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HumanmitochondrialtranscriptionfactorApossessesmultiplesubcellulartargetingsignals

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InnaShokolenko

UniversityofSouthAlabama

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BinWang

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MikhailFAlexeyev

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Human mitochondrial transcription factor A possessesmultiple subcellular targeting signalsViktoriya Pastukh1, Inna Shokolenko1, Bin Wang2, Glenn Wilson1 and Mikhail Alexeyev1,3

1 Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL, USA

2 Department of Mathematics and Statistics, University of South Alabama, Mobile, AL, USA

3 Institute of Molecular Biology and Genetics, Kyiv, Ukraine

Mitochondrial transcription factor A (TFAM, mtTFA)

is a member of a high-mobility group (HMG) of pro-

teins named on the basis of their electrophoretic mobil-

ity in polyacrylamide gels. This group is composed of

nonhistone chromatin proteins and transcription factors

that can bind DNA either nonspecifically or in a

sequence-dependent manner [1]. TFAM is encoded

in the nucleus and is synthesized on cytoplasmic

ribosomes as a precursor, which is converted, upon

mitochondrial importation, into a 24.4 kDa, 204 amino

acid mature form. The N-terminal sequence of the pre-

cursor has not been determined, and therefore it is possi-

ble that translation can start on either of two

N-terminal methionines, resulting in either 246 amino

acid (29 kDa) or 240 amino acid (28.4 kDa) precursors

[2]. The mature form contains two HMG boxes,

Keywords

chemotherapy; cisplatin; etoposide;

mitochondrial transcription factor A; nuclear

localization sequence

Correspondence

M. Alexeyev, Department of Cell Biology

and Neuroscience, University of South

Alabama, 307 University Blvd., MSB1201,

Mobile, AL 36688, USA

Fax: +1 251 460 6771

Tel: +1 251 460 6789

E-mail: malexeye@jaguar1.usouthal.edu

(Received 29 July 2007, revised 12 October

2007, accepted 25 October 2007)

doi:10.1111/j.1742-4658.2007.06167.x

The mitochondrial transcription factor A (TFAM) is a member of a high-

mobility group (HMG) family represented mostly by nuclear proteins.

Although nuclear localization of TFAM has been demonstrated in some

tumors and after treatment of tumor cells with anticancer drugs, the signifi-

cance of these observations has not been fully elucidated. Here we report

that both TFAM overexpression and impairment of its mitochondrial tar-

geting can result in nuclear accumulation of the protein. Both M1 and M7

methionines of human TFAM (hTFAM) can be used for translation initia-

tion with almost equal efficiency resulting in two polypeptides. The shorter

polypeptide, however, is not located in the nucleus, despite truncation in

the mitochondrial targeting sequence, and both isoforms are targeted to

mitochondria with similar efficiency. We further demonstrate that nuclear

TFAM confers significant cytoprotection against the chemotherapeutic

drugs etoposide, camptothecin, and cisplatin. Three regions of hTFAM

[HMG-like domain 1 (HMG1) and HMG-like domain 2 (HMG2), as well

as the tail region] can effect nuclear accumulation of enhanced green fluo-

rescent protein (EGFP) fusions. The HMG1 domain contains a bipartite

nuclear localization sequence whose identity is supported by site-directed

mutagenesis. However, this bipartite nuclear localization sequence is weak,

and both N-terminal and C-terminal flanking sequences enhance the

nuclear targeting of EGFP. Finally, several mutations in the HMG1

domain increased the mitochondrial targeting of the EGFP fusions, sug-

gesting that the mitochondrial targeting sequence of hTFAM may extend

beyond the cleavable presequence.

Abbreviations

EGFP, enhanced green fluorescent protein; HMG, high-mobility group; HMG1 and HMG2, HMG-like domains of human mitochondrial

transcription factor A; hTFAM, human mitochondrial transcription factor A; MTS, mitochondrial targeting sequence; NLS, nuclear localization

sequence; N ⁄ C, nucleus-to-cytoplasm; SOD2, manganese superoxide dismutase; Tc, tetracycline; TFAM, mitochondrial transcription

factor A.

6488 FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS

HMG-like domain 1 (HMG1) and HMG-like domain 2

(HMG2) (Fig. 1A), joined by a basic 36 amino acid lin-

ker and followed by a basic 27 amino acid tail. The gene

for TFAM spans about 10 kb and consists of seven ex-

onsandsixintrons[3,4].Inhumanandrat,exon 5cansplice

alternatively, resulting in two TFAM isoforms [4,5].

TFAM is required for mtDNA transcription and

maintenance. Inactivation of both TFAM alleles

results in embryonic lethality accompanied by severe

depletion of mtDNA [6]. Tissue-specific inactivation of

TFAM in cardiomyocytes, skeletal muscle cells, pan-

creatic b-cells and pyramidal neurons is associated

with mtDNA depletion, reduced levels of mitochon-

drial transcripts, and severe respiratory chain defi-

ciency [7–11]. TFAM levels generally correlate well

with mtDNA content, and upon transient depletion of

mtDNA with ethidium bromide, cellular TFAM con-

tent diminishes as well [12]. Conversely, both mtDNA

and TFAM levels are restored upon ethidium bromide

withdrawal, although TFAM appears to lag behind

mtDNA [12].

Like many other members of the HMG family,

TFAM can bind DNA in a nonsequence-specific man-

ner, although it appears to show a higher affinity for

mitochondrial heavy strand promoter and light strand

promoter [13]. TFAM binding to DNA induces

unwinding and bending [14], and the mitochondrial

TFAM content (approximately one TFAM molecule

per 10 bp) has been suggested to be high enough for

TFAM to cover mtDNA completely [15,16]. This,

together with TFAM’s high affinity for DNA contain-

ing cisplatin adducts and 8-oxo-7,8-dihydroguanine

raises the possibility of its involvement in recognition

and ⁄or repair of mtDNA damage [17].

TFAM effects could be modulated by its interaction

with p53 [18] and acetylation [19]. Another interesting

possibility is the regulation of the effects of TFAM by

its subcellular targeting. In the mouse and chicken, but

not in the human, expression of a special nuclear iso-

form of TFAM was demonstrated. This isoform is

generated by alternative splicing of the duplicated first

exons, resulting in a protein that lacks a mitochondrial

A

B

C

D

Fig. 1. Nuclear localization of hTFAM and hTFAM–EGFP fusion pro-

teins upon overexpression. (A) Structure of TFAM. The figure is

drawn to scale. The domain boundaries are in accordance with the

Entrez Protein Database entry Q00059. Numbers indicate the

amino acid position. (B) Structures of constructs 1760 and 2463

encoding constitutively expressed and Tc-inducible hTFAM–EGFP

fusion proteins, respectively. Numbers on the right indicate plasmid

designations. Crossed ATG, deleted or mutated translation initiation

site. (C) Top row: Flp-in T-Rex cells were transiently transfected

with constitutively expressed hTFAM–EGFP fusion construct (con-

struct 1760). Bottom two rows: inducible expression of hTFAM–

EGFP construct in stably transfected (single copy) Flp-in T-Rex

cells. Left images: green, EGFP fusion proteins. Middle images:

red, MitoTracker Red (mitochondrial stain). Right images: overlay;

yellow, regions of colocalization. – Tc and + Tc, cells were either left

uninduced, or induced with 2 lgÆmL)1 Tc for 48 h. (D) Accumula-

tion of hTFAM in the nuclei of transfected cells upon overexpres-

sion. The Flp-in T-Rex293 cells were stably transfected with

construct 2462, which encodes full-length hTFAM. Nuclear frac-

tions (12 lg of total protein) from the parental cell line (T-Rex), un-

induced (2462 unind) and induced (2462 ind) 2462 cell line as well

as the mitochondrial fraction (Mito) from the induced 2462 cell line

were subjected to western blot analysis, using antibodies against

nuclear lamin A ⁄ B (loading control) and SOD2 to verify the purity

of the fractions (top panel), as well as with antibodies against

lamin A ⁄ B (loading control) and antibody to hTFAM to determine

levels of hTFAM in the nuclei (the bottom panel).

V. Pastukh et al. Nuclear localization of hTFAM

FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6489

targeting sequence (MTS). However, the order of

‘nuclear’ and ‘mitochondrial’ exons in genomic DNA

of these species is opposite [20–22]. Nuclear localiza-

tion of TFAM was observed in rat hepatoma, where it

correlates with 10-fold overexpression of this protein

[23]. Also, TFAM was isolated recently from rat liver

nuclei, where it was found to be bound to chromatin

[24]. Presently, both the mechanism(s) and the physio-

logical consequences of the nuclear localization of

TFAM remain unclear. Here we identify the nuclear

localization sequences (NLSs) of human TFAM

(hTFAM), and demonstrate that nuclearly localized

hTFAM can exert significant sensitizing and cytopro-

tective effects in response to chemotherapeutic drugs.

Results and Discussion

HTFAM overexpression results in nuclear

localization of hTFAM–EGFP fusion proteins

As nuclear localization of TFAM correlates with ele-

vated levels of this protein in rat hepatoma cells [23],

we were interested in whether TFAM overexpression,

by itself, is sufficient for the relocalization of a fraction

of this protein to the nucleus. To this end, the con-

struct encoding TFAM–enhanced green fluorescent

protein (EGFP) fusion protein under the control of

the CMV promoter (construct 1760) was assembled

and introduced into HeLa and Flp-in T-Rex293 cell

lines by transient transfection. In both cases, a fraction

of the fusion protein accumulated in both the cyto-

plasm and the nucleus (Figs 1 and 2B). To confirm

that nuclear localization of the fusion protein was

indeed due to overexpression, we stably integrated an

identical fusion construct under the control of a CMV-

tet promoter (construct 2463) into the genome of the

Flp-in T-Rex293 cell line. The Flp-integrase-mediated

insertion occurs in a single defined site in the Flp-in

T-Rex293 genome. Therefore, our stable integrants,

unlike cells that received a similar construct by tran-

sient transfection, contained a single copy of the fusion

construct, and expressed lower levels of the fusion pro-

tein. In agreement with our hypothesis, the lower levels

of expression attained in the Flp-in T-Rex293-2463 cell

Fig. 2. Schematic diagrams (A) and subcellu-

lar localization (B) of hTFAM deletion con-

structs. The constructs were generated by

PCR and transfected using Polyfect reagent

as described in Experimental procedures.

Left images: green, EGFP fusion proteins.

Middle images: red, MitoTracker Red (mito-

chondrial stain). Right images: overlay;

yellow, the regions of colocalization.

EF1-alpha, a construct expressing unfused

EGFP.

Nuclear localization of hTFAM V. Pastukh et al.

6490 FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS

line in response to induction did not lead to nuclear

accumulation of the hTFAM–EGFP fusion protein, as

detectable by confocal microscopy, and instead com-

plete colocalization of the fusion protein and mito-

chondria was observed (Fig. 1, yellow color in the

overlay). To rule out the possibility that EGFP non-

specifically interferes with mitochondrial targeting, we

established a similar inducible stable cell line that

expressed unfused hTFAM (construct 2462), and used

subcellular fractionation techniques in combination

with more sensitive detection by western blotting. This

experiment also revealed accumulation of the hTFAM

in the nucleus in response to increased expression

(induction; Fig. 1D, lower panel). This accumulation

was not due to contamination of the nuclear fraction

with mitochondria, as shown by blotting for a mito-

chondrial marker, manganese superoxide dismutase

(SOD2; Fig. 1D, upper panel).

Translation of hTFAM can be initiated on both

N-terminal methionines with similar efficiency

The above observations suggest either that the two

specialized isoforms of hTFAM, nuclear and mito-

chondrial, are produced from a single cDNA, or that

a single hTFAM polypeptide possesses an intrinsic

nuclear localization signal and is unevenly partitioned

between the mitochondria and the nucleus. Indeed,

DNA ligase III has been shown to produce both

nuclear and mitochondrial isoforms by using alterna-

B

EF1alpha

1760

1788

1789

1790

1804

1805

2078

1819

1818

1817

1807

1806

Fig. 2. (Continued).

V. Pastukh et al. Nuclear localization of hTFAM

FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6491

tive translation initiation signals. In this case, a

shorter, nuclear isoform lacks the first 87 amino acids

encoding the MTS [25]. Similarly, hTFAM has two

methionines in its N-terminal region, M1 and M7, and

either one can potentially be used for translation initia-

tion. To verify whether this is indeed the case, the

5¢-region of hTFAM cDNA, including the 5¢-UTR,

was fused in frame with the luciferase gene, and either

M1 or M7, or both, were mutated to isoleucine (Fig. 3

and Experimental procedures). The luciferase assays

demonstrated that although translation initiation on

M7 occurs with somewhat lower efficiency as com-

pared to M1, these differences do not reach the level

of statistical significance (n ¼ 3, two-tailed t-test;

Fig. 3). To evaluate the subcellular distribution of the

shorter hTFAM variant with a truncated MTS, amino

acids 7–246 were fused to EGFP and the resulting con-

struct was transiently transfected into HeLa cells. No

substantial differences in the subcellular distribution

were detected between the full-length and the truncated

hTFAM variants (supplementary Fig. S1, 7–246). The

S12T polymorphism in the MTS of hTFAM has been

identified as a risk factor for Alzheimer’s disease [26].

We attempted to link this risk with altered nuclear tar-

geting of the shorter hTFAM variant containing the

S12T mutation. However, the patterns of subcellular

targeting of hTFAM(7–246) and hTFAM(7–246)S12T

were essentially identical (supplementary Fig. S1). As

both full-length and truncated [hTFAM(7–246)]

products are efficiently targeted to mitochondria, the

existence of a shorter hTFAM variant cannot account

for the nuclear accumulation of the EGFP fusion pro-

teins. Therefore, it is more likely that intrinsic NLS(s)

mediate the nuclear accumulation of hTFAM.

HTFAM possesses multiple NLSs

To determine whether hTFAM possesses intrinsic

NLS(s), a series of 5¢- and 3¢-deletions were introduced

into the hTFAM gene (Fig. 2). All three 3¢-deletions(constructs 1788, 1789, and 1790) retained both the

MTS and HMG1 domain and demonstrated promi-

nent mitochondrial localization of the fusion proteins

with some nuclear fluorescence. In contrast, all 5¢-dele-tions lacked the MTS and exhibited predominantly

nuclear and ⁄or cytoplasmic fluorescence. We further

fused individual hTFAM segments (HMG1, linker,

HMG2, tail) to EGFP to locate putative NLS(s). Sur-

prisingly, three of the four constructs tested (HMG1,

HMG2, and tail fusions) accumulated in the nucleus,

suggesting the presence of NLSs. The strength of these

signals can be ranked on the basis of nuclear ⁄ cyto-plasmic partitioning of the fusion proteins as

HMG1 > tail > HMG2 (Fig. 2B). Another unex-

pected result was that in approximately 2% of the cells

expressing the HMG1–EGFP fusion protein, a fraction

of the fusion protein was localized to mitochondria,

suggesting that mitochondrial targeting determinants

of hTFAM may extend beyond the cleavable MTS.

Lys96 and Lys97 are critical for the nuclear

targeting of HMG1

Human SRY protein, a nuclear transcription factor

expressed early in embryonic development, is arguably

the best studied member of the HMG family [27].

SRY contains two distinct NLSs, at either end of a

single HMG box. Both NLSs are highly conserved in

SRY among mammals and are believed to be required

for complete nuclear localization [28]. The N-terminal

NLS is bipartite and consists of two clusters of

Fig. 3. Translation initiation efficiency at M1 versus M7 of hTFAM.

(A) The structure of the reporter constructs. The bent arrow indi-

cates the initiating methionine. (B) Levels of luciferase activity

when initiated at either M1 or M7. Activities were normalized for

transfection efficiency using cotransfection with Renilla luciferase

and a dual luciferase assay system.

Nuclear localization of hTFAM V. Pastukh et al.

6492 FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS

positively charged residues separated by 12 amino

acids (Fig. 4B). Whereas the first of these clusters is

conserved in both HMG1 and HMG2 as well as in all

TFAMs aligned in Fig. 4B, the second cluster is absent

in mammalian TFAMs. As both HMG1 and HMG2

of hTFAM appear to possess NLSs, amino acid resi-

dues conserved between either HMG1 and HMG2

(Fig. 4A), or between HMG1 domains of TFAMs

from different species (Fig. 4B), were interrogated by

site-directed mutagenesis to identify residues that may

constitute the HMG1 NLS (Table 1; Fig. 4; supple-

mentary Fig. S2). Mutations in only four (K51, E63,

P73, and E106) of the 12 residues that are invariant

between HMG1 domains in all TFAMs aligned in

Fig. 4B had no effect on subcellular redistribution of

EGFP fusion proteins (Table 1; supplementary

Fig. S2). Of the eight remaining residues, mutations in

five (P50, P53, Y57, R104, and Y110) resulted in a sig-

nificant impairment of nuclear accumulation of fusion

proteins, implying involvement of these residues in

nuclear targeting. Interestingly, mutations in three

invariant residues (P66, W88, and K96) resulted in a

significant increase in the proportion of cells that

displayed mitochondrial partitioning of EGFP fusion

proteins (Table 1; Fig. 5A). On closer examination, a

putative bipartite NLS that consists of the R82-R83

duet and the K95-K96-K97 triplet separated by a

spacer of 11 amino acids (Fig. 4A) was found in the

HMG1 domain. Consistent with this observation, a

double mutation K96A + K97A completely elimi-

nated nuclear localization of the HMG1 domain

(Table 1). The nucleus-to-cytoplasm (N ⁄C) index in

cells transfected with this mutant was not statistically

different from that of the cells transfected with a con-

struct encoding unfused EGFP (results not shown).

However, this putative NLS, by itself, was unable to

effect nuclear accumulation of EGFP fusion proteins

(Fig. 4A, construct 2163), and both N-terminal and

C-terminal flanking sequences enhanced nuclear target-

ing of EGFP by this NLS (Fig. 4A, constructs 2208

and 2209, respectively). Unlike mutations W88R and

Y99A in HMG1, which resulted in increased mito-

chondrial localization of the fusion proteins, the corre-

sponding mutations W189R and Y200A in HMG2 did

not result in any detectable mitochondrial localization,

and led instead to increased nuclear accumulation

Fig. 4. Alignments of HMG domains from various sources. (A) Alignment of HMG1 and HMG2 domains of hTFAM. The invariant amino acid

residues are in bold and italic. The solid black lines below alignment indicate boundaries of deletion constructs, whose designations appear

to the left. The P-values, which appear in the brackets next to construct designations, are from one-way ANOVA with Dunnett’s post hoc test

comparisons with a construct expressing cytoplasmic EGFP. The statistically significant difference indicates nuclear accumulation of corre-

sponding EGFP fusion constructs. The HMG1 and HMG2 amino acid residues interrogated by site-directed mutagenesis are indicated by

arrows above and below the alignment, respectively. The brackets above the alignment designate components of the putative HMG1 NLS.

(B) Alignment of human SRY versus TFAMs from various species. The invariant amino acid residues are in bold and italic. Amino acid resi-

dues interrogated by site-directed mutagenesis are indicated by arrows below the alignment. The components of SRY bipartite NLS are indi-

cated by the brackets above the alignment, and corresponding amino acid residues in TFAMs from different species are indicated by

brackets below the alignment.

V. Pastukh et al. Nuclear localization of hTFAM

FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6493

(Table 1; Fig. 5; supplementary Fig. S3). In general,

unlike HMG1 mutations, none of the mutations in the

HMG2 domain led to detectable mitochondrial parti-

tioning of EGFP fusion proteins (Table 1). Mutations

D207A and E214A in HMG2, which affect residues cor-

responding to E106 and E113, respectively, behaved like

their HMG1 counterparts and did significantly affect

nuclear localization (Table 1; supplementary Fig. S3).

Mitochondrial targeting determinants of hTFAM

may extend beyond the cleavable MTS

Perhaps the most unexpected finding of the site-direc-

ted mutagenesis experiments was that several HMG1

mutations resulted in a significant increase in mito-

chondrial targeting of the HMG1–EGFP fusion

proteins. As mentioned above, in 2% of cells, HMG1–

EGFP fusion proteins are partially localized to mito-

chondria. Mutations P66R, P66A, W88R, W88A,

W88A + D93A, K96I and Y99A (Table 1; Fig. 5A)

significantly increased the fraction of cells with mito-

chondrial partitioning of the HMG1 fusion proteins.

The effect of the P66R and W88R mutations was addi-

tive, and the HMG1 P66R + W88R double mutant

localized to mitochondria in 60% of transfected cells,

as judged by subcellular distribution of its EGFP

fusion protein (Fig. 5A). Mitochondrial relocalization

of mutatnt HMG1–EGFP fusion proteins did not pre-

vent nuclear targeting of the same constructs, and dual

mitochondrial and nuclear localization was typically

observed (Fig. 5A). Interestingly, the P66E mutation,

unlike P66R and P66A, did not cause increased

mitochondrial localization of fusion proteins (Fig. 5A).

This is consistent with the notion that N-terminal posi-

tively charged amphiphilic a-helices, which are poor in

aspartic and glutamic acid residues, serve as mitochon-

drial targeting signals [29]. Further supporting the

notion that mutant HMG1 domains are targeted to

mitochondria using determinants similar to those

found in the N-terminal presequences, the placement

of HMG1 P66R + W88R at the C-terminus of EGFP

completely abolished the mitochondrial targeting effect

of the mutations, while having no effect on nuclear

targeting of this fusion protein (Table 1; Fig. 5A).

Finally, subcellular fractionation of cells transfected

with either wild-type HMG1–EGFP fusion construct

or with the double P66R + W88R mutant HMG1–

EGFP fusion construct has revealed increased mito-

chondrial accumulation of EGFP in cells transfected

with mutant construct. This accumulation was accom-

panied by the presence of putative processing products

in both whole cell lysates and in purified mitochondria

(Fig. 5B). Such products are characteristic of precursor

proteins cleaved by mitochondrial processing pepti-

dase, which removes presequences to produce mature

mitochondrial proteins. Collectively, these results

Table 1. Effects of mutations in HMG domains on their subcellular

distribution. ND, the N ⁄ C ratio was not determined for mutants dis-

playing mitochondrial retargeting, due to the existence of two dis-

crete populations of transfected cells; flN, decreased nuclear

accumulation; ›N, increased nuclear accumulation; ›M, mitochon-

drial redistribution of fusion proteins; ››M, strong mitochondrial

redistribution of fusion proteins; NS, not significantly different from

the wild type (WT).

Mutation(s)

N ⁄ C index

(mean ± SEM) P-value (n)

Trend in the

subcellular

redistribution

of mutants

EGFP (EF1-alpha) 1.01 ± 0.04

HMG1–EGFP fusion proteins

WT 6.3 ± 1.1

P50G 2.63 ± 0.31 P < 0.05 (8) flN

P50G + Y110C 2.71 ± 0.32 P < 0.05 (8) flN

P50G + E112A 8.17 ± 2.2 P > 0.05 (6) NS

K51A 3.81 ± 0.34 P > 0.05 (8) NS

K52A 0.37 ± 0.08 P < 0.01 (6) flN

P53A 3.33 ± 0.65 P < 0.01 (8) flN

Y57R 2.59 ± 0.4 P < 0.05 (8) flN

E63A 4.41 ± 0.84 P > 0.05 (8) NS

P66R ND ND ›M

P66A ND ND ›M

P66R + W88R ND ND ››M

P73A 3 ± 0.39 P > 0.05 (8) NS

P73E 2.7 ± 0.31 P > 0.05 (8) NS

K76A 2.12 ± 0.2 P > 0.05 (8) NS

W88R ND ND ›M

W88A ND ND ›M

W88A + D93A ND ND ›M

R89A 1.82 ± 0.1 P > 0.05 (8) NS

D93A 4.12 ± 0.41 P > 0.05 (8) NS

S94W 7.26 ± 0.75 P > 0.05 (6) NS

K96A + K97A 1.12 ± 0.08 P < 0.01 (8) flN

K96I ND ND ›M

Y99A ND ND ›M

R104A 2.02 ± 0.18 P < 0.01 (8) flN

E106A 6.05 ± 0.59 P > 0.05 (8) NS

E106A, E113A 2.24 ± 0.24 P < 0.01 (8) flN

Y110C 1.79 ± 0.2 P < 0.01 (8) flN

E113A 7.78 ± 0.47 P > 0.05 (6) NS

HMG2–EGFP fusion proteins

WT 1.48 ± 0.06

W189R 2.74 ± 0.18 P < 0.01 (8) ›N

E196K 1.91 ± 0.12 P < 0.05 (8) ›N

Y200A 1.78 ± 0.11 P > 0.05 (8) NS

K205A 1.38 ± 0.07 P > 0.05 (8) NS

D207A 1.77 ± 0.14 P > 0.05 (8) NS

E214A 1.55 ± 0.06 P > 0.05 (8) NS

D207A, E214A 1.63 ± 0.06 P > 0.05 (8) NS

EGFP–HMG1 fusion proteins

WT 5.51 ± 0.44

P66R + W88R 4.36 ± 0.68 P > 0.05 (8) NS

Nuclear localization of hTFAM V. Pastukh et al.

6494 FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS

A

B

Fig. 5. Increased mitochondrial partitioning

of some HMG1 mutants. (A) Partitioning as

observed by fluorescence microscopy. Cells

with mitochondrial localization of mutants

are indicated by white arrows. Note that

P66A and P66R mutations, but not P66E

mutations, increase mitochondrial targeting

of HMG1–EGFP fusion proteins. Left

images: green, EGFP fusions. Middle

images: red, MitoTracker Red (mitochondrial

stain). Right images: overlay; yellow,

regions of colocalization. (B) Partitioning as

observed by subcellular fraction-

ation ⁄ western blotting. HEK293FT cells

were transfected with either construct 1817

(wild-type HMG1–EGFP fusion construct,

Fig. 3) or construct 1925 [P66R + W88R

HMG1–EGFP fusion construct (A)], and 48 h

after transfection, cells were lysed to pro-

duce whole cell (wc) lysates, or mitochon-

dria were isolated using a Pierce

mitochondrial isolation kit. Twenty micro-

grams of wc lysates and 10 lg of mitochon-

drial fraction (mito) were separated by

SDS ⁄ PAGE and subjected to western blot-

ting with antibody to mitochondrial HSP60

(a-HSP60, loading control) or antibody to

GFP (a-GFP). Asterisk: putative processing

products cleaved by mitochondrial process-

ing peptidase, which removes MTS from

mitochondrial precursor proteins.

V. Pastukh et al. Nuclear localization of hTFAM

FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6495

indicate that a cryptic mitochondrial targeting determi-

nant may be present in the HMG1 domain. This deter-

minant is likely to play an accessory role in the

context of the full-length protein. Taken out of that

context, this signal, by itself, is insufficient to effect

mitochondrial localization of EGFP fusion proteins.

However, as a consequence of mutations in the HMG1

domain, this determinant can be strengthened, result-

ing in the retargeting of the EGFP fusion proteins to

mitochondria. The role of this cryptic determinant in

the mitochondrial import of hTFAM remains to be

determined. It is likely that it acts cooperatively with

the MTS to effect the mitochondrial localization of the

mature polypeptide. Importantly, we found no evi-

dence for the presence of a similar cryptic determinant

in the HMG2 domain.

Nuclearly targeted hTFAM exerts cytoprotective

effects

TFAM has been found to preferentially bind to DNA

damaged by the genotoxic drugs cisplatin and N-acet-

oxyacetylaminofluorene [17,24,30]. This, in combina-

tion with observations of TFAM accumulation in

transformed cells [23], and its presence in nuclear

extracts of normal liver cells [24], raises the possibility

of the involvement of hTFAM in cellular responses to

chemotherapy [31,32]. Indeed, HMG proteins have

been reported to both impede [33,34] and enhance [35–

37] repair of damaged DNA. Therefore, we established

a cell line with tetracycline (Tc)-inducible expression

of nuclear, MTS-less, hTFAM (construct 2476). Upon

the induction of nuclear hTFAM synthesis, the suscep-

tibility of this cell line to treatment with three different

chemotherapeutic drugs, etoposide, camptothecin, and

cisplatin, was tested. As compared to the similarly

treated parental cell line, the susceptibility to treatment

with etoposide, camptothecin and cisplatin was

decreased by 6.8%, 3.9% and 9.6%, respectively, in

the 2476 line (Table 2). Therefore, although nuclear

hTFAM may affect a tumor’s susceptibility to chemo-

therapy, and may represent a defensive mechanism, the

amplitude of this response with the drugs tested is too

low to be of practical significance.

The mitochondrial localization of hTFAM may rep-

resent an example of ‘eclipsed distribution’, the phe-

nomenon of uneven protein distribution between two

or more cellular compartments, where accumulation of

protein in one compartment impedes its detection in

another [38]. Nsf1 protein, which is involved in the

maturation of FeS proteins in mitochondria, represents

a prototypical example of such distribution. Similar to

that of hTFAM, the nuclear localization of Nsf1 pro-

tein is undetectable by physical means. However, it has

been demonstrated that Nsf1 possesses an internal

NLS, and that impairment of either nuclear or mito-

chondrial targeting of Nsf1 is lethal [39,40]. The

embryonic lethality of the TFAM knockout [6] appears

to extend the similarity between these two proteins.

However, more studies are needed to identify the exact

physiological role of nuclear TFAM.

Experimental procedures

Plasmids

pEF1a is a pcDNA3-derived plasmid in which the elonga-

tion factor 1a promoter drives expression of the EGFP

gene. The plasmid encoding full-length cDNA of hTFAM

was purchased from Open Biosystems (Huntsville, AL).

hTFAM fusion, deletion and mutant constructs were

assembled under the control of the CMV promoter. Con-

structs for generation of Tc-inducible cell lines were gener-

ated in a modified pcDNA5 ⁄FRT ⁄TO vector.

Site-directed mutagenesis and gene fusion

Site-directed mutagenesis was performed by an overlap

extension method [41] using Taq and Vent DNA poly-

merases. All mutations were verified by sequencing. For all

C-terminal fusions with HMG domains, an EGFP gene

lacking the initiating ATG codon was used. The ATG-less

EGFP gene was generated by PCR, cloned, and sequenced.

This was done to exclude expression of unfused EGFP by

means of leaky ribosomal scanning.

Cell culture and transfection

HeLa and Flp-in T-Rex293 cells were grown in DMEM

supplemented with 10% fetal bovine serum, 100 unitsÆmL)1

penicillin, and 100 lgÆmL)1 streptomycin. Cells were seeded

into 35 mm tissue culture dishes at a density of 3 · 105 cells

per dish, and transfections were performed using Polyfect

Table 2. Effect of nuclear hTFAM expression on susceptibility to

treatment with genotoxic drugs.

Drug

2476

Meana

(%)

SEM

(%)

P-value

(n ¼ 3)

Etoposide 106.8 2.1 < 0.0001

Camptothecin 103.9 1.7 0.0017

Cisplatin 109.6 2.6 < 0.0001

aViability as compared to the parental cell line.

Nuclear localization of hTFAM V. Pastukh et al.

6496 FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS

transfection reagent (Qiagen, Valencia, CA) according to

the manufacturer’s recommendations. Cells were observed

by confocal microscopy 40 h after transfection.

Generation of inducible cell lines

Cell lines with Tc-inducible expression of hTFAM or its

derivatives were generated with the help of a Flp-in T-Rex

system according to the manufacturer’s recommendations

(Invitrogen, Carlsbad, CA). Protein expression was induced

with 2 lgÆmL)1 Tc for 48 h.

Subcellular fractionation

Cells were collected by trypsinization, washed with NaCl ⁄Pi,

and resuspended in buffer A (10 mm Hepes, pH 7.9, 10 mm

KCl, 5 mm MgCl2), to which NP40 was added to a final con-

centration of 0.4%. Cells were vortexed for 1 min, and 1 m

sucrose in buffer A was added to a final concentration of

200 mm to make the solution isotonic. Nuclei were collected

by centrifugation at 850 g for 3 min at 4 �C and washed in

the same buffer with sucrose. The supernatant was centri-

fuged at 15 000 g for 10 min at 4 �C to pellet mitochondria.

Nuclei and mitochondria were lysed in 10 mm Tris (pH 8.0),

1 mm EDTA, and 0.5% SDS, and sonicated, and the protein

concentration was determined by the Bradford method.

Cell viability studies

The effect of expression of the hTFAM derivatives on cell

viability in response to various drug treatments was evalu-

ated using Alamar Blue fluorescence.

Microscopy

Confocal microscopy was performed on live cells using a

Leica DM RXE microscope and a TCS SP2 confocal system

(Leica Microsystems Inc., Bannockburn, IL) in combination

with a 63· water immersion objective. Prior to microscopy,

mitochondria were stained with 200 nm MitoTracker Red

(Invitrogen) for 15 min at 37 �C in an atmosphere of 5%

CO2. The nuclear accumulation of EGFP fusion proteins

was quantitated using the N ⁄C distribution index. To calcu-

late this index, average fluorescence intensities (pixel densi-

ties) in nuclear and cytoplasmic regions were determined

with the image j program (National Institutes of Health),

and nuclear fluorescence was divided by cytoplasmic fluores-

cence. Statistical analyses of N ⁄C indices were performed

using one-way anova with Dunnett’s post hoc test.

Luciferase assays

The pGL3 basic reporter plasmid was modified by intro-

ducing the CMV promoter and by removing the N-terminal

methionine of luciferase. The latter modification makes

luciferase expression dependent upon the upstream methio-

nine, which can be provided by a fusion partner. Then,

143 bp of hTFAM cDNA encompassing the 5¢-UTR and

the first 21 bp of the hTFAM gene was cloned upstream

of, and in frame with, the luciferase gene. Finally, three

constructs were generated by replacing either M1, M7 or

both with isoleucine (constructs 1969, 1970 and 1971,

respectively). Luciferase assays were performed using a

dual-luciferase reporter assay system (Promega, Madison,

WI). This system allows for the internal normalization of

results using cotransfection with a second plasmid encoding

Renilla luciferase. The light output was measured using a

TD-20 luminometer (Turner BioSystems, Inc., Sunnyvale,

CA).

Susceptibility to anticancer drugs

Flp-in T-Rex293 cells were stably transformed with con-

struct 2476, which encodes an MTS-less mature form of

hTFAM. The resulting cell line, 2476, accumulates hTFAM

in the nucleus in response to Tc induction. It was plated at

100 000 cells per well and pretreated with Tc for 24 h,

where necessary. Subsequently, cells were subjected to one

of four treatments: (a) carrier (dimethylsulfoxide) alone; (b)

Tc (2 lgÆmL)1) alone; (c) drug (etoposide, 20 lgÆmL)1;

camptothecin, 20 lgÆmL)1; or cisplatin 75 lgÆmL)1) alone;

and (d) drug plus Tc for 24 h. Viability was determined

using Alamar Blue fluorescence. The fluorescence readings

from each cell line that received the four different treat-

ments (triplicate wells) were normalized as follows: dividing

the reading obtained under treatments 2, 3 and 4 in each

experiment by the average of the triplicate readings

obtained under treatment 1. This normalized the readings

in different experiments on different days and made the

changes in readings comparable. A four-way anova was

used to evaluate the effects of nuclear hTFAM expression.

The mean changes, together with the SEMs, were also com-

puted and listed on the basis of the normalized data

obtained under treatment 4 between Flp-in T-Rex293 and

2476. The analyses were performed with the sas 9.1 soft-

ware package (SAS Institute Inc., Cary, NC).

Acknowledgements

The work in G. L. Wilson’s laboratory was supported

by National Institutes of Health Grants ES03456 and

AG19602.

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Supplementary material

The following supplementary material is available

online:

Fig. S1. Subcellular targeting of TFAM(7–246)–EGFP

and TFAM(7–246)S12T–EGFP fusion proteins in

transiently transfected HeLa cells.

Fig. S2. Subcellular distribution of mutant HMG1–

EGFP fusion proteins.

Fig. S3. The effect of HMG2 mutations on the subcel-

lular distribution of HMG2–EGFP fusion proteins.

This material is available as part of the online article

from http://www.blackwell-synergy.com

Please note: Blackwell Publishing is not responsible

for the content or functionality of any supplementary

materials supplied by the authors. Any queries (other

than missing material) should be directed to the corre-

sponding author for the article.

V. Pastukh et al. Nuclear localization of hTFAM

FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6499