Human mitochondrial transcription factor A possesses multiple subcellular targeting signals Viktoriya Pastukh1, Inna Shokolenko1, Bin Wang2, Glenn Wilson1 and Mikhail Alexeyev1,3 Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL, USA Department of Mathematics and Statistics, University of South Alabama, Mobile, AL, USA Institute of Molecular Biology and Genetics, Kyiv, Ukraine 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 highmobility 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 significance of these observations has not been fully elucidated Here we report that both TFAM overexpression and impairment of its mitochondrial targeting can result in nuclear accumulation of the protein Both M1 and M7 methionines of human TFAM (hTFAM) can be used for translation initiation 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 (HMG1) and HMG-like domain (HMG2), as well as the tail region] can effect nuclear accumulation of enhanced green fluorescent 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, suggesting that the mitochondrial targeting sequence of hTFAM may extend beyond the cleavable presequence Mitochondrial transcription factor A (TFAM, mtTFA) is a member of a high-mobility group (HMG) of proteins named on the basis of their electrophoretic mobility 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 precursor has not been determined, and therefore it is possible 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, 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 V Pastukh et al Fig Nuclear localization of hTFAM and hTFAM–EGFP fusion proteins 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 (construct 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 lgỈmL)1 Tc for 48 h (D) Accumulation of hTFAM in the nuclei of transfected cells upon overexpression The Flp-in T-Rex293 cells were stably transfected with construct 2462, which encodes full-length hTFAM Nuclear fractions (12 lg of total protein) from the parental cell line (T-Rex), uninduced (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) Nuclear localization of hTFAM A B C D HMG-like domain (HMG1) and HMG-like domain (HMG2) (Fig 1A), joined by a basic 36 amino acid linker and followed by a basic 27 amino acid tail The gene for TFAM spans about 10 kb and consists of seven exonsandsixintrons[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, pancreatic b-cells and pyramidal neurons is associated with mtDNA depletion, reduced levels of mitochondrial transcripts, and severe respiratory chain deficiency [7–11] TFAM levels generally correlate well with mtDNA content, and upon transient depletion of mtDNA with ethidium bromide, cellular TFAM content 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 manner, 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 containing 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 isoform of TFAM was demonstrated This isoform is generated by alternative splicing of the duplicated first exons, resulting in a protein that lacks a mitochondrial FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6489 Nuclear localization of hTFAM V Pastukh et al targeting sequence (MTS) However, the order of ‘nuclear’ and ‘mitochondrial’ exons in genomic DNA of these species is opposite [20–22] Nuclear localization 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 physiological 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 cytoprotective 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 elevated 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 construct 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 cytoplasm and the nucleus (Figs 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 CMVtet 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 transient transfection, contained a single copy of the fusion construct, and expressed lower levels of the fusion protein In agreement with our hypothesis, the lower levels of expression attained in the Flp-in T-Rex293-2463 cell Fig Schematic diagrams (A) and subcellular localization (B) of hTFAM deletion constructs 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 (mitochondrial stain) Right images: overlay; yellow, the regions of colocalization EF1-alpha, a construct expressing unfused EGFP 6490 FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS V Pastukh et al line in response to induction did not lead to nuclear accumulation of the hTFAM–EGFP fusion protein, as detectable by confocal microscopy, and instead complete colocalization of the fusion protein and mitochondria was observed (Fig 1, yellow color in the overlay) To rule out the possibility that EGFP nonspecifically 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 Nuclear localization of hTFAM with mitochondria, as shown by blotting for a mitochondrial 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 mitochondrial, 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 1806 1807 1760 1788 1817 1789 1818 1790 1819 1804 2078 1805 Fig (Continued) FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6491 Nuclear localization of hTFAM V Pastukh et al 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 targeting 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 proteins Therefore, it is more likely that intrinsic NLS(s) mediate the nuclear accumulation of hTFAM HTFAM possesses multiple NLSs Fig Translation initiation efficiency at M1 versus M7 of hTFAM (A) The structure of the reporter constructs The bent arrow indicates 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 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 initiation 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 and Experimental procedures) The luciferase assays demonstrated that although translation initiation on M7 occurs with somewhat lower efficiency as compared to M1, these differences 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 construct was transiently transfected into HeLa cells No 6492 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 prominent mitochondrial localization of the fusion proteins with some nuclear fluorescence In contrast, all 5¢-deletions 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) Surprisingly, 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 ⁄ cytoplasmic partitioning of the fusion proteins as HMG1 > tail > HMG2 (Fig 2B) Another unexpected 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 FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS V Pastukh et al Nuclear localization of hTFAM Fig 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 corresponding 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 residues interrogated by site-directed mutagenesis are indicated by arrows below the alignment The components of SRY bipartite NLS are indicated by the brackets above the alignment, and corresponding amino acid residues in TFAMs from different species are indicated by brackets below the alignment 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 residues 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; supplementary 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 significant 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 eliminated 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 construct 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 targeting of EGFP by this NLS (Fig 4A, constructs 2208 and 2209, respectively) Unlike mutations W88R and Y99A in HMG1, which resulted in increased mitochondrial localization of the fusion proteins, the corresponding mutations W189R and Y200A in HMG2 did not result in any detectable mitochondrial localization, and led instead to increased nuclear accumulation FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6493 Nuclear localization of hTFAM V Pastukh et al Table Effects of mutations in HMG domains on their subcellular distribution ND, the N ⁄ C ratio was not determined for mutants displaying mitochondrial retargeting, due to the existence of two discrete populations of transfected cells; flN, decreased nuclear accumulation; ›N, increased nuclear accumulation; ›M, mitochondrial 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) EGFP (EF1-alpha) HMG1–EGFP fusion WT P50G P50G + Y110C P50G + E112A K51A K52A P53A Y57R E63A P66R P66A P66R + W88R P73A P73E K76A W88R W88A W88A + D93A R89A D93A S94W K96A + K97A K96I Y99A R104A E106A E106A, E113A Y110C E113A HMG2–EGFP fusion WT W189R E196K Y200A K205A D207A E214A D207A, E214A EGFP–HMG1 fusion WT P66R + W88R 1.01 ± 0.04 proteins 6.3 ± 1.1 2.63 ± 0.31 2.71 ± 0.32 8.17 ± 2.2 3.81 ± 0.34 0.37 ± 0.08 3.33 ± 0.65 2.59 ± 0.4 4.41 ± 0.84 ND ND ND ± 0.39 2.7 ± 0.31 2.12 ± 0.2 ND ND ND 1.82 ± 0.1 4.12 ± 0.41 7.26 ± 0.75 1.12 ± 0.08 ND ND 2.02 ± 0.18 6.05 ± 0.59 2.24 ± 0.24 1.79 ± 0.2 7.78 ± 0.47 proteins 1.48 ± 0.06 2.74 ± 0.18 1.91 ± 0.12 1.78 ± 0.11 1.38 ± 0.07 1.77 ± 0.14 1.55 ± 0.06 1.63 ± 0.06 proteins 5.51 ± 0.44 4.36 ± 0.68 P-value (n) Trend in the subcellular redistribution of mutants P< P< P> P> P< P< P< P> ND ND ND P> P> P> ND ND ND P> P> P> P< ND ND P< P> P< P< P> 0.05 0.05 0.05 0.01 (8) (8) (6) (8) 0.01 0.05 0.01 0.01 0.05 (8) (8) (8) (8) (6) flN flN NS NS flN flN flN NS ›M ›M ››M NS NS NS ›M ›M ›M NS NS NS flN ›M ›M flN NS flN flN NS 0.01 0.05 0.05 0.05 0.05 0.05 0.05 (8) (8) (8) (8) (8) (8) (8) ›N ›N NS NS NS NS NS P > 0.05 (8) NS P P P P P P P < < > > > > > 0.05 0.05 0.05 0.05 0.01 0.01 0.05 0.05 (8) (8) (6) (8) (6) (8) (8) (8) 0.05 (8) 0.05 (8) 0.05 (8) (Table 1; Fig 5; supplementary Fig S3) In general, unlike HMG1 mutations, none of the mutations in the HMG2 domain led to detectable mitochondrial parti6494 tioning of EGFP fusion proteins (Table 1) Mutations D207A and E214A in HMG2, which affect residues corresponding 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-directed mutagenesis experiments was that several HMG1 mutations resulted in a significant increase in mitochondrial targeting of the HMG1–EGFP fusion proteins As mentioned above, in 2% of cells, HMG1– EGFP fusion proteins are partially localized to mitochondria Mutations P66R, P66A, W88R, W88A, W88A + D93A, K96I and Y99A (Table 1; Fig 5A) significantly increased the fraction of cells with mitochondrial partitioning of the HMG1 fusion proteins The effect of the P66R and W88R mutations was additive, 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 prevent 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 positively charged amphiphilic a-helices, which are poor in aspartic and glutamic acid residues, serve as mitochondrial 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 mitochondrial accumulation of EGFP in cells transfected with mutant construct This accumulation was accompanied 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 peptidase, which removes presequences to produce mature mitochondrial proteins Collectively, these results FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS V Pastukh et al Nuclear localization of hTFAM A Fig 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 fractionation ⁄ 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 produce whole cell (wc) lysates, or mitochondria were isolated using a Pierce mitochondrial isolation kit Twenty micrograms of wc lysates and 10 lg of mitochondrial fraction (mito) were separated by SDS ⁄ PAGE and subjected to western blotting with antibody to mitochondrial HSP60 (a-HSP60, loading control) or antibody to GFP (a-GFP) Asterisk: putative processing products cleaved by mitochondrial processing peptidase, which removes MTS from mitochondrial precursor proteins B FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6495 Nuclear localization of hTFAM V Pastukh et al Table Effect of nuclear hTFAM expression on susceptibility to treatment with genotoxic drugs 2476 Drug Meana (%) SEM (%) P-value (n ¼ 3) Etoposide Camptothecin Cisplatin 106.8 103.9 109.6 2.1 1.7 2.6 < 0.0001 0.0017 < 0.0001 a Viability as compared to the parental cell line indicate that a cryptic mitochondrial targeting determinant may be present in the HMG1 domain This determinant 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, resulting 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 evidence 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-acetoxyacetylaminofluorene [17,24,30] This, in combination 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 susceptibility 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 6496 hTFAM may affect a tumor’s susceptibility to chemotherapy, 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 represent an example of ‘eclipsed distribution’, the phenomenon 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 protein is undetectable by physical means However, it has been demonstrated that Nsf1 possesses an internal NLS, and that impairment of either nuclear or mitochondrial 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 elongation 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 Constructs for generation of Tc-inducible cell lines were generated 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 polymerases 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 unitsỈmL)1 penicillin, and 100 lgỈmL)1 streptomycin Cells were seeded into 35 mm tissue culture dishes at a density of · 105 cells per dish, and transfections were performed using Polyfect FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS V Pastukh et al 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 lgỈmL)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, mm MgCl2), to which NP40 was added to a final concentration of 0.4% Cells were vortexed for min, and 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 at °C and washed in the same buffer with sucrose The supernatant was centrifuged at 15 000 g for 10 at °C to pellet mitochondria Nuclei and mitochondria were lysed in 10 mm Tris (pH 8.0), 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 evaluated 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 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 calculate this index, average fluorescence intensities (pixel densities) in nuclear and cytoplasmic regions were determined with the image j program (National Institutes of Health), and nuclear fluorescence was divided by cytoplasmic fluorescence Statistical analyses of N ⁄ C indices were performed using one-way anova with Dunnett’s post hoc test Nuclear localization of hTFAM methionine of luciferase The latter modification makes luciferase expression dependent upon the upstream methionine, 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 construct 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 lgỈmL)1) alone; (c) drug (etoposide, 20 lgỈmL)1; camptothecin, 20 lgỈmL)1; or cisplatin 75 lgỈmL)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 treatments (triplicate wells) were normalized as follows: dividing the reading obtained under treatments 2, and in each experiment by the average of the triplicate readings obtained under treatment 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 computed and listed on the basis of the normalized data obtained under treatment between Flp-in T-Rex293 and 2476 The analyses were performed with the sas 9.1 software 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 References Luciferase assays The pGL3 basic reporter plasmid was modified by introducing the CMV promoter and by removing the N-terminal Bustin M (1999) Regulation of DNA-dependent 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corresponding author for the article FEBS Journal 274 (2007) 6488–6499 ª 2007 The Authors Journal compilation ª 2007 FEBS 6499 ... 3358–3367 Alam TI, Kanki T, Muta T, Ukaji K, Abe Y, Nakayama H, Takio K, Hamasaki N & Kang D (2003) Human mitochondrial DNA is packaged with TFAM Nucleic Acids Res 31, 1640–1645 Takamatsu C, Umeda S,... A, Mezzina M & Gadaleta G (2002) Human mitochondrial transcription factor A (mtTFA): gene structure and characterization of related pseudogenes Gene 291, 223–232 Tominaga K, Hayashi J, Kagawa... Yoshida Y, Izumi H, Ise T, Uramoto H, Torigoe T, Ishiguchi H, Murakami T, Tanabe M, Nakayama Y, Itoh H et al (2002) Human mitochondrial transcription factor A binds preferentially to oxidatively damaged