Identification of a 250 kDa putative microtubule- associated protein as bovine ferritin Evidence for a ferritin–microtubule interaction Mohammad R. Hasan 1 , Daisuke Morishima 1 , Kyoko Tomita 1 , Miho Katsuki 1 and Susumu Kotani 1,2 1 Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan 2 Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Kanagawa, Japan Although microtubules are heteropolymers of a- and b-tubulin, the diverse roles they play in different cellular processes, such as cell division, intracellular transport, cell motility and cytoplasmic morphogenesis, are largely dependent on various specific binding proteins [1]. These nontubulin components include the well-known micro- tubule-associated proteins (MAPs) that coassemble with tubulin, and are believed to regulate the microtubular properties in vivo [2]. To date, a considerably large number of MAPs have been reported, among which, the brain MAPs, such as MAP1, MAP2 and Tau, were shown to be responsible for neurite outgrowth in neuron cells [3,4]. On the other hand, the MAP4 proteins have a ubiquitous cellular distribution, and have been implica- ted in the regulation of both cytoplasmic and spindle microtubules in non-neuronal cells [5,6]. Previously, we reported the presence of MAP4 in bovine adrenal gland as the major MAP species. Moreover, an analysis of the adrenal MAPs that coex- isted with the tubulin after cycles of assembly and dis- assembly in vitro revealed several minor components in addition to MAP4 [7]. One of the minor components, Keywords ferritin; ferritin–microtubule interaction; microtubule; microtubule-associated protein Correspondence M. R. Hasan, Department of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502, Japan Fax: +81 948 29 7801 Tel: +81 948 29 7840 E-mail: c791009m@bio.kyutech.ac.jp (Received 22 October 2004, revised 6 December 2004, accepted 8 December 2004) doi:10.1111/j.1742-4658.2004.04520.x We reported previously on the purification and partial characterization of a putative microtubule-associated protein (MAP) from bovine adrenal cor- tex with an approximate molecular mass of 250 kDa. The protein was expressed ubiquitously in mammalian tissues, and bound to microtubules in vitro and in vivo, but failed to promote tubulin polymerization into microtubules. In the present study, partial amino acid sequencing revealed that the protein shares an identical primary structure with the widely distri- buted iron storage protein, ferritin. We also found that the putative MAP and ferritin are indistinguishable from each other by electrophoretic mobil- ity, immunological properties and morphological appearance. Moreover, the putative MAP conserves the iron storage and incorporation properties of ferritin, confirming that the two are structurally and functionally the same protein. This fact led us to investigate the interaction of ferritin with microtubules by direct electron microscopic observations. Ferritin was bound to microtubules either singly or in the form of large intermolecular aggregates. We suggest that the formation of intermolecular aggregates contributes to the intracellular stability of ferritin. The interactions between ferritin and microtubules observed in this study, in conjunction with the previous report that the administration of microtubule depolymerizing drugs increases the serum release of ferritin in rats [Ramm GA, Powell LW & Halliday JW (1996) J Gastroenterol Hepatol 11, 1072–1078], support the probable role of microtubules in regulating the intracellular concentration and release of ferritin under different physiological circumstances. Abbreviations MAP, microtubule-associated protein; PVDF, poly(vinylidene difluoride); RB, reassembly buffer; MDBK, Madin–Darby bovine kidney. 822 FEBS Journal 272 (2005) 822–831 ª 2005 FEBS with an approximate molecular mass of 250 kDa, was reported as a putative MAP, on the basis of various properties such as electrophoretic mobility, heat stabil- ity and immunoreactivity [8]. The protein was found to be distributed ubiquitously among various bovine organs. It coassembled with taxol-stabilized micro- tubules in vitro, and showed some association with the microtubular network in cultured cells. However, unlike the other common MAPs (MAP1, MAP2, Tau and MAP4), the 250 kDa protein lacked the ability to induce microtubule polymerization from purified tubu- lin molecules. The molecular shape of the protein, as determined by electron microscopy, was spherical, in contrast to the long, rod-like conformations of the other MAPs [8]. In this study, our attempts to further characterize the 250 kDa protein identified it as the ubiquitous iron stor- age protein, ferritin, as revealed by comparisons of the two proteins in terms of their amino acid sequences, immunoreactivity, molecular mass and shape, and iron storage ⁄ incorporation properties. As a widely distri- buted protein among bacteria, plants and animals, fer- ritin stores iron in the Fe(III) form, preventing the oxidative damage caused by Fe(II) atoms, and supplies cells with the necessary iron at an effective concentra- tion when required. Ferritin has a molecular mass of 450 kDa, and appears as a hollow, roughly spherical structure with an external diameter of about 12–13 nm. The inner cavity, which can accommodate up to 4500 Fe(III) atoms, has a diameter of about 8 nm [9–11]. Ferritin is a polymeric protein composed of 24 subunits, with two subunit types (H and L) in mammals that play distinct roles in iron homeostasis. The H subunit cata- lyzes the oxidation of Fe(II), the initial step in the iron storage process, and the L subunit is known to induce iron core nucleation [12–14]. Many studies have analyzed ferritin in terms of its structure, function and regulation, but no report has established a relationship between ferritin and micro- tubules. Here, based on microtubule cosedimentation assays and electron microscopic observations, we report a novel interaction between ferritin and micro- tubules in vitro, and hypothesize that microtubules might be involved in the stability, intracellular pool and release of ferritin. Results Determination of the primary structure of the 250 kDa protein Our first attempt to determine the N-terminal amino acid sequence of the purified 250 kDa protein in its native form was unsuccessful. Therefore, we thought that the protein might have chemical modifications at its N-terminus. Subsequently, we digested the protein with cyanogen bromide to cleave the protein at methio- nine residues for internal amino acid sequencing. Among the digested products, three fragments were selected and sequenced. The deduced amino acid sequences were identical to three sequences within the H subunit of bovine ferritin, as shown by the underlined sequences (a–c) in Fig. 1, corresponding to residues 39– 53, 72–87, and 102–117, respectively. As expected, all of the deduced sequences appeared after methionine resi- dues, suggesting the correctness of the procedures employed. The results obtained from amino acid sequencing gave us the first indication that the 250 kDa protein might actually be ferritin, although none of the sequences obtained matched the L subunit of ferritin. In addition, there was a great disparity between the known molecular mass of ferritin (450 kDa) and the apparent molecular mass of the 250 kDa protein on SDS⁄ PAGE. Therefore, further lines of evidence were necessary for a definitive conclusion. Comparison of the apparent molecular masses of ferritin and the 250 kDa protein To compare the molecular masses of ferritin and the 250 kDa protein, we checked the electrophoretic mobility of bovine liver ferritin, bovine adrenal cortex Fig. 1. Amino acid sequence analysis of the 250 kDa protein. A cyanogen bromide digest of the 250 kDa protein was separated by SDS ⁄ PAGE, and the fragments were electrophoretically transferred to a PVDF membrane for internal amino acid sequencing. Three fragments were selected and sequenced by automated Edman degradation. The three sequences were identical to three regions of the ferritin H subunit, as shown by the underlined sequences (a, b & c). M. R. Hasan et al. Identification of a putative MAP as ferritin FEBS Journal 272 (2005) 822–831 ª 2005 FEBS 823 ferritin and the 250 kDa protein by SDS ⁄ PAGE with or without heat treatment, for the monomeric and polymeric forms, respectively. As shown in Fig. 2A, all of the samples showed the same electrophoretic mobil- ity in either form. This eliminated the confusion about the molecular mass disparity between ferritin and the 250 kDa protein in the polymeric form. The globular nature of ferritin in the native form might be a reason for its faster movement in SDS ⁄ polyacrylamide gels. With respect to the monomeric forms, it should be noted here that, unlike other mammalian species where the molecular masses of the ferritin L and H subunits are 19 kDa and 21 kDa, respectively, the molecular mass of the L subunit (20.5 kDa) of bovine ferritin is larger than that of the H subunit (18.4 kDa), as observed by SDS ⁄ PAGE. This variation was attributed to differences in the binding affinity of SDS to the bovine L chains, rather than any insertions or dele- tions of amino acids in the bovine ferritin subunits [15]. We also observed similar properties with the bovine ferritin subunits in this study. In addition, the extremely low content of L subunits in adrenal ferritin and in the 250 kDa protein, as compared to that of liver ferritin, is also consistent with previous reports that the H and L subunit contents may differ in mam- mals, depending on the organs and their iron require- ments. Again, the abundance of the L subunit in the liver is important for its iron storage functions [11]. The extremely low abundance of the L subunit in the 250 kDa protein now explains why no sequences homologous to the L subunit were detected in the sequencing experiment (Fig. 1). Immunocrossreactivity of ferritin and the 250 kDa protein The immunological properties of ferritin and the 250 kDa protein were investigated using an anti-(horse spleen ferritin) IgG, which recognizes the L subunit of ferritin, and an anti-(250 kDa protein) Ig. For subunit- specific detection, we used the monomeric forms of liver ferritin, adrenal ferritin, and the 250 kDa protein. Fig- ure 2B(a) shows that the anti(horse spleen ferritin L subunit) IgG reacted with all of the samples, revealing the distinct L subunit band for each sample. When the same samples were allowed to react with the anti- (250 kDa protein) Ig, it clearly recognized both the H and L subunits in liver ferritin, adrenal ferritin and the 250 kDa protein [Fig. 2B(b)], indicating that the anti- serum raised against the 250 kDa protein is a mixture of antibodies to the H and L subunits of ferritin. Because the L subunit was hardly detectable in the adre- nal cortex ferritin and in the 250 kDa protein, the sam- ples were overloaded to visualize the L subunit band. Detection of iron in the 250 kDa protein To determine whether the 250 kDa protein possesses the iron storage property of ferritin, we added potas- sium ferrocyanide to the gel filtration column fractions [8], which should cause the color of the solution to turn blue if iron is present. Figure 3A clearly demon- strates the presence of iron in the 250 kDa protein peak fractions (Fractions 11–14). Iron was not detec- ted in any of the fractions that lacked the 250 kDa protein, eliminating the chance that the detected iron was an artifact of the purification procedure. The Fig. 2. Electrophoretic patterns and immunocrossreactivity of the 250 kDa protein and ferritin. (A) Electrophoretic mobility of the 250 kDa protein and ferritin: SDS ⁄ PAGE was carried out with or without heat treatment of the samples prior to loading in the pres- ence of SDS detergent, for the monomeric (b) and polymeric (a) forms, respectively. Lane 1, bovine liver ferritin; lane 2, bovine adrenal gland ferritin; lane 3, the 250 kDa protein; and lane M, molecular mass standards (myosin heavy chain, 220 kDa; myosin light chain 1, 26 kDa; myosin light chain 2, 18 kDa). (B) Immuno- crossreactivity: monomeric 250 kDa protein and ferritin were trans- ferred to a PVDF membrane after SDS ⁄ PAGE, and the blots were incubated with either an anti-(horse spleen ferritin) IgG (Sigma- Aldrich Japan K.K.) that recognizes the L subunit of ferritin (a) or an anti-(250 kDa protein) IgG (b). The bound antibodies were detected by an incubation with horseradish peroxidase-conjugated anti-(rabbit IgG) IgG (Sigma-Aldrich Japan K.K.). The subsequent staining proce- dures are described in Experimental procedures. Lanes 1 and 4, bovine liver ferritin; lanes 2 and 5, bovine adrenal gland ferritin; and lanes 3 and 6, the 250 kDa protein. Identification of a putative MAP as ferritin M. R. Hasan et al. 824 FEBS Journal 272 (2005) 822–831 ª 2005 FEBS absorbance data shown in the upper panel of Fig. 3A represent the total protein concentrations of the gel fil- tration column fractions. The SDS ⁄ PAGE patterns of the corresponding fractions (Fig. 3A: middle panel) revealed that the protein peak at 280 nm is different from the peak concentrations of the 250 kDa protein, because of the presence of contaminating proteins. Therefore, as shown in the lower panel of Fig. 3A, the presence ⁄ absence of iron in the gel filtration column fractions was compared with the presence ⁄ absence of a visible band corresponding to the 250 kDa protein in the SDS ⁄ PAGE. Comparison of iron uptake by ferritin and the 250 kDa protein The iron uptake activity was measured by considering the results of a previous report: when ferritin was incu- bated with ferrous iron and molecular oxygen in vitro, an amber colored product [Fe(III)] was formed that could be monitored by a change in absorbance at 310 nm [16]. The progression plot in Fig. 3B indicates that the uptake rates of ferritin and that of the 250 kDa protein were almost the same, and were higher than both controls. The apparent increase in A B Fig. 3. Iron storage and uptake activity of the 250 kDa protein. (A) Detection of iron in the 250 kDa protein: potassium ferrocyanide was added to a final concentration of 10 m M to all of the fractions obtained from the gel filtration column chromatography, which was the final step of the 250 kDa protein purification procedure [8]. The presence of iron was detected by the appearance of a blue color, and was com- pared with the electrophoretic patterns and the spectrophotometric observations of all fractions. Upper panel, plot showing the absorbance of all of the fractions from the gel filtration column chromatography at 280 nm, reflecting the total protein contents. Middle panel, SDS ⁄ PAGE profile of the gel filtration chromatography fractions. The lanes are aligned to the fraction numbers of the plot, in the upper panel. Lower panel, + ⁄ ) signs are given to indicate the presence ⁄ absence of the 250 kDa protein (first row) and to indicate the pres- ence ⁄ absence of iron (second row) in the aligned fractions. An increased number of + signs reflects a higher concentration of the 250 kDa protein as well as the greater intensity of the blue color in the iron detection assay. (B) Iron uptake activity: proteins (either ferritin or the 250 kDa protein) and ferrous sulfate (Fe 2+ ) were mixed in 20 MEM to final concentrations of 1.5 mM and 10 nm, respectively, and the increase in absorbance was monitored at 310 nm for up to 10 min. The data were compared with the progression curves derived from con- trol 1 (1.5 m M ferrous sulfate in 20 MEM only) and control 2 (1.5 mM ferrous sulfate + 10 nM BSA). M. R. Hasan et al. Identification of a putative MAP as ferritin FEBS Journal 272 (2005) 822–831 ª 2005 FEBS 825 the absorbance in control 1 is due to the auto-oxida- tion of ferrous iron upon reaction with molecular oxy- gen. Control 2, which contained an unrelated protein (BSA) at the same concentration, was included to observe the effect of a protein in general on the absorbance data. Morphological appearances of ferritin and the 250 kDa protein by electron microscopy Previously, the 250 kDa protein was reported to appear as a hollow sphere with a diameter of about 12 nm, as determined by electron microscopic observa- tions [8]. To compare the molecular dimensions of both bovine adrenal ferritin and the 250 kDa protein, we observed the negatively stained samples by higher resolution microscopy, operating at 200 kV. Both of the proteins appeared to be the same, with an external diameter of 13 nm and an internal diameter of about 6–7 nm (Fig. 4: A, ferritin; B, 250 kDa protein). The dark region in the center of each molecule might repre- sent the iron cores of the ferritin molecules. Interaction of ferritin with microtubules To identify whether ferritin binds to microtubules, as reported for the 250 kDa protein, we examined the binding of ferritin with taxol-stabilized microtubules by an in vitro microtubule-binding assay. When tubulin was excluded from the reaction mixture, the ferritin remained in the supernatant fraction, but in the pres- ence of tubulin, a significant portion of the ferritin sedimented with the microtubule pellet (Fig. 5: lanes 3 and 1). We also found by immunoblotting experiments that ferritin was present in the mammalian brain microtubule protein fractions (data not shown). To clarify the ferritin–microtubule interaction further and Fig. 4. Electron micrographs of negatively stained 250 kDa protein and ferritin. Purified ferritin (A) and the 250 kDa protein (B) were fixed by 2.5% glutaraldehyde on carbon coa- ted grids and negatively stained with 2% uranyl acetate before observation. Fig. 5. Microtubule binding activity of ferritin. Horse spleen ferritin (Sigma-Aldrich Japan K.K.) (5 l M) and tubulin (15 lM) were mixed in RB containing 30 l M taxol and 0.5 mM GTP, incubated at 37 °C for 30 min and centrifuged at 16 000 g for 30 min. The resultant supernatant and pellet were analyzed by SDS ⁄ PAGE (lane 1). Two control experiments included preparations without ferritin (lane 2) or without tubulin (lane 3), respectively. s, Supernatant; p, pellet. Identification of a putative MAP as ferritin M. R. Hasan et al. 826 FEBS Journal 272 (2005) 822–831 ª 2005 FEBS to demonstrate the binding architecture of ferritin on microtubules, we made direct observations by electron microscopy. To exclude the possibility that, on the electron micrographs, the ferritin might appear in association with microtubules by chance, we collected the ferritin–microtubule complex by centrifugation and redissolved the pellet, while preparing the samples for electron microscopy. Figure 6 shows the various types of interactions between ferritin and microtubules. Ferritin was found to interact randomly with micro- tubules, either single or in the form of large inter- molecular aggregates (Fig. 6A–I). Therefore, both the sedimentation data and the electron microscopy results clearly demonstrate an association between ferritin and microtubules in vitro. To investigate whether the iron within ferritin plays any role in mediating the ferritin– microtubule interaction, we determined the microtubule binding ability of apoferritin, the protein shell of fer- ritin that lacks iron, by an in vitro microtubule-binding assay. A portion of the apoferritin sedimented with the microtubule pellet, similar to the iron-containing ferritin (data not shown), suggesting that the ferritin– microtubule interaction occurs independently of iron. We then considered the possibility that the tubulin preparation, used for the in vitro microtubule binding assay, might contain trace amounts of residual MAPs or other noncytoskeletal proteins, and the ferritin might have interacted with the microtubules, indirectly, through one or more of these proteins. Therefore, we observed the effect of total MAP fractions on the ferr- itin–microtubule interaction. The total MAP fraction was prepared so that it contained most of the proteins of the microtubule protein fraction, other than tubulin. We observed that the addition of increasing concentra- tions of the total MAP preparation to the reaction mixtures, containing the same concentrations of tubu- lin and ferritin, caused a reduction in the amount of ferritin in the microtubule pellet (Fig. 7: lanes 2–4). Similar results were obtained, when heat-treated MAPs (predominantly MAP2 and Tau) were added to the ferritin ⁄ tubulin reaction mixtures, instead of the total MAPs (data not shown). Furthermore, Katsuki et al. [8] showed that the addition of an excess amount of a MAP4 fragment, containing the microtubule-binding region, prevented ferritin from binding with micro- tubules. Altogether, these observations suggest that ferritin directly interacts with microtubules, and MAPs or other microtubule associated noncytoskeletal pro- teins seem to inhibit, rather than facilitate, the ferritin– microtubule interaction. Discussion In this paper, we have described our detailed charac- terization of a protein that we reported previously on as a putative MAP, with a relative molecular mass of 250 kDa, which binds microtubules both in vitro and in vivo. Determination of the primary structures of cer- tain regions of the protein gave us the first clue that this protein and the iron storage protein ferritin might be the same (Fig. 1). Although the absence of the L subunit sequence and the discrepancy in the molecular Fig. 6. Electron micrographs showing ferritin–microtubule inter- action. Ferritin was added to a microtubule preparation reassem- bled in vitro and the mixture was observed by electron microscopy. Samples were fixed using 2.5% glutaraldehyde on carbon-coated grids and negatively stained with 2% uranyl acetate. Ferritin mole- cules appeared singly (short arrows) or in the form of intermole- cular aggregates (long arrows) on the microtubule surface. M. R. Hasan et al. Identification of a putative MAP as ferritin FEBS Journal 272 (2005) 822–831 ª 2005 FEBS 827 masses suggested that the 250 kDa protein could be a degradation product or a premature form of ferritin, further investigations on the molecular masses of the proteins made it clear that both have the same elec- trophoretic mobility on SDS ⁄ polyacrylamide gels. Moreover, the 250 kDa protein was found to dissoci- ate into two subunits that were indistinguishable from the ferritin H and L subunits, in terms of their elec- trophoretic mobility. Although the subunit content of the 250 kDa protein differed from that of bovine liver ferritin, it resembled that of adrenal cortex ferritin. These observations rule out the possibilities that the 250 kDa protein is a degradation product or a precur- sor form of ferritin. Subsequently, we showed that antibodies against ferritin and the 250 kDa protein crossreact with each other, and that the 250 kDa protein conserves the iron storage ⁄ incorporation properties of ferritin. Finally, electron microscopic observations revealed the identical morphological appearance of both proteins, leaving us with no doubt that ferritin and the 250 kDa protein are structurally and functionally the same protein. The interaction of the 250 kDa protein with micro- tubules was first described by Katsuki et al. [8]. Although the protein lacked the ability to polymerize tubulin into microtubules and was structurally distinct from the other common MAPs, the microtubule bind- ing characteristics of the protein, as well as the salt sensitivity and the competition with other MAPs for microtubule binding, led the authors to conclude that the protein is a distinct MAP subspecies. However, the in-depth analysis of the protein, in this study, identi- fied it as the ubiquitous iron homeostatic protein fer- ritin, suggesting that the protein should no longer be considered as belonging to the group of MAPs. Never- theless, a very important outcome of characterizing the putative 250 kDa MAP as ferritin is that a novel inter- action between two very important components of the cell, namely, microtubules and ferritin, has now been revealed. The previous report of the binding of the putative MAP [8], which we have now identified as ‘ferritin’, to microtubules (both in vitro and in vivo)is further supported by our in vitro sedimentation data, and our direct observation of ferritin in association with microtubules by electron microscopy. Moreover, we found that the addition of a mixture of MAPs and other microtubule-associated, noncytoskeletal proteins decreased the extent of the association between ferritin and microtubules, precluding any chance that the ferri- tin–microtubule interaction is mediated by another protein within the microtubule protein fraction. Subsequently, we noted that apoferritin, which lacks iron, cosediments with microtubules in a manner sim- ilar to that observed for the iron-containing ferritin, indicating that the ferritin–microtubule interaction is not mediated by the iron stored in the ferritin mole- cules. Therefore, it is conceivable that the protein por- tion of the ferritin molecule is responsible for this interaction, which is most likely to be ionic, as the addition of salt prevented the ferritin from cosediment- ing with microtubules in vitro [8]. Thus, the neutraliza- tion of the anionic microtubule surface by MAPs could account for the observed inhibition of the ferri- tin–microtubule interaction by MAPs in vitro. The intermolecular aggregates of ferritin associated with microtubules, which we observed by electron micros- copy, correspond well with the microtubule associated punctuate structures observed by Katsuki et al. [8] in Madin–Darby bovine kidney (MDBK) and 3Y1 fibro- blast cells stained with anti-tubulin mAb and anti- (250 kDa protein) Ig. The formation of intermolecular aggregates and their association with microtubules Fig. 7. Effect of total MAPs on the microtubule binding activity of ferritin. A total MAP preparation (lane 1) was added to a reaction mixture (15 l M tubulin and 5 lM ferritin in RB with 30 lM taxol and 0.5 m M GTP), to final concentrations of 0 mgÆmL )1 (lane 2), 2mgÆmL )1 (lane 3) and 6 mgÆmL )1 (lane 4), incubated at 37 °C for 30 min and centrifuged at 16 000 g for 30 min. The contents of the microtubule pellets were analyzed by 7.5% SDS ⁄ PAGE. Identification of a putative MAP as ferritin M. R. Hasan et al. 828 FEBS Journal 272 (2005) 822–831 ª 2005 FEBS might be physiologically significant, in terms of the stabilization of ferritin molecules from degradation and the prevention of unwanted iron release into the cell. Unfortunately, the nature of this aggregation is unclear at present. It might be an intrinsic feature of ferritin molecules to aggregate on the microtubule surface. Alternatively, ferritin molecules might form cross-bridges between each other through interactions with free tubulin. In support of the latter explanation, Katsuki et al. [8] also suggested an interaction between ferritin and free (nonmicrotubular) tubulin. Further analyses of the properties and mechanisms of the association between ferritin and microtubules are cur- rently underway. Ferritin exists in a variety of cells and tissue types, and plays central roles in iron metabolism. In addition, the presence of ferritin in serum was reportedly corre- lated with the tissue ferritin and body iron stores. Although some differences were detected between the serum ferritin and the intracellular ferritin, the serum ferritin was shown to be tissue-derived through secre- tion. Under normal circumstances, equilibrium is maintained between intracellular and extracellular fer- ritin, but the concentration of ferritin in serum and other biological fluids may rise, depending on the iron status of the body, and under various physiological cir- cumstances [17–19]. In a couple of studies, Ramm et al. [20,21] showed that the administration of the microtubule depolymerizing drugs colchicine and vin- blastine, in normal and iron-loaded rats, inhibited fer- ritin uptake and significantly increased the release of endogenous ferritin in both the serum and bile, sug- gesting that disturbed microtubule function could account for these results. These findings also agree with the fluorescent microscopic observations of Kat- suki et al. [8], who found that, when MDBK and 3Y1 fibroblast cells were treated with the microtubular inhibitor nocodazole before staining with anti-tubulin mAb and anti-(250 kDa protein ⁄ ferritin) Ig, the ferri- tin that was once associated with the microtubule net- work to some extent disappeared from the cytoplasm and accumulated towards the periphery of the cells. Based on these facts, we presume that the interaction between ferritin and microtubules and its possible rela- tionship with microtubule dynamics might be import- ant in the regulation of ferritin release under different physiological conditions. On the other hand, the microtubule-related punctate structures that gathered around the remaining microtubules after nocodazole treatment [8] might represent the essential intracellular pool of ferritin, which remained in the cytoplasm by forming large intermolecular complexes and interacting with microtubules. Further studies are required to reveal the detailed in vivo role of ferritin binding with microtubules. Experimental procedures Chemicals and protein preparations Taxol was a generous gift from N Lomax (Division of Can- cer Treatment, National Cancer Institute, Bethesda, MD, USA). Other reagents used in the study were of reagent grade, unless otherwise mentioned. The 250 kDa protein was purified from bovine adrenal cortex, according to Katsuki et al. [8]. Preparation of ferr- itin from bovine liver and bovine adrenal cortex was carried out by following the method described by Ishitani et al. [22]. Tubulin was purified by phosphocellulose column chromatography, from a twice-cycled porcine brain micro- tubule protein fraction, as described previously [23,24]. After the collection of tubulin fractions, the column bound proteins were eluted by 20 MEM buffer [20 mm Mes pH 6.8, 0.1 mm EGTA, and 0.5 mm MgCl 2 ] containing 0.8 m KCl. The peak fractions were combined, concentra- ted and dialyzed for subsequent use as the total MAP fraction. Amino acid sequence analysis The purified 250 kDa protein was digested with cyanogen bromide in 70% (v ⁄ v) formic acid for 24 h at room tem- perature. The digested products were separated by SDS ⁄ PAGE and transferred to a poly(vinylidene difluoride) (PVDF) membrane (Millipore, Bedford, MA, USA) in transfer buffer [100 m m Tris ⁄ HCl, 192 mm glycine, 20% (v ⁄ v) methanol, 0.05% SDS, pH 8.3], using an electroblot- ting system (ATTO, Tokyo, Japan) at 2 mAÆcm )2 for 90 min. The membrane was stained with 0.1% (w ⁄ v) Ponc- eau-3R. Three distinct bands were selected, and excised for sequencing. Sequencing was performed by automated Edman degradation in a PROCISE TM protein sequencer (Applied Biosystems, Foster City, CA, USA). Immunoblotting After SDS⁄ PAGE, the proteins were transferred to a PVDF membrane, as described above, which was blocked in blocking buffer [10 mm Tris ⁄ HCl, 100 mm NaCl, 0.1% (v ⁄ v) Tween 20, 5% (w ⁄ v) skimmed milk, pH 7.5] for 1 h at room temperature. The blot was then incubated over- night with either an anti-(250 kDa protein) Ig [8] or an anti-(horse spleen ferritin) IgG (Sigma-Aldrich Japan K.K., Tokyo, Japan) in blocking buffer at 4 °C, washed with wash buffer [10 mm Tris ⁄ HCl, 100 mm NaCl, 0.1% (v ⁄ v) Tween 20, pH 7.5], and incubated with a horseradish per- oxidase-conjugated anti-(rabbit IgG) IgG (Sigma-Aldrich M. R. Hasan et al. Identification of a putative MAP as ferritin FEBS Journal 272 (2005) 822–831 ª 2005 FEBS 829 Japan K.K.) for 1 h at room temperature. The membrane was washed, and the bound antibodies were detected by a staining solution [0.01% (w ⁄ v) O-dianisidine, 0.03% (w ⁄ v) 4-chloro-1-napthol, 0.01% (v ⁄ v) H 2 O 2 ,50mm sodium acet- ate buffer, pH 5.5]. Detection of iron in the 250 kDa protein The presence of iron in the 250 kDa protein was detected by adding potassium ferrocyanide, to a final concentration of 10 mm, to all fractions obtained from the gel filtra- tion column chromatography, performed as described by Katsuki et al. [8]. Fractions that turned blue were consid- ered to be positive for the presence of iron. Iron uptake assay Iron uptake reactions were carried out in 20 MEM buffer, with 1.5 mm Fe 2+ (ferrous sulfate) and 10 nm protein con- centrations. Iron uptake by the 250 kDa protein and ferritin was monitored by measuring the increase in absorbance at 310 nm at room temperature in a UV spectrophotometer (U 2000, Hitachi, Tokyo, Japan) for up to 10 min. Microtubule-binding assay The microtubule-binding assay was carried out in 100 lL reaction mixtures by adding horse spleen ferritin (5 lm; Sigma-Aldrich Japan K.K.) to tubulin (15 lm) in reassem- bly buffer (RB: 100 mm Mes, 0.1 mm EGTA, 0.5 mm MgCl 2 , pH 6.8) containing 30 l m taxol and 0.5 mm GTP. The mixture was then incubated at 37 °C for 30 min and was centrifuged at 16 000 g for 30 min. The pellet was resuspended in the same volume of RB, and both the super- natant and the pellet were analyzed by electrophoresis on a 10% SDS ⁄ polyacrylamide gel. Control experiments were performed in the same way, except that either ferritin or tubulin was excluded from the preparation. The binding of ferritin with microtubules in the presence of the total MAP fraction was also assayed under the same conditions, except that the total MAP fraction was added to the reaction mix- tures at concentrations of 0, 2 and 6 mgÆmL )1 . The con- tents of the microtubule pellets were then analyzed by 7.5% SDS ⁄ PAGE. Electron microscopy Protein samples were mounted on carbon coated grids (JEOL substrated grids), fixed with 2.5% glutaraldehyde, and negat- ively stained with 2% uranyl acetate. Microtubule containing samples were prepared by adding purified tubulin (15 lm)to RB containing 20 lm taxol and 0.5 mm GTP. The prepar- ation was incubated at 37 °C for 10 min, and ferritin was added to a final concentration of 10 nm. The mixture was further incubated at 37 °C for 10 min, and centrifuged at 12 000 g for 5 min at 37 °C. The pellet was dissolved in the same buffer and incubated for 10 min at 37 °C before fix- ation and staining. Observations were made with a Hitachi EF-2000 electron microscope operating at 200 kV. Miscellaneous SDS ⁄ PAGE was carried out as described by Laemmli [25]. Polymeric 250 kDa protein and ferritin were loaded onto the gel without heat treatment. To obtain the monomeric forms, samples were heated at 100 °C for 5 min before loading. Protein concentrations were determined by the conventional Lowry method [26], using bovine serum albu- min as the standard. Acknowledgements We would like to thank Dr T. Yasunaga for advice about electron microscopy. We are grateful to T. Koga, K. Miyoshi and H. Fujita for generous technical assist- ance. Thanks are also due to Dr B. Guthrie (SKYBAY Scientific Editing) for reading the manuscript. References 1 Wiche G, Oberkanins C & Himmler A (1991) Molecular structure and function of microtubule associated pro- teins. Int Rev Cytol 124, 217–273. 2 Alberts B, Bray D, Lewis J, Raff M, Roberts K & Wat- son JD (2000) Molecular Biology of the Cell, 3rd edn. Garland Publishing Inc., New York. 3 Knops J, Kosik KS, Lee G, Pardee JD, Cohen-Gould L & McConlogue L (1991) Overexpression of tau in a non-neuronal cell induces long cellular processes. 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