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.
Trang 1multiple subcellular targeting signals
Viktoriya Pastukh1, Inna Shokolenko1, Bin Wang2, Glenn Wilson1and 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.
Trang 2HMG-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 isoiso-form 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)1Tc 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 un-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).
Trang 3targeting 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.
Trang 4line 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).
Trang 5tive 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 (n¼ 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.
Trang 6positively 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 indiindi-cated by brackets below the alignment.
Trang 7(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
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
P50G + Y110C 2.71 ± 0.32 P < 0.05 (8) flN
P50G + E112A 8.17 ± 2.2 P > 0.05 (6) NS
K96A + K97A 1.12 ± 0.08 P < 0.01 (8) flN
E106A, E113A 2.24 ± 0.24 P < 0.01 (8) flN
HMG2–EGFP fusion proteins
D207A, E214A 1.63 ± 0.06 P > 0.05 (8) NS
EGFP–HMG1 fusion proteins
P66R + W88R 4.36 ± 0.68 P > 0.05 (8) NS
Trang 8B
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.
Trang 9indicate 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 retargetresult-ing 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 observacombina-tions of TFAM accumulacombina-tion 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)1streptomycin Cells were seeded into 35 mm tissue culture dishes at a density of 3· 105cells per dish, and transfections were performed using Polyfect
Table 2 Effect of nuclear hTFAM expression on susceptibility to
treatment with genotoxic drugs.
Drug
2476 Mean a
(%)
SEM (%)
P-value (n ¼ 3)
a
Viability as compared to the parental cell line.
Trang 10transfection 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)1Tc 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 4C and washed in
the same buffer with sucrose The supernatant was
centri-fuged at 15 000 g for 10 min at 4C 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 37C 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
References
1 Bustin M (1999) Regulation of DNA-dependent activi-ties by the functional motifs of the high-mobility-group chromosomal proteins Mol Cell Biol 19, 5237–5246