Báo cáo khoa học: Identification of a 250 kDa putative microtubuleassociated protein as bovine ferritin Evidence for a ferritin–microtubule interaction pdf

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

associated protein as bovine ferritin

Evidence for a ferritin–microtubule interaction

Mohammad R Hasan1, Daisuke Morishima1, Kyoko Tomita1, Miho Katsuki1and Susumu Kotani1,2

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.

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 immunoreactivstabil-ity [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 micromicro-tubules

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).

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 proceproce-dures Lanes 1 and 4,

bovine liver ferritin; lanes 2 and 5, bovine adrenal gland ferritin; and

lanes 3 and 6, the 250 kDa protein.

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 m M 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 m M ferrous sulfate + 10 n M BSA).

the absorbance in control 1 is due to the

auto-oxida-tion of ferrous iron upon reacauto-oxida-tion 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 l M ) were mixed in

RB containing 30 l M taxol and 0.5 m M 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.

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.

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 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 l M ferritin in RB with 30 l M taxol

and 0.5 m M GTP), to final concentrations of 0 mgÆmL)1(lane 2),

2 mgÆmL)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.

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 CanCan-cer 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 MgCl2] 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 mm 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 PROCISETM 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

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) H2O2, 50 mm 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 Fe2+(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

MgCl2, pH 6.8) containing 30 lm 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 Observfix-ations 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

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