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Originally published In Press as doi:10.1074/jbc.M210050200 on April 15, 2003

J. Biol. Chem., Vol. 278, Issue 26, 23963-23970, June 27, 2003
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Ferritoid, a Tissue-specific Nuclear Transport Protein for Ferritin in Corneal Epithelial Cells*

John M. Millholland {ddagger}, John M. Fitch, Cindy X. Cai, Eileen P. Gibney, Kelly E. Beazley and Thomas F. Linsenmayer §

From the Department of Anatomy and Cellular Biology, Tufts University Medical School, Boston, Massachusetts 02111

Received for publication, October 1, 2002 , and in revised form, April 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of ~50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
UV light constitutes a major environmental insult to all exposed tissues of the body, damaging a wide variety of macromolecular components ranging from DNA to proteins and lipids (1, 2). This damage can be direct, or it can be indirect through the generation of reactive oxygen species. The resulting damage to DNA is thought to be a major factor in epidermal cancers (3). However, corneal epithelial (CE)1 cells seem to be refractory to such damage. Although these cells are constantly exposed to UV light and to other reactive oxygen species-generating sources such as molecular O2, primary cancers of these cells are rare (4). This suggests that CE cells have defense mechanisms that prevent damage to their DNA. Our previous studies (5) suggest that one of these mechanisms involves ferritin, a molecule known to decrease the deleterious effects of reactive oxygen species by sequestering iron (6). This sequestration minimizes the Fenton reaction-mediated conversion of hydrogen peroxide to hydroxy radicals that are capable of damaging DNA (7, 8) and other cellular components (9), with the damage to DNA generating at least 50 degradation products (10, 11).

In most cell types, ferritin is cytoplasmic. However, we (12) have observed that in avian CE cells, but in no other cell type examined, ferritin is predominantly, if not exclusively, nuclear. In this location, it seems to be especially effective in diminishing damage by UV irradiation to DNA (5). Ferritin is a multimeric protein comprised of 24 subunits that undergo supramolecular assembly to form the complex (13). In mammals, this complex contains both H- and L-chain subunits (14). However, in chicken, the H-chain (which we will refer to as the ferritin monomer or subunit) is the only one that we and others (15, 16) have detected, suggesting that chicken ferritin is homopolymeric. The monomer is comprised of five {alpha}-helical regions (A–E) (17) that are involved in the supramolecular assembly of the complex and contribute to its functional characteristics, such as the ability to sequester iron (18, 19).

In the present study, we have examined how this normally cytoplasmic molecule undergoes nuclear transport in a tissue-specific manner. Proteins pass into the nucleus through the nuclear pore complex with most small ones entering by free diffusion (20) and large ones employing a nuclear localization signal (NLS)-dependent mechanism. The best characterized NLS is the "basic type" (21), which consists of one or two clusters of basic amino acids. However, other NLSs also exist, such as the M9 signal (22, 23). Ferritin, however, contains neither of these NLS, nor, if present, would they provide the tissue specificity required to restrict transport to CE cells. Also, our recent transfection analyses with deletion constructs for ferritin (24) failed to detect any discrete region that might function as a non-consensus NLS. Instead, the results suggested that, for nuclear transport to occur, most (>85%) of the monomer must be present.

That much of the molecular structure of ferritin is required for nuclear transport suggests that some aspect(s) of molecular conformation is involved, possibly through binding and/or recognition by another cellular component, such as a tissue-specific nuclear transporter. In the present study, we present evidence for the involvement of such a molecule (which we have termed ferritoid) that: 1) is expressed only in CE cells, 2) has one domain with a functional SV40-type NLS and another (likely to be involved in binding to ferritin) that is structurally similar to ferritin, and 3) can facilitate the transport of ferritin into the nucleus.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
White Leghorn eggs were obtained from Charles River/SPAFAS (North Franklin, CT). Chemicals were purchased from Fisher Biochemicals unless otherwise noted. Oligonucleotides were synthesized by IDT (Coralville, IA), and sequencing was performed by the Tufts University Core Facility.

Subtractive Hybridization and Library Screening—To isolate cDNA clones for genes up-regulated in cornea, subtractive hybridization (25, 26) was performed using cDNA from 12-day cornea versus sclera and eyelid. Clones were screened by colony PCR, sequenced, and analyzed by BLAST (27) search of GenBankTM. One clone, 12C1-A2, which had a ferritin-like amino acid composition, was used to screen a CE cDNA library (12), and positive colonies were sequenced. One (6–309) was characterized as being full-length for the coding region (see "Results").

Sequence Analysis—BLAST (27) sequence homology analysis of clone 6–309 was performed using the GenBankTM non-redundant protein and nucleotide databases. The theoretical open reading frame was determined with the Map program of the GCG Wisconsin software package, version 10.1 (Genetics Computer Group). PredictProtein (28) and 123D+ (29) were used to determine secondary structure and features, and PSORT II (30) was used to detect potential cellular sorting signals. Comparisons of three-dimensional structures were made using the Cn3D 3.0 program, with the crystal structure of human ferritin-H (Protein Data Bank code, 2FHA [PDB] (31)) as a template.

Northern Blot Analysis—Tissues were dissected from chick embryos, rinsed in diethylpyrocarbonate-treated PBS, and stored at –20 °C in RNALater (Ambion). Total RNA was isolated using the RNEasy Maxi kit (Qiagen). 10 µg of RNA from each tissue was electrophoresed for 3 h through a 1.2% denaturing formaldehyde agarose gel; RNA was transferred to a Hybond-N+ nylon membrane (Amersham Biosciences) and immobilized by UV cross-linking. Ferritoid clone 6–309 was labeled with [32P]dCTP (ICN) using the High Prime DNA labeling system (Roche Diagnostics), and hybridization was carried out according to Ausubel et al. (32). The integrity of the RNA used in the Northern blot was assessed by visualizing the 28 S ribosomal RNA with ethidium bromide.

In Situ Hybridization—In situ hybridization was performed (33) on frozen tissue sections (14 µm) of O.C.T. embedded anterior eyes (including cornea, sclera, and retina from 14-day embryos). The sections were thawed and fixed in 3.2% paraformaldehyde/PBS followed by dehydration and permeabilization in ethanol. Digoxigenin-labeled RNA probes were made from clone 12C1-A2 and others (see below) using a Genius 3 RNA labeling kit (Roche Biochemicals). Tissue was hybridized for 16 h at 52.5 °C in 50% deionized formamide, 10% dextran sulfate, 4x SSC, 1x Denhardt's solution (Sigma), 1 mg/ml Escherichia coli tRNA, and 10 mM dithiothreitol with the labeled antisense 12C1-A2 RNA probe (100–200 µg/ml). Also used were the sense probe (a negative control), and as a positive control, a probe for type I collagen, which was a 330-nucleotide fragment corresponding to positions 45–374 of the chicken pro {alpha}-2 chain of type I collagen (GenBankTM accession number X02657 [GenBank] ). Following hybridization, the sections were treated with RNase A (8.25 µg/ml) and were washed sequentially in 2x SSC, 1x SSC, and 0.5x SSC (15 min each at room temperature) followed by 0.1x SSC (55 °C for 30 min). Then the sections were blocked in 0.05% Triton X-100, 2% sheep serum (Sigma), 2x SSC (2 h), washed twice in 100 mM Tris-HCl, pH 7.5, and 150 mM NaCl (5 min each), and incubated overnight in a 1:1000 dilution of a mouse anti-digoxigenin alkaline phosphatase-conjugated antibody (Roche Biochemicals). Tissue was equilibrated in GB3 (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2, 1 mg/ml levamisole (Sigma)) and incubated in the chromagen (GB3 plus nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Biochemicals)) at room temperature in the dark until adequate color had developed. The reaction was stopped with 1x TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) (15 min).

Cell Culture, Plasmid Construction, and Transfection—COS-1 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Hyclone) and penicillin/streptomycin (Invitrogen). Primary cultures of chicken CE cells were initiated as described previously (12) and cultured in Dulbecco's modified Eagle's medium:F12 (1:1) (Invitrogen) plus 10% heat-inactivated fetal calf serum, 5 µg/ml insulin (Sigma), 5 ng/ml recombinant human epidermal growth factor (Invitrogen), and penicillin/streptomycin.

For transfections, confluent COS-1 cells were passaged from one 10mm plate into four- or eight-chamber culture slides (BD Biosciences); the CE cells were plated onto 100-mm dishes. An expression construct for full-length ferritoid (FTD-FL) was generated by PCR from clone 6–309 using oligonucleotide primers Fdf10 (5'-ACGAGCATGGCCGAGCCGCGC-3') and Fdr10 (5'-AAGTACCTCCCTCCCCTGTT-3'). A ferritoid construct from which the putative NLS was deleted (FTDNLS–) was similarly generated using primers Fdf11 (5'-ACGAGCATGGCCGACGTCACGCTG-3') and Fdr10. PCR products were ligated into the eukaryotic expression vector pCDNA3.1 TOPO V5-His (Invitrogen). The inserts were confirmed by sequencing, and plasmid preparations were made using the Endo-free Plasmid Maxi Prep kit (Qiagen). COS-1 cells were transfected (using LipofectAMINE Plus (Invitrogen)) with the ferritoid constructs, either singly or in co-transfections with a c-Myc-tagged construct for full-length chicken ferritin-H (construct HM) (12). For single transfections in four-chamber slides, 1.0 µg of plasmid was transfected per chamber; for co-transfections, 1.0 µg of the ferritoid plasmid plus 0.5 µg of the ferritin plasmid were used. For transfections in eight-chamber slides, the quantities of DNA were halved. 20–24 h after transfection, the cells were fixed and processed for immunofluorescence. Primary cultures of CE cells were transfected with 8 µg of each plasmid using FuGENE 6 (Roche Biochemicals), and 48 h later, these cultures were harvested for immunoprecipitation.

Immunofluorescence Analyses—Immunofluorescence was performed as described previously (5). The transfected cells were washed twice with cold PBS, fixed in 4% paraformaldehyde (10 min at 4 °C), washed again in PBS, and permeabilized with 100% methanol (10 min at –20 °C). Cells were washed again with PBS, and free aldehydes were quenched (50 mM lysine, 50 mM glycine in PBS; 10 min) followed by blocking with 1% bovine serum albumin (10 min). Cells were then incubated in primary antibody overnight at 4 °C, washed in PBS, and incubated in secondary antibody for 1.5 h at room temperature. For double labeling, isotype-specific secondary antibodies were used. Cells were then washed (3 x 5 min) in PBS and mounted in 95% glycerol/PBS containing 1 µg/ml Hoechst 33258 (Sigma). The cells were visualized using epifluorescence on a Nikon Fluophot microscope and photographed with a SPOT room temperature real-time CCD camera (Diagnostic Instruments, Inc.).

The primary monoclonal antibodies used were: 6D11 against chick ferritin (34), 9E10 against the c-Myc epitope tag (35) located at the carboxyl terminus of the ferritin construct, and anti-V5 (Invitrogen) against the V5 epitope tag of the ferritoid construct. The secondary antibody used for single labeling immunolabeling was rhodamine-conjugated goat anti-mouse IgG (Pierce); for double immunolabeling, the antibodies used were goat anti-mouse IgG1 (TRITC-labeled) for ferritin and goat anti-mouse IgG2a (fluorescein isothiocyanate-labeled) for ferritoid (Southern Biotechnology Associates).

Immunoprecipitation and Western Blotting—Transfected CE cells (100-mm dishes) were lysed for 10 min in radioimmune precipitation buffer (10 mM Tris, pH 7.4, 300 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100) containing EDTA-free Complete protease inhibitors (Roche Biochemicals) followed by scraping and gentle pipetting. Cell debris was removed by centrifugation (10 min at 14,400 RPM), and the supernatant was stored at –80 °C. To prevent non-specific binding, lysates were incubated with 1.0 µg of mouse IgG and 20 µl of Protein G plus/Protein A-Agarose (Oncogene) (30 min, 4 °C) with gentle rocking followed by centrifugation (2500 rpm, 5 min). The supernatant was incubated with primary antibody (100 µl of hybridoma supernatant for 6D11 and AC9 (anti-chick collagen X) monoclonal antibodies and 4 µlof anti-V5 antibody (Invitrogen)) (1 h, 4 °C with rocking). Then 20 µl of Protein G plus/Protein A-Agarose was added and incubated overnight at 4 °C with rocking. The immunoprecipitates were collected by centrifugation (2500 RPM for 5 min at 4 °C), washed four times in PBS, and resuspended in 40 µl of 2x electrophoresis sample buffer (32). Samples were heated (95 °C, 5 min), spun briefly, and electrophoresed (15% SDS-PAGE).

Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride membranes (Bio-Rad). Western blots were performed using the following protocol: after brief treatment with 100% methanol, the polyvinylidene difluoride membranes were treated with blocking solution (10% non-fat dry milk in Tris-buffered saline (TBS)) for 1 h at room temperature with shaking. Primary antibody was diluted in blocking solution (1:5000 for anti-V5, 1:2 for 9E10 and AC9) and incubated for 1.5 h at room temperature with shaking. Membranes were then washed (5 x 10 min) with TTBS (TBS plus 0.5% Tween 20) to remove unbound antibody. Secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce)) was diluted 1:20,000 in blocking solution and placed onto membranes for 30 min at room temperature. Membranes were washed again in TTBS as above and incubated in chemiluminescence substrate (PerkinElmer Life Sciences) for 2 min prior to exposure to photographic film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of a Ferritoid cDNA and Tissue Specificity—Ferritoid was initially identified as an up-regulated, 300-bp clone in a subtracted 12-day embryonic chicken corneal cDNA library. In analysis of the up-regulated clones by DNA sequencing and computer BLAST searches (27), one clone (12C1-A2) contained an insert that, at the amino acid level, showed similarity to ferritin. Since ferritin monomers themselves undergo association during self-assembly of the ferritin complex (36, 37), we considered the possibility that this clone might represent the ferritin-binding region of a nuclear transporter for ferritin in CE cells, as hypothesized in our previous studies (24).

If so, the encoded molecule should be specific for the CE or at least highly selective for this tissue. This was examined and confirmed by in situ hybridization and by Northern blotting. In situ hybridization of sections of entire anterior eyes (14-day embryos) localized ferritoid to the CE (Fig. 1A). (The dark linear structure present in all panels of Fig. 1 is not an in situ signal but the pigment of the pigmented epithelium (PE) of the iris (i) and ciliary body (cb) indicated in Fig. 1B.) The in situ signal for ferritoid ended at the corneal-scleral junction (designated by the asterisks) and was not detected in the numerous tissues/cell types present in the other structures present, which, as shown in the corresponding phase contrast micrograph (Fig. 1B), included the corneal stroma (cs), ciliary body (cb), and iris (i), among others. The specificity of the in situ hybridization was further confirmed by the extensive reaction observed with a positive control probe for the pro {alpha}2 chain of type I collagen (Fig. 1C), which is expressed in both mesenchymal and epithelial cells of ocular and non-ocular tissues and by the absence of any reaction with a negative control probe (the sense strand for ferritoid, Fig. 1D). Consistent with this specificity, Northern blots (Fig. 2) of total RNA from embryonic cornea (lane 1), brain, heart, liver, and skin (lanes 2–5), when probed with radiolabeled ferritoid cDNA, produced a signal of ~1.6 kb only with the RNA from cornea.



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  		FIG. 1.
In situ hybridization of day 14 anterior eye probed with digoxigenin-labeled RNA antisense probes for: ferritoid (A) and a positive control (C), the pro {alpha}2 chain of type I collagen. D, a negative control, is probed with the sense strand for ferritoid. B is a phase contrast micrograph of the same section shown in A. In A, ce denotes the corneal epithelium, and the asterisks demarcate the corneal-scleral junction. In B, additional labels identify the corneal stroma (cs), iris (i), and ciliary body (cb). The dark linear structure in each panel is the pigmented epithelium associated with the iris and ciliary body. All panels are the same magnification, as shown by the bar in D (500 µm).

 


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  		FIG. 2.
A Northern blot of total RNA isolated from embryonic chick tissues (all day 14 except skin, day 8) probed with radiolabeled ferritoid cDNA. Lanes are as follows: 1, cornea; 2, brain; 3, heart; 4, liver; 5, skin. The positions of the molecular size markers are shown along the left margin, and the integrity of the RNAs is shown by the presence of 28 S ribosomal RNA in ethidium bromide-stained samples (bottom panel).

 

Isolation of a Full-length Coding cDNA and Structural Analysis—To obtain a larger cDNA for ferritoid, clone 12C1-A2 (Fig. 3, single overline) was used to screen a chicken CE library (12). This resulted in several clones, with the largest (1175 bp) having characteristics suggesting that it contains a full-length open reading frame. The 5'-end of the open reading frame contains a consensus sequence for a translation start site (Fig. 3, double overline), which is in-frame with an uninterrupted coding region of 830 bp, terminating at the 3'-end with a polyadenylation signal (Fig. 3, double underline) and a poly-A tail. The conceptual translation product contains two regions that fit with its proposed role as the nuclear transporter for ferritin. The first region contains a consensus SV40-like NLS (Fig. 3, light gray). The second region, located downstream of the NLS, is ferritin-like (Fig. 3, dark gray) and thus could be involved in the interaction with/binding to ferritin. This region: 1) is overall 55% similar to ferritin; 2) contains short sequences of similarity to ferritin (Fig. 4, yellow); and 3) is similar in size and structure to ferritin (described below). Also present are several potential phosphorylation sites (single underline).



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  		FIG. 3.
Nucleotide sequence and conceptual translation product of ferritoid clone 6–309. The NLS sequence (PRSKRPR) is blocked in light gray, and the ferritin-like region is in dark gray. Also designated in the amino acid sequence are potential phosphorylation sites (single underline). Designated in the nucleotide sequence are the initiator methionine (double overline), the stop codon (box), the polyadenylation signal (double underline) and the original clone 12C1-A2 (single overline). These sequence data are available from GenBankTM/EMBL/DDBJ under accession number AF447376 [GenBank] .

 


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  		FIG. 4.
A comparison of the amino acid sequence of FTD and chicken FTN. Areas of similarity are highlighted in yellow. Amino acid similarities are shown between the two sequences with identity denoted by the amino acid and conservative substitutions with a plus sign. The amino acids shown in green are those conserved in ferritoid that have been shown previously by Santambrogio et al. (37) to be involved in the ferritin/ferritin interactions that occur during assembly of the ferritin supramolecular complex. The amino acid shown in pink has also been shown previously (37) to be involved in ferritin/ferritin interactions but is not conserved between ferritin and ferritoid. The helical regions in ferritin (30) are shown as bracketed regions designated A–E. The predicted helical regions in ferritoid are shown as bracketed regions designated A'–E'.

 

Ferritoid structure was analyzed further using computer programs that predict secondary and tertiary structures. The secondary structure of ferritoid, determined using the 123D+ program (29), showed the ferritin-like region to have structural similarities to the H-chain of ferritin itself, the chief one being the presence of the five helical domains (Fig. 4, labeled A'–E'). When the helical domains of ferritoid were aligned with those of ferritin (using the PredictProtein program (28)), the only difference was helix D, which in the ferritin-H chain is a single domain but in ferritoid is predicted to be divided into two (in Fig. 4, designated D1' and D2').

As mentioned above, a characteristic that might be expected for a molecule that assists in the nuclear transport of another molecule is an interaction between the two. Consistent with this, the conserved amino acids in the helical regions of ferritoid and ferritin include 9 (Fig. 4, green) of the 10 amino acids that have been demonstrated previously by mutation analysis (37) to be involved in certain of the interactions between ferritin subunits during assembly of the supramolecular ferritin complex.

Other structural evidence for such an interaction between ferritoid and ferritin comes from a three-dimensional analysis in which we inserted the sequence of ferritoid into the known crystal structure of the human ferritin-H monomer (using the Cn3D 3.0 program). The results suggest that the short ferritin-like sequences in ferritoid (Fig. 5, shown in red) are not distributed randomly along the helices but are located preferentially at the surface, again where they may interact with/bind to ferritin.



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  		FIG. 5.
Conservation of key amino acids between ferritin and ferritoid. In this figure, the amino acid sequence of ferritoid is superimposed on four different views of the three-dimensional crystal structure of human ferritin-H (GenBankTM 2FHA [PDB] (31)) using the program Cn3D 3.0. Conserved amino acids are colored red, and non-conserved amino acids are colored blue. Note that many of the conserved amino acids in ferritoid are located along the outer faces of {alpha}-helices. For reference points, the amino-(arrows) and carboxyl-terminal (arrowheads) ends of the protein are depicted.

 

Ferritoid/Ferritin Interaction—To test experimentally whether ferritoid and ferritin associate with one another, coimmunoprecipitations were employed to determine whether an antibody against one component, either anti-ferritoid or anti-ferritin, would co-precipitate the other component. For this, CE cells were co-transfected with constructs for both components, and then lysates of transfected cells were co-immunoprecipitated and subsequently analyzed for ferritoid and ferritin by immunoblotting. Since no antibody currently exists against ferritoid, the CE cells were transfected with V5-tagged ferritoid, which allowed for the use of an antibody against the V5 tag for subsequent immunoprecipitations and immunoblotting. In addition, to ensure that only newly synthesized proteins from the transfected constructs were analyzed, a c-Myc-tagged ferritin construct was employed that allowed the ferritin component to be detected in immunoblots with an antibody against the Myc epitope (9E10). The immunoprecipitations were performed either with the anti-V5 antibody for ferritoid or with the anti-chicken ferritin-H antibody (6D11) for ferritin because in our hands, the anti-Myc antibody (9E10) was not able to coimmunoprecipitate tagged ferritin and ferritoid (see ``Discussion''). Subsequent identification of the transfected products by Western blotting was performed with the antibody against the V5 tag for ferritoid and the antibody against the Myc tag for ferritin.

The results of these analyses are shown in Fig. 6. As a positive control, cultures were singly transfected with the constructs for either V5-tagged ferritoid (FTD) or Myc-tagged ferritin (FTN). When extracts of these cultures were immunoprecipitated with antibodies against either ferritoid (IP: {alpha}-V5 (FTD)) or ferritin (IP: {alpha}-FTN (6D11)) and subsequently analyzed by Western blotting with antibodies against ferritoid (WB: {alpha}-V5 (FTD)) or ferritin (WB: {alpha}-Myc (FTN)), in each case, the only detectable band was for the component that had been transfected. When, however, the lysates of cultures co-transfected with both ferritoid and ferritin (FTD/FTN) were immunoprecipitated with either antibody (IP: {alpha}-V5 (FTD) or IP: {alpha}-FTN (6D11)), the Western blots showed bands for both ferritoid (WB: {alpha}-V5 (FTD)) and ferritin (WB: {alpha}-Myc (FTN)). Thus, ferritoid and ferritin associate with one another with an affinity strong enough to withstand immunoprecipitation. Negative controls, which by Western blotting failed to show bands for either ferritin or ferritoid, included lysates of cells that: 1) had been singly transfected with one component (FTD) and immunoprecipitated with an antibody against the other component (IP: {alpha}-FTN (6D11)); 2) were immunoprecipitated with an unrelated antibody (anti-type X collagen) (data not shown); and 3) had been transfected without DNA (MOCK) and immunoprecipitated with antibodies against either ferritoid (IP: {alpha}-V5 (FTD)) or ferritin (IP: {alpha}-FTN (6D11)).



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  		FIG. 6.
Co-immunoprecipitation of ferritin and ferritoid. CE cells were transiently transfected with no plasmid (MOCK) as a control, V5-tagged ferritoid alone (FTD), Myc-tagged ferritin alone (FTN), or both plasmids (FTD/FTN). Transfected cell lysates were then immunoprecipitated (IP) with the {alpha}-v5 antibody against ferritoid ({alpha}-V5 (FTD)) or with the 6D11 antibody against chicken ferritin ({alpha}-FTN (6D11)). Immunoprecipitates were then separated on a 15% SDS-PAGE gel, transferred to polyvinylidene difluoride membrane, and detected by WB with either {alpha}-V5 (FTD) or {alpha}-Myc (FTN) antibodies.

 

Ferritoid-mediated Transport of Ferritin into the Nucleus— To test directly the ability of ferritoid to transport ferritin into the nucleus, we performed immunofluorescence on cells transfected with ferritoid and ferritin. For these experiments, we employed COS-1 cells, which is a cell type that has been used by others in transfection studies of ferritin (38) and which we found has a much higher transfection efficiency than the corneal fibroblasts we employed in our previous studies (12). In describing these studies, the terms ``nuclear ferritin'' and ``nuclear ferritoid'' refer to cells in which the localization of the transfected gene product(s) is predominantly or exclusively nuclear, and the term ``uniform distribution'' refers to cells in which the signal is distributed throughout the cytoplasm and nucleus. Both single transfections and co-transfections were performed. The data from these are summarized in Table I, but the actual fluorescence micrographs are shown only for the co-transfections (Fig. 7).


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  		TABLE I
Summary of ferritoid/ferritin transfections in COS-1 cells

 


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  		FIG. 7.
Immunofluorescence micrographs of COS-1 cells cotransfected with a full-length construct for Myc-tagged ferritin and a V5-tagged construct for ferritoid that is either full-length (NLS +) (frames A–C) or one from which the NLS has been deleted (NLS–) (frames D–F). Ferritin was detected with a monoclonal antibody ({alpha}-Ferritin) against its Myc tag and visualized with a TRITC-conjugated, isotype-specific secondary antibody ({alpha}-IgG1). Ferritoid was detected with a monoclonal antibody ({alpha}-Ferritoid) against its V5 tag and visualized by a fluorescein isothiocyanate-conjugated, isotype-specific secondary antibody ({alpha}-IgG2a). For visualization of all nuclei, the cells were also stained with Hoechst 33258 (C and F). In D–F, the position of the nucleus is shown with an asterisk. C and F show small flecks of Hoechst-stained material that are found scattered throughout the culture, most likely representing residue from the transfected LipofectAMINE/DNA complex. All panels are the same magnification as shown by the bar in F (10 µm).

 

Preliminary studies employing single transfections were performed to verify that: 1) the NLS of ferritoid is functional and 2) ferritin itself has no inherent tendency to accumulate in the nucleus of COS-1 cells. To examine whether the NLS is functional, cells were singly transfected with ferritoid constructs, either full-length for the coding region (FTD-FL) or from which the NLS had been removed (FTDNLS–). For subsequent immunofluorescence analyses, both constructs contained a V5 epitope tag, and Hoechst dye was included (in the mounting medium) to delineate nuclei. As summarized in Table I, transfections with the FTD-FL construct showed nuclear ferritoid in most of the transfected cells (73.5%); in the remaining cells (26.5%), the ferritoid was uniformly distributed throughout the cell. Conversely, in the cultures transfected with the FTDNLS– construct, none of the transfected cells showed nuclear ferritoid (i.e. in 100% of the cells, the distribution was uniform). In cultures transfected with a full-length construct for ferritin itself (FTN) (12), 100% of the transfected cells had ferritin with a uniform distribution; thus, the molecule has no inherent tendency for nuclear localization.

To examine whether ferritoid is capable of transporting ferritin into the nucleus, co-transfections were performed with full-length constructs for V5-tagged ferritoid and Myc-tagged ferritin. IgG isotype-specific secondary antibodies were used to discriminate between the V5 and Myc tags on the two constructs. (Ferritin was also detected using the anti-ferritin antibody 6D11. This is specific for chicken and gave results identical to those with the Myc tag.) When the construct for ferritin was co-transfected along with the full-length construct for ferritoid (Table I, FTD/FTN), in 44.7% of co-transfected cells, both the ferritin and ferritoid were nuclear (Table I, N/N), as is also shown in Fig. 7 (A and B, NLS +). 34.9% of the cells had nuclear ferritoid with the ferritin uniformly distributed throughout the cell (Table I, N/U), and the remaining 20.4% showed uniform distributions of both the ferritin and ferritoid (Table I, U/U). Most importantly, all cells with nuclear ferritin also had nuclear ferritoid.

However, when the co-transfections were performed using the ferritoid construct from which the NLS had been removed (Fig. 7, NLS–; Table I, FTDNLS–/FTN), none of the cells showed either nuclear ferritin or nuclear ferritoid, as can be seen in the co-transfected cell in Fig. 7, D and E. Instead, in all cells, both components were uniformly distributed throughout. Controls, using singly transfected cells, showed that no cross-reactivity occurred between the different primary or secondary antibodies (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous studies (5, 12) demonstrated that avian CE cells are rich in ferritin and that most, if not all, of this ferritin is nuclear. This nuclear localization of ferritin is highly selective, if not specific, for the CE cell, at least for normal cell types in vivo. A number of lines of evidence suggest that this nuclear ferritin is the same molecule as the ferritin that is cytoplasmic in other cell types. These include (12) sequencing of cDNA clones, which shows identity with chicken reticulocyte ferritin (39); immunoblotting of nuclear and cytoplasmic fractions of CE cells; immunofluorescence with a number of monoclonal and polyclonal antibodies against the H-chain of ferritin; and electrophoretic behavior of the supramolecular complex under non-denaturing conditions. The only other reports of nuclear ferritin have been in pathological conditions and in K562 cells, in which a ferritin-like protein has been suggested to function in gene regulation (42, 43). Thus, to our knowledge, the CE-nuclear ferritin is the only example in normal cells in which a cytoplasmic molecule undergoes nuclear transport in a tissue-specific and essentially quantitative manner.

Ferritoid as the Nuclear Transport Protein—In the present study, the data suggest that ferritoid has the properties predicted for the nuclear transporter of ferritin in CE cells, including tissue specificity, binding to ferritin, and the ability to transport ferritin into the nucleus. By in situ hybridization on sections of anterior eyes, ferritoid was localized to the CE and was not detected in any of the other large number of epithelial, stromal, vascular, and nervous cell/tissue types present. Consistent with this, Northern blotting showed ferritoid in the cornea but not in any of the other tissues examined.

The ability of ferritoid to facilitate the transport of ferritin into the nucleus was determined by co-transfections with constructs for ferritoid and ferritin into a test cell type, the COS-1 cell. (Similar results were observed with CE cells and corneal stromal cells, but these had lower transfection efficiencies.)

In control, single transfections with ferritin alone, the transfected cells had ferritin distributed throughout the cytoplasm and nucleus, confirming that ferritin has no inherent tendency to undergo nuclear transport or to preferentially accumulate within the nucleus by other mechanisms such as passive diffusion followed by nuclear sequestration. Conversely, control transfections with ferritoid alone showed the majority of the transfected cells (74%) to have the molecule predominantly, if not exclusively, nuclear. The remainder of the cells had the molecule throughout the cell. At present, we have no definitive explanation why, for this latter group of cells, the ferritoid fails to show a preferential nuclear localization. However, two possibilities are (a) that the nuclear transport machinery of some cells has become saturated by the rapid synthesis of protein from the transfected constructs or (b) that as part of the normal transport process, ferritoid undergoes reversible shuttling between the cytoplasm and nucleus, and the rate at which this occurs may differ depending on the physiological state of the cell.

In the co-transfections with full-length constructs for ferritin and ferritoid, a high proportion of the cells expressing both constructs (45%) showed nuclear localization of both the ferritoid and ferritin, strongly supporting the ferritoid-mediated nuclear transport of ferritin. Another 20% of the cells had both ferritoid and ferritin distributed uniformly throughout the cell, and the remaining cells (35%) had nuclear ferritoid, but the ferritin was uniformly distributed throughout the cell (Table I). Again, we have no definitive explanation for the differences observed in these subpopulations of cells, but they may be attributable, at least in part, to the possibilities mentioned above for the single transfectants. In addition, in the co-transfections, other variables may exist, such as the relative transfection efficiencies of the ferritoid and ferritin constructs and the relative rates at which the constructs synthesize their respective proteins. Empirically, we have observed that the transfection efficiency with ferritin is appreciably higher than that with ferritoid, but we have no information on their relative rates of synthesis once transfection has occurred. Nonetheless, the results show most importantly that no cell had nuclear ferritin, unless the ferritoid was also predominantly in that location.

Mechanism(s) of the Ferritoid-mediated Transport of Ferritin—We do not yet know many of the details by which ferritoid facilitates the nuclear transport of ferritin. However, from the work described here, it seems that: 1) the NLS of ferritoid is involved and 2) at some step in the process, an interaction/binding occurs between ferritoid and ferritin. A requirement for the NLS in transport is shown by the observations that when the NLS of the ferritoid construct is deleted, single transfections with this construct (FTDNLS–) show a loss of the nuclear localization of ferritoid, and co-transfections along with the ferritin construct show a loss of nuclear localization of both components.

In addition, a direct interaction/binding between ferritoid and ferritin is shown by their co-immunoprecipitation with antibodies against either of the individual components. In performing these assays, we observed that the monoclonal antibody against ferritin effected co-immunoprecipitation of the ferritin-ferritoid complex, but the one against the Myc-epitope tag on the ferritin did not. At present, we do not know why this occurs, but at least two possibilities exist. One is that during formation of the ferritoid-ferritin complex, the binding of ferritoid to the Myc-tagged ferritin masks the Myc epitope. The other is that binding of the anti-Myc antibody competes with ferritoid for binding to ferritin, thus dissociating the ferritin/ferritoid complex.

An interaction between ferritoid and ferritin is also indicated in preliminary work employing the yeast two-hybrid system. In these ongoing studies, when ferritoid and ferritin were used as bait and prey, or vice versa, an interaction was detected as evidenced by the plating efficiency of the clones obtained. When ferritin was used as both bait and prey, an even stronger interaction was detected, most likely reflecting the interaction that occurs between ferritin subunits during assembly of the ferritin supramolecular complex.

Although experimental evidence concerning the details of the mechanism(s) through which ferritoid and ferritin bind is not yet available, the structural information on ferritoid suggests that the interaction may occur between its ferritin-like regions and similar regions in ferritin (monomers and/or multimers) in a manner analogous to the interactions that occur during assembly of the supramolecular ferritin complex. Investigations by others (37) on the interactions that occur during ferritin supramolecular assembly have identified amino acid residues that are involved in the interactions between the monomers. In the ferritin-like region of ferritoid, most of these residues are conserved and, as suggested by computer modeling, are likely to be sterically positioned such that they can interact with ferritin subunits.

For the actual nuclear transport, a number of possible scenarios can be envisioned. For example, cytoplasmic ferritoid may bind a ferritin monomer/multimer, with the complex being transported into the nucleus where it subsequently dissociates. Then the ferritoid could be either degraded or recycled back into the cytoplasm, with the ferritin monomers undergoing assembly into the supramolecular complex, the form in which our previous studies suggest that nuclear ferritin exists. In support of this model, our (24) previous transfection analyses with deletion constructs for ferritin suggest that the monomeric subunit of ferritin can undergo nuclear transport. The ferritin deletion construct on which this conclusion is based (missing 30 amino acids from COOH end) is unable to undergo supramolecular assembly, but it can undergo nuclear transport. Although this result suggests that ferritin monomers can be transported into the nucleus, it does not rule out the possibility that multimeric units of ferritin may also undergo transport. It is even possible that the ferritoid within the nucleus remains associated with partially or fully formed ferritin complexes, but this remains to be tested.

In the ferritoid sequence, we have found several consensus serine/threonine phosphorylation sites that may play a role in regulating its behavior (Fig. 3, single underline). Several examples exist for phosphorylation-regulated nuclear transport, at the level of either import or export (reviewed in Ref. 40). In ferritoid, several of these putative phosphorylation sites fall near, or within, the NLS. In Drosophila protein Dorsal, serine phosphorylation sites located near an NLS have been implicated in regulating nuclear transport (41). For ferritoid, it remains to be tested whether phosphorylation/dephosphorylation is involved in modulating its nuclear transport activity.

Lastly, the sequence and structural similarities between ferritoid and ferritin suggest that ferritoid is likely to be a member of the ferritin family of molecules. If so, ferritoid provides an interesting example of a transport molecule that evolved from the molecule (ferritin) for which it facilitates intracellular translocation, in this case into the nucleus.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to GenBankTM/EBI Data Bank with the accession number(s) AF447376 [GenBank] .

* This work was supported by National Institutes of Health Grant EY 13127 (to T. F. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Department of Pathology and Laboratory Medicine, University of Pennsylvania, 421 Curie Blvd., Philadelphia, PA 19104. Back

§ To whom correspondence should be addressed: Dept. of Anatomy and Cellular Biology, Tufts University Medical School, Jaharis 329, 150 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6695; Fax: 617-636-6536; E-mail: thomas.linsenmayer{at}tufts.edu.

1 The abbreviations used are: CE, corneal epithelial; NLS, nuclear localization signal; FTD, ferritoid; FTD-FL, full-length FTD; FTN, ferritin; PBS, phosphate-buffered saline; IP, immunoprecipitate; WB, Western blot; TRITC, tetramethylrhodamine isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Bryan Toole for providing the COS-1 cells.



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