequences were compared to those in the SWISS-PROT data base.
Immunoabsorption of IP4BP2b (PLP) Using Protein G-Sepharose-The AA3-IgG solution which was prepared by general ammonium sulfate-precipitation method, or normal rat IgG solution (Inter-Cell Technologies Inc.) were diluted with PBS to give a final protein concentration of 2 mg/ml. To each 500 l of the above IgG solution (1 mg of protein), an equal volume of binding solution (1 M acetate buffer, pH 4.6, containing 3 M NaCl) was added, and incubated with 200 l of packed Protein G-Sepharose (Sigma) equilibrated with the binding solution overnight at 4°C on a rotator. Each of these solutions was poured into a column ( 5 mm), and washed with 2 ml of the washing solution containing 0.15 M NaCl, 20 mM HEPES-NaOH, pH 7.5. The washed gel particles were transferred into sample tubes and incubated with 20 l of the purified IP4BP2b (PLP) (13.6 g) for 1 h at 4°C on a rotator. After incubation, these were re-poured into columns ( 5 mm) and washed four times with 100 l of washing solution. Nonadherent proteins were collected at approximately 100 l/fraction. A control experiment was also performed by the same method without IgG. Each of these four fractions was used for [ 3

H]InsP 4 -binding assay or SDS-PAGE analysis.
Purification of Myelin-Myelin was purified from the medulla oblongata and the spinal cord of adult male ddY mouse by the procedure of Lucas et al. (29). . Nonadherent proteins were collected and discarded. PLP was eluted with 8 times of 0.2 ml of 1.0 M NaCl/Buffer 6, and peak fractions of PLP detected by immunoblot analysis were pooled and stocked as "PLP-containing fraction" at Ϫ70°C.

Preparation of PLP-containing and DM-20-containing Fractions-
Stocked CM-52 flow-through fraction containing DM-20 (100 ml) was incubated with 1.0 ml of packed heparin-agarose for 1 h at 4°C on a rotator. The heparin-agarose was poured into a column ( 0.5 cm) washed with 5 ml of Buffer 6. Nonadherent proteins were collected and discarded. DM-20 was eluted with 10 times of 0.2 ml of 1.0 M NaCl/ Buffer 6, and peak fractions of DM-20 detected by immunoblot analysis were pooled and stocked as "DM-20-containing fraction" at Ϫ70°C.

RESULTS
During the sequential purification process of the receptor protein for InsP 3 (IP3R) from mouse cerebella (27), we noticed that some fractions contained [ 3 H]InsP 4 binding activity, which indicated the existence of InsP 4 -binding proteins. We have already purified and identified two InsP 4 -binding proteins (IP4BPs). One is IP4BP1/synaptotagmin II (30) and the other is IP4BP2a/aldolase A. 2 We purified and identified another IP4BP (IP4BP2b). The sequential purification procedure is depicted as a flow chart (Fig. 1).
Purification of IP4BP2b-IP4BP1/synaptotagmin II and the other concomitant proteins (IP3R and phosphatases) were separated from IP4BP2 (a and b) by the first anion-exchange chromatography on DE-52. Since the volume of the DE-52 flow-through fraction was large, InsP 4 binding activity was concentrated by heparin-agarose chromatography. At the heparin-agarose chromatography step, the detergent in the sample and purification buffers was changed from Triton X-100 to CHAPS because Triton X-100 inhibited the InsP 4 binding activity of IP4BP2b more than CHAPS and because the concomitant proteins had been effectively separated from IP4BP2b.
IP4BP2a/aldolase A was separated from IP4BP2b by washing the heparin-agarose with 0.25 M NaCl ( Fig. 2A). The IP4BP2b was eluted with a 0.25-1.0 M NaCl linear gradient. The InsP 4 binding activity of this fraction seemed to be expressed by only IP4BP2b. After dilution to lower the NaCl concentration and pH, the InsP 4 binding activity was concentrated and enriched by cation-exchange chromatography on CM-52 (Fig. 2B). The final step was Sephacryl S-300 gel filtration (Fig. 2C). Since the solubilizing detergent had been changed and the InsP 4 binding assay modified (described below), the purification process cannot be summarized in a figure. Approximately 0.5 mg of IP4BP2b was obtained from 40 g of mouse cerebella.
The protein profile of each purification step was characterized by SDS-PAGE (Fig. 3A). Particular attention was given to not boiling the sample mixtures in SDS solution, but rather allowing them stand at room temperature. Since the IP4BP2b appeared to be extremely hydrophobic, IP4BP2b protein aggregated and did not enter the separation gel after boiling (data not shown).
Many contaminating proteins which appeared after heparinagarose chromatography were efficiently eliminated by CM-52 chromatography (Fig. 3A, lanes 2 and 3). After the final step on Sephacryl S-300, a single protein band with a molecular weight of 26,000 was detected (Fig. 3A, lane 4). While the molecular weight of IP4BP2b was 26,000 on SDS-PAGE, the apparent molecular weight was estimated to be 440,000 -669,000 by gel filtration chromatography (Sephacryl S-300) (Fig. 3B). Consequently, IP4BP2b was expected to be a homomultimer or to aggregate. The pattern of InsP 4 binding activity and that of the intensities of Coomassie Brilliant Blue R-250 staining of this 26,000 protein differed slightly (Fig. 2C and Fig. 3B). The results below describe the binding activity resides in this 26,000 protein.
Identification of IP4BP2b as PLP by NH 2 -terminal Sequencing Analysis and Immunochemical Analysis-The NH 2 -terminal sequence of purified IP4BP2b after Sephacryl S-300 gel filtration chromatography was determined with a gas-phase protein sequencer. The NH 2 -terminal sequence of IP4BP2b was checked against the SWISS-PROT data base. All 10 amino acid residues identified out of 14 NH 2 -terminal amino acid residues of IP4BP2b were identical with mouse myelin proteolipid protein (PLP) ( Table I). With the sequencing method used, cysteine (C) and arginine (R) are undetectable.
Purified IP4BP2b after Sephacryl S-300 gel filtration chromatography and purified myelin sample as a positive control were applied to SDS-PAGE, and the proteins were electrotransferred to nitrocellulose membranes. The membranes were either stained with Amido Black (Fig. 4, lanes 1 and 4) or analyzed immunochemically using monoclonal antibodies against PLP (epitope: amino acid residues number 209 -217 (AH7-2a) or 264 -276 (AA3)). Fig. 4 (lanes 2 and 3) shows that monoclonal antibodies against PLP recognized the 26,000 molecules. However, these monoclonal antibodies have also been shown to recognize DM-20 (26), an alternative splicing variant of PLP. Immunoblot analysis of the purified myelin containing both PLP and DM-20 revealed that the 26,000 band comigrated with the band corresponding to PLP (Fig. 4). These observations suggested that IP4BP2b (26,000 band) is PLP.
To further confirm that IP4BP2b is PLP, we determined whether the InsP 4 binding activity of IP4BP2b could be immunoabsorbed by anti-PLP antibody. The purified sample obtained by Sephacryl S-300 gel filtration chromatography was incubated with Protein G-Sepharose resin coupled with AA3-IgG or normal rat IgG, or non-coupled. The resins were poured into columns and washed with washing solution (100 l ϫ 4). The nonadherent fractions were collected, and assayed for their [ 3 H]InsP 4 binding activity and analyzed on SDS-PAGE with silver staining (Fig. 5, A and B). The InsP 4 binding activity and 26,000 protein bands were immunoabsorbed by AA3-Protein G-Sepharose (Fig. 5, A and B; ϩAA3, Fraction No. 2), but not by non-coupled Protein G-Sepharose or normal rat IgG-Protein G-Sepharose (Fig. 5, A and B, ϪIgG and ϩRat IgG; Fraction No. 2). These results together with the NH 2 -terminal sequences indicate that the purified IP4BP2b is in fact mouse PLP.
Characterization of the Inositol Polyphosphate (InsP X )-Binding Activity of PLP-Analysis of the InsP 4 binding described thus far was performed under the hypotonic conditions. To investigate its physiological significance, we measured InsP 3 , InsP 4 , and InsP 6 binding activities of purified IP4BP2b (PLP) in an isotonic buffer containing 0.15 M KCl, 20 mM HEPES-KOH, at pH 7.2. Binding activity was detectable only against InsP 6 (data not shown).
These findings suggested that InsP 6 is the true ligand for PLP. Therefore, the K d and B max under isotonic conditions were 2 Y. Yamaguchi, M. Niinobe, and K. Mikoshiba, unpublished result.  B) were concentrated and applied to a gel filtration column on Sephacryl S-300 (C). Aliquots of each fraction were assayed for protein concentration (q---q) and for InsP 4 binding activity (qOq). The NaCl concentration was indicated by the solid line. determined for [ 3 H]InsP 6 binding. Since the purified PLP (Sephacryl S-300 fraction) was unstable and occasionally showed two types of binding sites (high and low affinity) (data not shown), we determined the K d and B max of heparin-agarose fraction, which showed only one type (high affinity) of binding site and in which most, if not all, InsP 6 binding activity is attributable to PLP. Scatchard analysis of InsP 6 binding to the heparin-agarose fraction showed that the K d was 52 nM, the B max 6.5 pmol/g of protein (Fig. 6). This value of K d was nearly the same as that of high affinity binding site of the purified PLP.
Preparation of PLP and DM-20 from DE-52 Flow-through Fraction-All of the results obtained thus far clearly indicate that PLP has InsP 6 binding activity. However, it is not un-   (lanes 2 and 3)) and myelin samples (2.1 g (lane 4) and 0.42 g (lane 5)) were applied to SDS-PAGE on a 15% Laemmli gel. Proteins were transferred to nitrocellulose membranes, and the blots were stained with Amido Black (lanes 1 and 4) and probed with monoclonal antibody against PLP, AA3 (lanes 2 and 5), or AH7-2a (lane 3). Peroxidase-coupled detection was performed by the diaminobenzidine staining method by using a Vectastain ABC kit. Molecular weight markers used were 10-kDa Protein Ladder (Life Technologies, Inc.). Sizes of markers are shown at the left. The band indicated by the arrow seems to be aggregates of two PLP molecules. known whether DM-20 has this activity. During purification of IP4BP2b/PLP, DM-20 separated from PLP at the heparin-agarose step. DM-20 was recovered from the heparin-agarose flowthrough fraction, although we could not use this fraction to study the InsP 6 binding activity of DM-20 because it also contained PLP as revealed by immunoblot analysis (data not shown). To separate DM-20 from PLP, we devised several modifications of the purification method. First, we changed the pH of the DE-52 flow-through fraction from 8.0 into 5.0, by dilution with acetate buffer to achieve pH 5.0. The DE-52 flow-through fraction used was the same as that of the IP4BP2b/PLP purification procedure. DM-20 separated from PLP and was recovered from the flow-through fraction after CM-52 chromatography at pH 5.0. The adsorbed fraction did not contain DM-20. Because both PLP and DM-20 are extremely hydrophobic and the InsP 6 binding activity of PLP was apparently stable in the solution containing 1% Triton X-100, we performed the CM-52 chromatography with this solution. PLP separated from the other concomitant proteins (including IP4BP2a/aldolase A) by elution with a 0.05-0.5 M NaCl gradient. Both the CM-52 flow-through fraction containing DM-20 and the CM-52 adsorbed fraction containing PLP were concentrated by heparin-agarose chromatography and the buffer detergent was changed from Triton X-100 to CHAPS because the InsP 6 binding activity was inhibited more by Triton X-100 than by CHAPS. After these steps, we obtained fractions containing either PLP or DM-20. PLP or DM-20 was the major protein in PLP-or DM-20-containing fractions, respectively (Fig. 7A). Importantly, as shown by immunoblot analysis (Fig. 7B), PLP-containing fraction did not contain a detectable amount of DM-20 and either DM-20-containing fraction did not contain detectable amounts of PLP.
Comparison of InsP 6 Binding Activity of PLP and DM-20 -We measured the InsP 6 binding activity of PLP-containing and DM-20-containing fractions at equal volumes. Since CHAPS also inhibited the InsP 6 binding activity, although to a lesser extent than Triton X-100, we had to make the same dilution of the samples. The concentration of DM-20 was similar or slightly higher than that of PLP as shown semiquantitatively by immunoblot analysis (Fig. 7B). However, the InsP 6 binding activity of the DM-20-containing fraction was not de- 2) buffer. Nonspecific binding was determined by removing the sample. Samples were incubated for 10 min at 0°C and binding activity was measured by the polyethylene glycol precipitation method described under "Experimental Procedures." Each column represents the mean from three experiments. SDS-PAGE was carried out on a 15% gel using the buffer system of Laemmli. The gel was visualized by silver staining. Molecular weight markers with corresponding M r values are shown at the left as described in the legend to Fig. 3.

Inhibition of specific [ 3 H]InsP 6 -binding by various inositol phosphates
Binding assay mixtures contained 0.17 g of the purified IP4BP2b/ PLP, 2.4 nM [ 3 H]InsP 6 , 50 or 200 nM inositol phosphates, 20 g of ␥-globulin in 50 mM HEPES-KOH at pH 7.2 containing 0.15 M KCl (100 l) (isotonic condition). Nonspecific binding was determined by removing the sample. Samples were incubated for 10 min at 0°C and binding activity was measured by the polyethyleneglycol precipitation method described under "experimental procedures." Each value represents the mean from two independent duplicated experiments.  55 12 tectable and only the PLP-containing fraction showed InsP 6 binding activity (Fig. 7C).

DISCUSSION
Identification of IP4BP2b as PLP-Recently, inositol polyphosphates (InsP X ), such as InsP 4 , InsP 5 , InsP 6 , and PP-InsP 5 have been shown to accumulate intracellularly in response to several stimuli (15,16). We identified three InsP 4binding proteins (IP4BP); one, an IP4BP from the DE-52 adsorbed fraction, was called IP4BP1 and the others, from the DE-52 flow-through fraction, were called IP4BP2a and IP4BP2b (Fig. 1). We previously showed that IP4BP1 is synaptotagmin II (30). We also purified IP4BP2a and identified it as aldolase A, which is one of the three isoforms of fructose 1,6bisphosphate aldolase. 2 A similar observation had already been reported by Koppitz et al. (31). In this study, we succeeded in purifying IP4BP2b and identifying it as PLP.
Evidence for identifying IP4BP2b as PLP includes: (i) identity of their NH 2 -terminal amino acid sequences (Table I); (ii) nearly the same molecular weights of IP4BP2b, as determined by SDS-PAGE (26K), and of PLP (Fig. 3); (iii) immunoreactivity of 26,000 molecules against anti-PLP monoclonal antibodies (AA3 and AH7-2a) (Fig. 4), and immunoabsorption of InsP 4 binding activity of IP4BP2b by AA3 antibody (Fig. 5). From these results, we concluded that the purified IP4BP2b is mouse PLP. In addition, the further analysis of InsP X binding activity of PLP under the isotonic condition demonstrated that PLP is not really IP4BP but InsP 6 -binding protein.
PLP is a major integral membrane protein of central nervous system myelin. The amino acid and nucleotide sequences of cow, rat, mouse, and human PLP are closely homologous (32)(33)(34)(35)(36)(37)(38)(39)(40), and mutations within the PLP gene cause severe dysmyelination (8). Thus, PLP seems to play a significant role in central nervous system myelination, presumably by promoting the apposition of extracellular surfaces of the myelin lamellae. However, many PLP mutations also result in profound abnormalities in premyelinating oligodendrocytes. These include (a) a decrease in the number of mature oligodendrocytes, (b) premature cell death of oligodendrocytes, (c) abnormal oligodendrocyte inclusions and organelle distentions, and (d) increased oligodendrocyte proliferation (41). Therefore, it is important to understand the premyelinating functions of PLP. We have shown that expression of the PLP gene results in secretion of a factor influencing oligodendrocyte development (42). The present results indicate that another function of PLP is the binding of InsP 6 .
Other InsP X -binding Proteins-Recently, some InsP X -binding proteins have been reported by several groups. Partial amino acid sequencing of one protein has revealed that it is clathrin assembly protein 2 (AP-2), which is possibly an essential protein in the endocytotic or recycling pathway of all cells (17)(18)(19)(20). The K d of InsP X -binding protein toward InsP 6 reported by Theibert et al. (17) is 12 nM and that reported by Chadwick et al. (18) is 120 nM. Dependence of the InsP X binding on the salt concentration in the assay system seems to account for this difference. Another clathrin assembly protein, AP-3, has also been reported to have InsP X binding activity (23,24). AP-3 is expressed in neurons and localized to synapses. It has been suggested that AP-3 is involved in synaptic vesicle biogenesis and recycling (43 (24) 239 nM. In addition, coatomer, which is a cytosolic protein complex containing subunits of non-clathrin-coated Golgi intercisternal transport vesicles, was shown to have the InsP X binding activity (K d for InsP 6 ϭ 0.2 nM, K d for InsP 4 ϭ 0.1 nM) by Fleischer et al. (25).
We also found that synaptotagmin II is an InsP X -binding protein (30). Synaptotagmin is an integral membrane protein of synaptic vesicles considered to play a significant role in the docking and fusion of synaptic vesicles at presynaptic release sites (44,45).
From the viewpoint of InsP X affinity, the IP4BP2b/PLP purified in this study resembles IP4BP1/synaptotagmin II and several proteins involved in vesicular transport (AP-2, AP-3, and coatomer), whereas IP4BP2a/aldolase A does not fit into any groups because of its comparatively lower affinity and different specificities for InsP X (31). 2 PP-InsP 5 is a newly discovered pyrophosphorylated derivative (46,47). The inhibition of InsP 6 binding activities of AP-3 and coatomer by PP-InsP 5 was stronger than by InsP 6 (24,25). PP-InsP 5 also inhibited InsP 6 binding to PLP more strongly than InsP 6 . This suggests that PLP is one of the member of a family of InsP X -binding proteins, including AP-2, AP-3, and coatomer. . Nonspecific binding was determined by removing the sample. Samples were incubated for 10 min at 0°C and binding activity was measured by the polyethylene glycol precipitation method described under "Experimental Procedures." Each column represents the mean from triplicate experiments.
All of these reports indicate that InsP X -binding protein is involved in vesicular transport, suggesting the involvement of PLP in vesicular transport. Abnormal transport of PLP found in the PLP mutants further supports this hypothesis (see Introduction).
InsP 6 -binding Site-The current model of PLP topology in the plasma membrane was proposed by Popot et al. (48) and Weimbs et al. (49). In this model, PLP has four ␣-helical transmembrane regions, two pairs of disulfide bonds (Cys 183 -Cys 227 , Cys 200 -Cys 229 ), and six cysteine residues (in positions 5, 6, 9, 108, 138, and 140) acylated with long-chain fatty acids. The NH 2 terminus and the COOH terminus of PLP are on the cytoplasmic side. Therefore, highly basic residues (amino acid residues number 115-150) specific for PLP are on the cytoplasmic side between the second and third loop, which is spliced out in the DM-20 molecule. Thus far, the structure of DM-20 is thought to be the same as PLP with a small cytoplasmic domain between the second and third loop. Since we detected InsP 6 binding activity in PLP but not in DM-20 (Fig. 7), it is suggested that the PLP-specific domain contains the InsP 6binding site. Therefore, the conclusion can be drawn that the ancestral DM-20 molecule acquired the PLP-specific domain as an InsP 6 -binding site (or at least changed conformation of other part of DM-20 to create InsP 6 -binding site).
The InsP X -binding site of synaptotagmin II was determined to be in the C2B region (amino acid residues, 315-346) (50,51). Although the C2A domain also contains a lysine-rich sequence, InsP 4 bound only to the C2B domain. This observation indicated that the C2A and C2B domains of synaptotagmin II have different conformations and functions. The C2B domain of synaptotagmin is highly conserved from C. elegans to humans (52)(53)(54)(55), suggesting that the InsP X binding capacity has also been maintained during evolution. On the other hand, Voglmaier et al. (20) reported that the InsP 6 -binding site of AP-2 lies on an ␣-subunit of AP-2 as shown by a specific photoaffinity label. The details of the InsP 6 -binding site of AP-2 have not yet been clarified. In addition, Ye and Lafer (56) showed that the InsP 6 -binding site of AP-3 exists in the 33,000 amino terminus of AP-3. This NH 2 -terminal region is known to be a basic and clathrin-binding domain (57). However, since there are few similarities among these InsP 6 -binding proteins (including PLP) in their primary structure, the consensus sequence of the InsP 6 -binding site is still unknown. Furthermore, the InsP 6binding sites of proteins other than synaptotagmin II (PLP, AP-2, AP-3, and coatomer) have not been precisely determined. In all likelihood, the InsP 6 -binding sites are in the basic region and are governed by as yet unknown mechanisms.
In conclusion, we identified PLP as an InsP 6 -binding protein and showed that DM-20 does not have InsP 6 binding activity. InsP 6 might regulate the vesicular transport of PLP having PLP-specific domain as InsP 6 -binding site. We are currently investigating how InsP 6 affects the transport of PLP to the myelin membrane.