The C-terminal fragment of hSuv3p that interacts with HBXIP is important for hSuv3p mitochondrial import In order to verify whether deletion of the C-terminal fragment of hSuv3p could ha
Trang 1interacts with the cofactor of survivin HBXIP
Michal Minczuk1, Seweryn Mroczek1, Sebastian D Pawlak1,* and Piotr P Stepien1,2
1 Department of Genetics, University of Warsaw, Pawinskiego 5A, Warsaw, Poland
2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, Warsaw, Poland
The NTP-dependent RNA⁄ DNA helicase Suv3p
belongs to the Ski2 class of DExH-box RNA helicases
and its orthologues have been found in bacteria, yeast,
plants and animals [1] The product of the SUV3 gene
was described for the first time in Saccharomyces
cere-visiae [2], where it functions in mitochondrial RNA
surveillance Yeast Suv3p is one of the two subunits of
a protein complex called mitochondrial degradosome
or MtEXO, which displays an NTP-dependent
exo-ribonucleolytic activity [3–5] The second component
of the degradosome is Dss1p, a single-strand specific
exoribonuclease with motifs similar to bacterial
RN-ase II [6] The RNA-degrading activity of the
degrado-some complex is necessary for maintaining proper
mitochondrial RNA metabolism in yeast Mutations in either of the two degradosome subunits result in over-accumulation of excised group I introns [7], distur-bances in processing at 5¢ and 3¢ ends of mtRNA precursors, lack of mitochondrial translation and in changes in steady-state levels of mature mt mRNAs; yeast strains bearing deletions of SUV3 or DSS1 genes are respiratory incompetent but are viable on ferment-able carbon sources [4,8,9]
In contrast to yeast, much less is known about the human SUV3 and its physiological functions Our recent report indicated that the hSUV3 exhibits typical characteristics for a nuclear-encoded mitochondrial gene, which is constitutively expressed [10] The human
Keywords
apoptosis; helicase; intracellular localization;
mitochondria; mitochondrial import
Correspondence
M Minczuk, Department of Genetics,
University of Warsaw, Pawinskiego 5A,
02-106 Warsaw, Poland
Fax: +48 22 5922244
Tel: +48 22 5922240
E-mail: mminczuk@ibb.waw.pl
*Present address
Laboratory of Bioinformatics and Protein
Engineering, International Institute of
Molecular and Cell Biology, Ks Trojdena 4a,
02-109 Warsaw, Poland
(Received 4 July 2005, revised 5 August
2005, accepted 10 August 2005)
doi:10.1111/j.1742-4658.2005.04910.x
The human SUV3 gene encodes an NTP-dependent DNA⁄ RNA DExH box helicase predominantly localized in mitochondria Its orthologue in yeast is a component of the mitochondrial degradosome complex involved
in the mtRNA decay pathway In contrast to this, the physiological func-tion of human SUV3 remains to be elucidated In this report we demon-strate that the hSuv3 protein interacts with HBXIP, previously identified as
a cofactor of survivin in suppression of apoptosis and as a protein that binds the HBx protein encoded by the hepatitis B virus Using deletion analysis we identified the region within the hSuv3 protein, which is respon-sible for binding to HBXIP The HBXIP binding domain was found to be important for mitochondrial import and stability of the Suv3 protein
in vivo We discuss the possible involvement of the hSuv3p–HBXIP inter-action in the survivin-dependent antiapoptotic pathway
Abbreviations
aa, amino acids; IAP, inhibitor of apoptosis; BIR, baculovirus IAP repeat; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein;
HA, hemagglutinin; hSUV3, human SUV3; IVT, in vitro translation; TAP, tandem affinity purification.
Trang 2Suv3p enzyme expressed in Escherichia coli had a
strong preference for a double stranded DNA, while
also displaying an NTP-dependent RNA helicase
activ-ity [11] Recently, Shu et al [12] confirmed the multiple
substrate unwinding activity of the human Suv3
pro-tein, being able to unwind DNA, RNA and
hetero-duplex substrates The unwinding reaction was found
to depend on conformational change of the protein
induced by pH The human Suv3p has a bona fide
mitochondrial leader sequence and using
immunofluo-rescence analysis, an in vitro mitochondrial uptake
assay and subfractionation of human mitochondria we
showed that hSuv3p is a soluble protein localized in
the mitrochondrial matrix [11] However, intracellular
localization studies using polyclonal antibodies, raised
against the heterologously expressed protein, revealed
that in HeLa cells endogenous hSuv3p also exhibits a
faint nuclear localization signal in addition to a strong
mitochondrial signal [11]
Interestingly, recent data have shown that a fraction
of the Suv3p helicase may indeed be localized in the
nucleus Such a suggestion was made by Bader [13],
who employed in silico analysis of the network of
yeast protein–protein interactions Employing this
method he identified yeast Suv3p as a potential
mem-ber of proliferating cell nuclear antigen-like complex
In addition, high-throughput analysis of yeast
pro-tein–protein interactions has revealed several nuclear
protein partners of the yeast Suv3p, most of them
being involved in DNA replication, repair and
recom-bination [14] Among the identified Suv3p interactors
the following proteins have been reported: (a) the
SGS1 helicase, involved in maintaining genome
stabil-ity, homologous to E coli RecQ and human WRN
helicase (defective WRN helicase leads to premature
aging disorder Werner syndrome); (b) the RFC4
pro-tein, a DNA binding ATPase that acts as a processivity
factor for DNA polymerase delta and epsilon and
loads proliferating cell nuclear antigen; (c) MEC3
pro-tein, involved in checkpoint control and DNA repair;
and (d) DDC1 protein, involved in the DNA damage
checkpoint In agreement with above observations Shu
et al [12] have recently suggested the nuclear
localiza-tion of a fraclocaliza-tion of cellular human Suv3p, but no
data were presented The authors proposed that
hSuv3p has multiple physiological roles in the cell,
including telomere maintenance, DNA repair and cell
cycle checkpoint control
In this paper we show the results of the yeast
two-hybrid system in screening for interactors of the
human SUV3 gene product We demonstrate that
hSuv3p interacts with HBXIP, which was previously
identified as a cofactor of survivin in apoptosis
suppression and as a protein binding to the hepatitis B viral protein X
Results
Identification and characterization of the HBXIP– hSuv3p interaction in the two-hybrid system
In order to screen the cDNA library derived from HeLa cells in two-hybrid system as described by Finley
& Brent [15] we constructed baits by linking N-ter-minal or C-terN-ter-minal part of hSuv3p (residues 1–479 and 380–786, respectively) to the LexA DNA binding domain LexA-hSuv3p 380–786 fusion was chosen for further two-hybrid experiments after our initial tests have shown the lack of its self-activation ability, proper nuclear import and operator binding ability in yeast cells (supplementary Appendix S1, Fig S1) The
6· 106 library clones were screened and 57 positive yeast colonies were identified and subjected to sequen-cing Among 57 positive colonies 18 appeared to be independent clones and HBXIP (HBx interacting pro-tein) proved to be the most frequently occurring cDNA among the isolates (seven out of 18 of the hSuv3p interacting clones; sequence characterization of the clones is shown in supplementary Appendix S1, Fig S2) In order to rule out the possibility of nonspe-cific interaction of HBXIP, different nonrelevant baits including bicoid, CD4 and IC-LexA fusion proteins [15] were tested with all positive interacting cDNA clones In order to identify and partially characterize the hSuv3p domain interacting with HBXIP prey clones, several deletion mutants of the C-terminal hSuv3p bait were used in the two-hybrid test As depicted in Fig 1A, the hSuv3p fragment necessary for interaction with HBXIP is contained within the
136 C-terminal amino acids of hSuv3p (amino acids 650–786)
HBXIP interacts with hSuv3p in vitro
We employed an in vitro binding test in order to exclude the possibility that the interaction between the HBXIP and hSuv3p proteins occurred through a yeast-derived bridging protein(s) and to provide evi-dence of direct binding of the proteins in a different system We constructed HBXIP fusion protein contain-ing TAP tag [16] at the C-terminus We purified HBXIP-TAP fusion on IgG-agarose resin after hetero-logous expression in E coli and studied the interaction with an in vitro translated (IVT) [35S]methionine labe-led hSuv3p In this assay two versions of hSuv3p were used: full-length protein (hSuv3p 1–786) and a protein
Trang 3lacking the 136 C-terminal amino acids (hSuv3p 1–
650); both of them contained a c-myc epitope-tag at
the C-terminus As illustrated in Fig 1B only IVT of
full-length hSuv3p interacted with HBXIP-TAP
immo-bilized on IgG-agarose No interaction was detected
in the case of heterologously purified TAP-tag or the
IgG-agarose resin only (Fig 1B) This result is consis-tent with the finding that the 136 amino acid-long C-terminal part of hSuv3p is responsible for forming a complex with the HBXIP protein
HBXIP shows nucleo-cytosolic localization
in human cells The exact subcellular localization of HBXIP has not been studied up to date To address this issue we tried
to develop the anti-HBXIP polyvalent antibodies We purified HBXIP-TAP fusion in the two step procedure
as described by Rigaut et al [16] after heterologous expression in E coli but we failed to obtain high-affin-ity antibodies against the small hydrophobic HBXIP protein in rabbit Therefore, to determine the subcellu-lar distribution of HBXIP, the protein was C-termin-ally tagged with c-myc or HA epitope and its subcellular localization was studied in transiently transfected HeLa cells The cells were stained with the primary anti-myc (or anti-HA) monoclonal antibodies and visualized with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies In addition, the cells were stained with nuclear marker (DAPI) and mitoch-ondrial marker (MitoTracker CMXRos) As presented
in Fig 2A,B for HeLa cells the HBXIP-myc fusion showed double nucleo-cytosolic localization and practi-cally no colocalization with mitochondria was observed The same result was obtained for the HBXIP-HA fusion and in the case of simian COS-1 cells (data not shown)
Next, in order to provide further evidence on intra-cellular localization of HBXIP, a subintra-cellular fraction-ation experiment was performed As depicted in Fig 2C the HBXIP protein was confirmed to reside in the cytosolic fraction of HeLa cells and it was not found in the mitochondrial fraction
The C-terminal fragment of hSuv3p that interacts with HBXIP is important for hSuv3p mitochondrial import
In order to verify whether deletion of the C-terminal fragment of hSuv3p could have an effect on protein function in vivo we transiently expressed C-terminally truncated (136 amino acids) hSuv3p (hSuv3p-myc 1– 650) in COS-1 cells First, we analyzed the subcellular distribution of the truncated mutant protein using myc monoclonal antibodies visualized with anti-mouse secondary antibodies conjugated with FITC In the case of hSuv3p-myc 1–650 fusion colocalization with the mitochondrial marker MitoTracker was substantially reduced, as compared to the wildtype
A
B
Fig 1 Interaction of hSuv3p with HBXIP (A) Schematic
representa-tion of the hSuv3p baits used in this study with the relative binding
affinities to HBXIP prey clone In order to screen the HeLa
cell-derived cDNA library by the yeast two-hybrid screening, stable bait
was generated by fusing the LexA DNA binding domain with the
C-terminal part of hSuv3p (hSuv3p 380–786) The yeast bait vector
carrying the full-length hSuv3p was also tested under the same
experimental conditions Several hSuv3p deletion mutants were
used to specifically identify and partially characterize the hSuv3p
domain that is necessary for interaction with HBXIP (B) In vitro
interaction of hSuv3p with HBXIP The SDS ⁄ PAGE analysis of the
binding of wildtype hSuv3p or its mutant devoid of the C-terminal
136 amino acids (hSuv3p 1–650) to IgG-agarose-HBXIP is shown.
Both variants of hSuv3p have been synthesized using IVT in the
presence of [ 35 S]methionine Lanes 1 and 2 show hSuv3p (WT)
and the hSuv3p 1–650 mutant, respectively, bound to HBXIP-TAP
immobilized on IgG-agarose Lanes 3 and 4 show the binding of
hSuv3p (WT) and the hSuv3p 1–650 mutant, respectively, bound to
IgG-agarose resin alone Lanes 5 and 6 show the binding of hSuv3p
(WT) and the hSuv3p 1–650 mutant, respectively, to heterologically
expressed TAP-tag immobilized on IgG-agarose Lanes 7 and 8
show 20% of the input of the IVT [ 35 S]methionine labeled hSuv3p
(WT) and the hSuv3p 1–650 mutant, respectively.
Trang 4hSuv3-myc protein expressed under exactly the same conditions, and a significant fraction of the protein was retained in the cytosol (Fig 3) It therefore appears that, in addition to the N-terminal mitochond-rial leader peptide, the 136 amino acid-long C-terminal part of hSuv3p is involved in targeting of the protein
to mitochondria It is worth mentioning, however, that the 136 amino acid-long C-terminal part of hSuv3p alone cannot serve as a bona fide mitochondrial target-ing signal because hSuv3p 650–786-TAP protein con-struct (see below) is not localized in mitochondria (data not shown)
The C-terminal fragment of hSuv3p that binds HBXIP is important for hSuv3p stability The experiment described above also showed that the truncated form of hSuv3p, lacking the domain respon-sible for HBXIP binding, was expressed in a signifi-cantly lower number of cells as compared to the wildtype form of the protein The transfection effi-ciency was 21% and 1.5% for full-length and trun-cated hSuv3p, respectively (although the transfection conditions and plasmid DNA preparations were the same in both cases) Such a difference could be the result of mRNA instability, accelerated protein degra-dation or cell toxicity of the truncated hSuv3 protein
In order to discriminate between those possibilities we measured the mRNA steady-state levels by Northern hybridization As illustrated in Fig 4A there was no significant difference in mRNA level for hSuv3p wild-type and truncated construct In order to exclude that lowered expression frequency observed for the trun-cated hSuv3p results from cellular toxicity, the wild-type and 1–650 forms were coexpressed with green fluorescence protein (GFP) as a internal marker (GFP was encoded within the vector backbone, therefore all cells expressing either form of hSuv3p expressed GFP
as well) First, we measured the transfection efficiency for the constructs encoding either version of hSuv3p
by counting GFP-positive cells; in both cases the effi-ciency was 17% Then, after transient expression, wildtype and truncated forms of hSuv3p, the proteins were visualized by anti-myc monoclonal antibodies and anti-mouse Texas Red conjugated secondary antibod-ies Furthermore, we studied the correlation between red (hSuv3p-derived) and green (GFP-derived) fluores-cence for full-length and truncated forms of hSuv3p as described in Experimental procedures As presented in Fig 4B,C the correlation observed was 84 ± 12% and
9 ± 6% for wildtype hSuv3p and hSuv3p 1–650 trun-cated protein, respectively This result inditrun-cated that the hSuv3p version lacking HBXIP binding domain
A
B
C
Fig 2 Intracellular localization of HBXIP in mammalian cells (A)
HeLa cells were grown on coverslips and transiently transfected
with cDNA encoding c-myc tagged HBXIP (HBXIPmyc) After
incu-bation with MitoTracker red, fixation and permeabilization as
des-cribed in Experimental procedures the cells were immunostained
with anti-(c-myc) monoclonal antibody (9E10), which was then
visu-alized with fluorescein isothiocyanate-conjugated antibody At the
final stage, nuclei were stained with DAPI present in the mounting
medium The figure shows representative fluorescent image of
cells stained with DAPI (blue), c-myc-tagged HBXIP (green) and
MitoTracker (red) taken by a confocal microscope Similar results
were obtained for cDNA encoding HA tagged HBXIP expressed in
HeLa cells as wells as for the HBXIPmyc and HBXIP-HA expressed
in COS-1 cells (B) Magnified confocal microscope images prepared
as in (A) for HeLa cells transfected with cDNA encoding HBXIPmyc
are shown (C) HeLa cells were transiently transfected with cDNA
encoding c-myc tagged HBXIP (HBXIPmyc) Cell lysates from
unfractionated HeLa cells (T), the cytoplasmic (C) and the
mitoch-ondrial fraction (M) were immunoblotted with anti-myc monoclonal
antibodies The fractions were verified using anti-hSuv3p serum
used here as a marker for the mitochondrial fraction.
Trang 5might be less stable in comparison to wildtype hSuv3p
protein
In order to address the question of whether the
hSuv3p 1–650 truncated protein is less stable than the
wildtype form in vivo, protein synthesis was inhibited in
transfected cells by cycloheximide and protein decay
rates were measured at various time points thereafter
In these studies, COS-1 cells were transiently transfected
with expression vectors encoding either TAP-tagged
wildtype hSuv3p or the 1–650 truncated form of
hSuv3p At 24 h following the transfection, the protein
synthesis was inhibited with cyclohexamide treatment
and total cell extracts were prepared at 0, 2, 4, 6 or 8 h
after cycloheximide addition The protein levels at
var-ious time points were analyzed by western blot using
the PAP antibody (i.e antibody against protein A
which is encompassed within the TAP tag) to measure
protein decay rates As presented in Figure 5A the
TAP-taged hSuv3p 1–650 form lacking the protein
frag-ment responsible for hSuv3p–HBXIP interaction was
significantly less stable as compared to the wildtype
hSuv3TAP The absence of HBXIP binding domain
resulted in 50% decline in the truncated protein levels
within 4 h following inhibition of protein synthesis by
cycloheximide We next wanted to examine whether the presence of HBXIP binding protein fragment of the hSuv3 protein by itself can increase the protein stability For these studies the protein decay rates were measured for the COS-1 cells transfected with the expression vec-tors encoding the HBXIP binding domain of hSuv3p fused to TAP (construct hSuv3TAP 650–786; Fig 5B)
or the TAP protein alone Figure 5B illustrates that the protein stability of TAP fused to the HBXIP binding domain of the hSuv3p protein is much higher in com-parison to TAP alone These results suggest that the hSuv3p C-terminal fragment (residues 650–786), which was also found to bind HBXIP, plays an important role
in regulation of the hSuv3 protein stability
Discussion
The data presented in this paper indicate that human hSuv3p helicase interacts with HBXIP protein The human HBXIP is a small protein of 91 amino acids that was discovered by Melegari et al [17], as the result of yeast two-hybrid screening for the interactors of hepati-tis B virus-encoded protein HBx The viral HBx protein seems to be the major cause of hepatocarcinogenesis
Fig 3 Localization of the truncated form of hSuv3p lacking the HBXIP interacting C-terminal domainCOS-1 were grown on coverslips and transiently transfected with cDNA encoding c-myc tagged hSuv3p (hSUV3myc WT) or the hSuv3p mutant lacking the C-terminal 136 amino acids (hSuv3myc 1–650) After incubation with MitoTracker red, fixation and permeabilization the cells were immunostained with anti-(c-myc) monoclonal antibody, which was then visualized with fluorescein isothiocyanate-conjugated antibody (9E10) Fluorescent images of mito-chondria stained with MitoTracker (red) and c-myc-tagged variants of hSuv3p (green) were taken by a confocal microscope Colocalization of either forms of hSuv3p with mitochondria appears in yellow ⁄ orange in digitally overlaid images.
Trang 6[18] and is a multifuctional regulator of transcription,
cell responses to genotoxic stress, protein degradation
and signaling pathways [19] Recent data indicate that
HBx localizes in mitochondria, and its overexpression
induces a perinuclear mitochondrial distribution and
loss of a mitochondrial membrane potential [20,21]
Studies with the mutant HBx proteins revealed that its
mitochondrial targeting sequences are important for
mitochondrial localization, mitochondrial membrane
potential disruption and cell death [21,22]
Further-more, it has been reported that HBx interacts with at
least two mitochondrial proteins: (a) VDAC3, which is
confined to the outer mitochondrial membrane [22];
and (b) the heat shock protein 60 predominately
locali-zed in the mitochondrial matrix [23] However, the
exact physiological significance of the
intramitochond-rial localization of HBx and of the HBx–HBXIP
inter-action remains unknown
Recently it has been shown that HBXIP is a neces-sary cofactor of survivin in the process of suppression
of apoptosis in cancer cells Survivin is a small protein (16.5 kDa) that contains N-terminal zinc binding bacu-lovirus inhibitor of apoptosis repeat (BIR) domain linked to a C-terminal amphipathic helix [24] Under normal physiological conditions survivin is involved in coordinating the chromosomal and cytoskeletal events
of mitosis [25] In most cancer cells survivin is strongly up-regulated, forms a complex with HBXIP and inhibits apoptosis The mechanism of the inhibition is mediated
by binding of the survivin-HBXIP to Apaf1 and pre-venting the activation of procaspase 9 [26] Thus, HBXIP has an important function in apoptosis suppres-sion The siRNA inhibition of either survivin or HBXIP results in restoration of apoptotic ability of cancer cells
To the best of our knowledge no other interactors
of the HBXIP have been reported, nor has its exact
A
B
C
Fig 4 Expression of hSuv3p lacking the HBXIP interacting domain A Northern blot analysis of the steady-state levels of the hSuv3myc and hSuv3myc 1–650 mRNA COS-1 were transiently transfected with the pcDNA3.1(–) vector (lane 1), cDNA encoding c-myc tagged, wildtype form of hSuv3p (lane 2, expected transcript length 2690 nt) or the hSuv3p mutant lacking the C-terminal 136 amino acids (lane 3, expected transcript length 2282 nucleotides) Total RNA from the cells was isolated and subjected to the northern blot analysis as described in the Experimental procedures In the conditions applied the hybridization signal corresponding to the endogenous hSUV3 mRNA is not visible (lane 1) (B) Coexpression of GFP with either the c-myc tagged wildtype hSuv3p or the hSuv3p mutant lacking the C-terminal 136 amino acids COS-1 were grown on coverslips and transiently transfected with cDNA encoding c-myc tagged hSuv3p (hSUV3myc WT) or the hSuv3p mutant (hSuv3myc 1–650) After fixation and permeabilization the cells were immunostained with anti-(c-myc) monoclonal antibody, which was then visualized with TexasRed-conjugated antibody The panel shows representative fluorescent images of c-myc-tagged variants
of hSuv3p (red) coexpressed with GFP (green) (C) Quantitative analysis of the correlation between the expression of the hSuv3p variants and GFP Black columns represent the correlation between hSuv3p-derived (red column) and GFP-derived (green column) fluorescence in the cells coexpressing GFP with either the wildtype form of hSuv3p (hSuv3myc WT) or the truncated version lacking of the C-terminal 136 amino acids (hSuv3p 1–650) calculated as described in Experimental procedures from the three independent experiments.
Trang 7subcellular localization been analyzed In this work the
fragment of the hSuv3 protein encompassing 380–786
of its total 786 amino acids has been used as the bait
in a yeast two-hybrid system Out of 18 bona fide
clones representing interacting human proteins, seven
clones were found to encode the HBXIP protein The
results of our two-hybrid screen have been confirmed
by pull-down assays
We constructed a series of deletions of the hSUV3
cDNA and demonstrated that only the 136 amino acid
long C-terminal domain of the hSuv3 protein is
responsible for the observed interaction with HBXIP
This domain has no obvious homology to the domain
within the hepatitis B virus protein X, which is known
to bind HBXIP as well [17] Nevertheless, the 136
amino acid domain seems to be of importance for
functioning of the hSuv3 protein Its deletion not only
abolishes interactions with HBXIP, but leads to
delo-calization of the hSuv3 protein: a significant portion of
it has been found in the cytosol In addition, the
trun-cated hSuv3 protein is less stable
Initially we assumed that because hSuv3p was
shown to be a mitochondrial protein, the site of the
discovered hSuv3p–HBXIP interaction should be a
mitochondrion In contrast to this, our data on
subcellular distribution of HBXIP indicated mainly cytosolic or nuclear localization Therefore, the inter-action of hSuv3p with HBXIP in vivo might occur out-side mitochondria, for instance: (a) in the cytosol before Suv3p translocates through mitochondrial mem-branes or (b) in the nucleus, where a fraction of hSuv3p has been recently suggested to reside [12] This result is in agreement with studies of Marusawa et al [26], which suggested that the HBXIP–survivin interac-tion is not localized in mitochondria On the other hand, owing to limited detection limits of the methods used by both us and others, it cannot be excluded that
a vary small amount of HBXIP is localized in mito-chondria Another possibility is that HBXIP may change its cellular localization and, for example, could
be translocated to mitochondria in certain physiologi-cal conditions
What could be the physiological significance of the observed hSuv3p–HBXIP interaction? Two hypotheses can be put forward First, HBXIP can serve as a chap-erone for Suv3p, necessary for its proper import into mitochondria after being translated on cytosolic ribo-somes Because a fraction of truncated Suv3p, i.e lack-ing the C-terminal HBXIP bindlack-ing domain, can be found in the cytosol, the binding of HBXIP may
Fig 5 The role of the C-terminal, HBXIP interacting, domain of hSuv3p in protein stability (A) The protein stability of the wildtype hSuv3TAP and the hSuv3TAP variant lacking the C-terminal 136 amino acids (hSuv3TAP 1–650) in mammalian cells COS-1 cells were transfected with pcSUVTAP or pcSUVTAP-1–650 and after 24 h the protein synthesis was inhibited by addition of cycloheximide to the culture medium The protein levels were examined by Western blot in the indicated time points (B) The protein stability of the fusion protein containing the C-ter-minal 136 amino acids of hSuv3p fused to TAP (hSuv3TAP-650–786) and TAP alone in mammalian cells COS-1 cells were transfected with pcSUVTAP-650–786 or pcTAP and treated as described in (A) The graphs illustrate the densitometric quantification of the amounts of pro-tein for a representative experiment presented as a percentage of propro-tein in the time point 0 h ‘P’ and ‘M’ indicates the precursor and mature mitochondrial form of hSuv3p, respectively The asterisk indicates an unidentified degradation product.
Trang 8promote mitochondrial localization of hSuv3p
Analy-sis of recent reports on mitochondrial localization⁄
function of HBx [27,28] suggests that the interaction of
HBx and HBXIP might also be necessary for the
import of HBx into mitochondria The region within
HBx responsible for the binding of HBXIP [17]
coin-cides with the domain necessary for the mitochondrial
localization of HBx (supplementary Appendix S1,
Fig S3) Therefore, this hypothesis would assume
similarity with the functions of HBXIP in transport of
viral hepatitis B protein X into mitochondria It
should be stressed, however, that the HBx terminal
domain is only one of the determinants of
mitochond-rial localization: similarly as for hSuv3p, the
N-term-inal leader sequence is required for the import as well
The chaperone hypothesis also seems to be in
agree-ment with our data that the hSuv3 protein devoid of
the 136 amino acid C-terminal domain is significantly
less stable This in turn is consistent with the report by
Zhao et al [29], which shows that mutations of critical
amino acid residues within the BIR domain of
survi-vin, which is responsible for interaction with HBXIP
[26], sensitize survivin to degradation
Our second hypothesis assumes that the interaction
of hSuv3p and HBXIP plays a role in the suppression
of apoptosis by the survivin–HBXIP complex
Accord-ingly, hSuv3p would interact with this complex, which
prevents binding of Apaf1 to procaspase 9 [26]
Because HBXIP was shown to be a necessary cofactor
in this process, by binding to survivin, the ability of
hSuv3p to interact with HBXIP would constitute an
important regulatory mechanism in apoptosis
suppres-sion in cancer cells Our preliminary data indicate that
this possibility cannot be excluded, as siRNA
inhibi-tion of hSUV3 in HeLa cells resulted in apoptosis (A
Dmochowska, unpublished data, Warsaw, Poland)
Interestingly, recent reports have shown that survivin
also localizes in mitochondria, and in response to cell
death stimulation, the mitochondrial pool of survivin
is displaced into cytosol, where it prevents casapase
activation [30,31] Such trafficking of the proteins in
and out of mitochondria might constitute an important
element in apoptosis control It is clear that more
research is needed to test the involvement of hSUV3 in
this pathway and our experiments are in progress
Experimental procedures
Plasmid construction
The bait plasmids, used in the two-hybrid screen, encoded
LexA DNA binding domain fused to various fragments
of hSuv3p and were constructed using pEG202 [15] as
described below The schematic representation of all the bait fusion proteins is shown in Fig 1A Numbers in the LexA fusion names correspond to amino acid positions in the hSuv3 protein fragments All enzymes used for cloning were purchased from Fermentas (Vilnius, Lithuania) The pEGhSUV3-1–479 plasmid encoding the LexA-hSuv3p 1–479 fusion was constructed by PCR amplification
of the appropriate hSuv3p cDNA fragment using the fol-lowing primers: CCGGAATTCTCGATGTCCTTCTCCC GTGC (forward; incorporating EcoRI site, underlined) and GCGGGATCCGAAACCGTGAGCTGAATCTGCC (reverse, incorporating BamHI site, underlined) The result-ing fragment was cloned into pEG202 usresult-ing EcoRI and BamHI
The pEGhSUV3-380–786 plasmid encoding the LexA-hSuv3p 380–786 fusion was constructed by PCR amplifica-tion of the appropriate hSuv3p cDNA fragment using the following primers: GCGGAATTCTCTGTGAGTCGGCA GATTGAA (forward; incorporating EcoRI site, under-lined) and CATGCCATGGCTAGTCCGAATCAGGTTC
CT (reverse, incorporating NcoI site, underlined) The resulting fragment was cloned into pEG202 using EcoRI and NcoI
The pEGhSUV3-1–786 plasmid encoding the LexA-hSuv3p 1–786 fusion was constructed by PCR amplification
of the appropriate hSuv3p cDNA fragment using the for-ward primer as in case of pEGhSUV3-1–479 and the reverse primer as in case of pEGhSUV3-380–786 The resulting fragment was cloned into pEG202 using EcoRI and NcoI
The pEGhSUV3-380–786D393–506 plasmid encoding LexA-hSuv3p 380–786D393–506 fusion was constructed by excision of the PvuII-PvuII form pEGhSUV3-380–786 and religation
The pEGhSUV3-380–735, pEGhSUV3-380–650 and pEGhSUV3-380–580 plasmids encoding the LexA-hSuv3p 380–735, 380–650 and 380–580 fusions, respectively, were constructed by PCR amplification of the appropriate hSuv3p cDNA fragments using the forward primer as in case of pEGhSUV3-380–786 and the following reverse primers: CCATCCATGGCTAGGAAGCAAGGGACAGC TCTCC, GGATCCATGGTCATGGAAACATATCCATA AATCGG and CCATCCATGGTCAGTTGATAGGAGC TGTGAAGAAAAC, respectively (all incorporating NcoI site, underlined) The resulting fragments were cloned into pEG202 using EcoRI and NcoI
The pEGhSUV3-650–786 and pEGhSUV3-650–735 plas-mids encoding LexA-hSuv3p 650–786 and 650–735 fusions, respectively, were constructed by PCR amplification of the appropriate hSuv3p cDNA fragments using the following forward primer CCTGAATTCGATGCCAGCCTTATTCG AGATCTCC (EcoRI site underlined) and the reverse prim-ers as in the case of 380–786 and pEGhSUV3-380–735, respectively The resulting fragments were cloned into pEG202 using EcoRI and NcoI
Trang 9The pcHBXIPmyc and pcHBXIP-HA constructs used for
immunoflourescence analysis that encode HBXIP fused to
C-terminal epitope tags c-myc and HA, respectively, were
constructed as follows: the cDNA fragment encoding
HBXIP of full-length was PCR amplified using the
follow-ing reverse primers: for pcHBXIPmyc CCATAAGCTTCA
CAGGTCCTCCTCGGAGATCAGCTTCTGCTCAGAGGC
CATTTTGTGCACTGCC introducing c-myc epitope
cod-ing sequence (italic) and HindIII site (underlined); for
pcHBXIP-HA CCATAAGCTTCAGAGGCTAGCGTAATC
CGGAACATCGTATGGGTAAGAGGCCATTTTGTGCAC
TGCC introducing HA epitope coding sequence (italic) and
HindIII site (underlined) In both cases the forward BCO1
primer (CCAGCCTCTTGCTGAGTGGAGATG) was used,
which binds upstream of the multiple cloning site (MCS) in
the cDNA library pJG4-5 plasmid [15] The 3–54 clone
(supplementary Appendix S1, Fig S2) selected from the
cDNA library in the yeast two-hybrid system was used as a
template for both constructs The resulting fragment was
cloned into EcoRI and HindIII sites of pcDNA3.1(–) vector
(Invitrogen, Carlsbad, CA, USA)
The pET15HBXIP-TAP construct used for
overexpres-sion of the HBXIP-TAP fuoverexpres-sion in E coli was constructed
as follows: the BamHI and NcoI fragment encoding
TAP-tag was excised from pBS1539 [16] and inserted into the
pET15b bacterial expression vector (Novagen, Madison,
WI, USA) The resulting plasmid was named pET15TAP
and expressed TAP-tag only Then the fragment encoding
HBXIP was PCR-amplified using the 3–54 clone template
(supplementary Appendix S1, Fig S2) and the following
primers: CGATCCATGGAGGCGACCTTGGAGCAG
(forward) and GACTCCATGGAGGCCATTTTGTGC
ACTG (reverse), both incorporating NcoI site The
obtained PCR fragment was cloned into pET15TAP using
NcoI site
The pchSUV3myc plasmid used for expression of
wild-type hSuv3p in a c-myc-tagged form in mammalian cells
was as described previously [11] The pchSUV3-1–650myc
construct encoding hSuv3p lacking the 136 C-terminal
amino acids with a c-myc epitope (named hSuv3p 1–650)
was constructed as follows: a DNA fragment encoding the
first 650 amino acids of hSuv3p was PCR amplified using
the following primers: GCATCTAGACACGATGGCCTT
CTCCCGTGCCCTATTGTGG (forward) introducing XbaI
site (underlined) and CGTGAATTCACAGGTCCTCCTCG
GAGATCAGCTTCTGCTCTGGAAACATATCCATAAAT
CGGTAGC (reverse) introducing c-myc epitope coding
sequence (italic) and EcoRI site (underlined) The
pchSUV3myc (see above) plasmid served as a template The
resulting fragment was cloned into XbaI and EcoRI sites of
the pcDNA3.1(–) vector (Invitrogen)
The pTRhSUV3myc and pTRhSUV3-1–650myc
con-structs used for coexpression of the wildtype form of
hSuv3p or the hSuv3p 1–650 mutant with GFP were
con-structed by subcloning of the NheI-EcoRI fragments from
pchSUV3myc and pchSUV3-1-650myc, respectively, into pTRACER CMV⁄ Bsd (Invitrogen)
In order to obtain the pchSUV3TAP plasmid, encoding the full-length hSuv3p as a C-terminal fusion with TAP-tag, the hSUV3 cDNA was amplified using the following primers: TACCCATGGGCATCTGCTCTGCCCTTCG – forward and CATGCCATGGCTAGTCCGAATCAGGT TCCT – reverse, both incorporating NcoI site (underlined) The resulting fragment was cloned into NcoI site of the pET15TAP plasmid (see above) Then the BamHI-BamHI fragment was subcloned into the pchSUV3myc vector The pchSUV3TAP-1–650 plasmid encoding the truncated form of hSuv3p (lacking 136 C-terminal amino acids) fused
to TAP-tag was constructed as described for pchSUV3TAP with the exception of using the following reverse pri-mer: GGATCCATGGTCATGGAAACATATCCATAAA TCGG
The pchSUV3TAP-650–786 plasmid encoding the C-ter-minal part of hSuv3p as a fusion with TAP-tag was obtained by the PCR amplification of the appropriate cDNA fragment from the pchSUV3TAP plasmid with the following primers: CCTCTCGAGATGGATGCCAGCCTT ATTCGAGATCTCC – forward (incorporating XhoI site, underlined) and GCTGAATTCTCAGGTTGACTTCCCC GCGGAGTTCG – reverse (incorporating EcoRI site, underlined) The resulting fragment was cloned into the pcDNA3.1(–) vector Please note: letter in boldtype in the reverse primer represents the AfiG mutation introduced in order to disrupt the EcoRI site present in the original TAP sequence
The pcTAP plasmid encoding TAP-tag only was gener-ated similarly to pchSUV3-650–786-TAP with the exception that the forward primer had the following sequence: CGTCTCGAGATGGAAAAGAGAAGATGGAAAAAG AATTTC (XhoI site is underlined)
Two-hybrid screening Before conducting the two-hybrid screening, the LexA-hSuv3p 1–479 and LexA-LexA-hSuv3p 380–735 baits were tested
in order to verify whether the fusion proteins are able to enter the nucleus, bind LexA operators, and not activate transcription of the reporter genes by themselves The test was performed as described previously [15] Additionally, it was verified by immunoblotting with the anti-hSuv3p serum described in [11] whether the full-length bait proteins were made by the yeast cells transformed with the pEGhSUV3-1–479 or pEGhSUV3-380–786 bait plasmids
The yeast two-hybrid screening was performed according
to the sequential method described previously [15] with a HeLa-derived cDNA library cloned into the yeast pJG4-5 shuttle vector [32] Briefly, the EGY48 yeast strain (contain-ing LEU2 reporter gene) was transformed with the HIS pSH18-34 LacZ reporter plasmid [15] and the URA pEG-hSUV3-380–786 bait plasmid (this work) Then the TRP
Trang 10library plasmids were transformed into this strain using
high-efficiency transformation as described previously [15]
and the transformants ( 3 · 107
) were counted, harvested from the 24· 24 cm plates (Nunc, Wiesbaden, Germany)
and frozen for storage at )70 C Aliquots of the
library-transformed pellets were thawed and plated onto selective
medium (containing galactose and lacking leucine –Gal⁄ Raf
ura-his-trp-leu-) following 4 h of amplification In the next
step, single yeast resistant colonies were replicated onto the
following media: Gal⁄ Raf trp-leu-, Gal ⁄ Raf
ura-his-trp- X-gal, Glu ura-his-ura-his-trp-leu- and Glu ura-his-ura-his-trp-X-gal
Galactose dependent LEU+ blue colonies were identified
and subjected to a plasmid DNA isolation procedure as
described previously [15] Library cDNA inserts were then
PCR amplified with the BCO1 and BCO2 primers [15] and
subjected to restriction analysis in order to identify
repetit-ive clones, and then sequenced Human proteins encoded
by the library inserts were identified by blastx [33]
Plas-mids of independent clones encoding different clones of
HBXIP (supplementary Appendix S1, Fig S2) were rescued
using the E coli KC8 strain [34] Next, the HBXIP prey
plasmids were retransformed into the yeast EGY48 strain
carrying plasmids encoding nonspecific baits, i.e fusions of
LexA with either bicoid, CD4, CD4D85 or IC [15]
Addi-tionally, one of the HBXIP clones (supplementary
Appen-dix S1, Fig S2, 3–54) was transformed into the EGY48
strain harbouring several deletion mutants of the
hSUV3-380–786 C-terminal bait (Fig 1A) The resulting strains
were tested on the selective media as described above
Protein purification
In order to overexpress the HBXIP-TAP fusion or the
TAP-tag control the E coli BL21-CodonPlus(DE3)-RP
strain (Stratagene, Kirkland, WA, USA) transformed with
pET15HBXIP-TAP or pET15TAP, respectively, was grown
to D600¼ 0.6 and induced for 20 h in 16 C with 1 mm
iso-propyl thio-b-d-galactoside Bacterial pellets were incubated
in the IPP150 buffer without NP40 (10 mm Tris⁄ HCl
pH 8.0, 150 mm NaCl, 1 mm EDTA) supplemented with
proteinase inhibitor cocktail (Roche, Mannheim, Germany),
1 mm phenylmethanesulfonyl fluoride and 100 mm
lyso-syme After the incubation, NP40 was added to 0.1% (w⁄ v)
and the samples were sonicated Insoluble material was
pel-leted (26 000 g), the supernatant was loaded on the
IgG-agarose column equilibrated with IPP150 and the sample
was rotated for 2 h at 4C Following the incubation,
unbound proteins were eluted with IPP150 and a portion of
the resin with bound HB-XIP or TAP-tag was mixed with
the SDS loading buffer and boiled for 5 min The
IgG-agarose immobilized proteins were then resolved using
SDS⁄ PAGE, stained with Coomassie and subjected to
den-sitometry in order to assay the purity of the sample In
addition, the purified proteins were immunobloted and
probed with PAP antibodies (Sigma, Steinheim, Germany)
In vitro protein–protein interaction The in vitro interaction between HBXIP and hSuv3p was studied as follows: the wildtype form of hSuv3p or the hSuv3p 1–650 mutant lacking the C-terminal 136 amino acids were in vitro translated (IVT) in the presence of [35S]Met using the TNT Quick coupled transcription⁄ trans-lation system (Promega, Madison, WI, USA) and the pchSUV3myc or pchSUV3-1–650myc plasmid as a tem-plate The [35S]Met labeled proteins were incubated with purified and IgG-agarose immobilized HBXIP-TAP (or TAP-tag) in 0.1 m phosphate buffer (pH¼ 8.1) for 1 h at
4C After the incubation, the IgG-agarose resin was inten-sively washed with 0.1 m phosphate buffer, mixed with the SDS loading buffer, boiled for 5 min and resolved in the SDS⁄ PAGE gel After electrophoresis the gel was dried and subjected to autoradiography
Immunofluorescence experiments and cell fractionation
For the immunofluorescence studies of HBXIP, hSuv3myc and its hSuv3myc 1–650 mutant form in HeLa or COS-1 the cells were plated in 6-well cluster dishes with a cover slip placed at the bottom of the well and grown overnight
in DMEM (Sigma, St Louis, MO, USA) supplemented with 10% FCS and 4 mm glutamine The cells were then trans-fected using FuGene6 reagent (Roche, Indianapolis, IN, USA) At 24 h after the transfection staining of the mito-chondria and the immunodetection of the tagged proteins was carried out as described previously [11] The primary antibodies against c-myc and HA as well as the secondary anti-mouse antibodies conjugated with fluorescein isothio-cyanate or TexasRed were purchased form Santa Cruz Bio-technology (Santa Cruz, CA, USA) In some experiments, where indicated, cell nuclei were stained with DAPI present
in the Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA)
In order to study the correlation between expression levels
of the hSuv3p variants and GFP cells were transfected with pTRhSUV3myc or pTRhSUV3-1–650myc and prepared for immunofluorescence analysis as described above Then, for
100 randomly selected cells intensity of red fluorescence, derived form TexasRed conjugated secondary antibody bound to either form of hSuv3p, and green fluorescence, derived from GFP, were calculated using imagej software (W Rosband http://rsb.info.nih.gov/ij) and expressed as rel-ative units The ‘correlation TexasRed vs GFP’ as presen-ted on Fig 4C was obtained by dividing the sum of the red fluorescence by the sum of green fluorescence
For cellular fractionation experiments three to four 6-well cluster dishes of HeLa cells were transfected with pcHBXIP-myc as described above At 24 h after the transfection cell fractionation and isolation of mitochondria on sucrose gra-dient were performed as described previously [11] The