From the Mount Sinai School of Medicine, Ruttenberg
Cancer Center, ¶ Howard Hughes Medical Institute, New York, New
York 10029
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ABSTRACT |
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In this paper, we present the molecular cloning
and characterization of a murine homolog of the Escherichia
coli chaperone ClpX. Murine ClpX shares 38% amino acid sequence
identity with the E. coli homolog and is a novel member of
the Hsp100/Clp family of molecular chaperones. ClpX localizes to human
chromosome 15q22.2-22.3 and in mouse is expressed tissue-specifically
as one transcript of ~2.9 kilobases (kb) predominantly within the
liver and as two isoforms of ~2.6 and ~2.9 kb within the testes.
Purified recombinant ClpX displays intrinsic ATPase activity, with a
Km of ~25 µM and a
Vmax of ~660 pmol min The Hsp100/Clp family of ATPases constitutes a group of molecular
chaperones that participate in a broad range of biological processes in
both prokaryotes and eukaryotes. Sequence similarity and conservation
of structural features among the over 70 known family members define
two classes, which are further subdivided into eight subfamilies (1).
All Hsp100/Clps examined have been demonstrated to assemble into
homo-oligomeric ring-shaped structures and to modulate substrates in an
ATP-dependent manner (1, 2). Specific substrate recognition
occurs through protein-protein interactions directed by the PDZ-like
domains of the Hsp100/Clp family members (3). Members of the family
participate in the disaggregation of improperly folded and damaged
proteins, the facilitation of DNA transposition, the selective
coordination of substrate degradation, the regulation of the
inheritance of prion-like factors, and the modulation of gene
expression (reviewed by Schirmer et al. (1)). Despite the
involvement of Hsp100s in such diverse processes, it is the conserved
structural organization of the members that suggests that these varied
functions may involve a common mechanism governing disassembly of
high-order quartenary protein complexes (1, 4-6).
E. coli ClpX is a heat-shock protein (7-9) of the class II
Hsp100/Clp subfamily (1) and can act alone as a molecular chaperone. It
is an essential component of the Mu transposase life cycle where it
mediates dissociation of stable MuA tetramer-DNA complexes (10, 11).
Deletion of ClpX blocks the growth of Mu by arresting transposition at
the transition between the recombination and DNA replication stages
(10-12). The molecular chaperone properties of E. coli ClpX
are further supported by its capacity to prevent the heat inactivation
of the bacteriophage While E. coli ClpX can function alone as a bona
fide molecular chaperone, it also contributes to a number of
processes as a regulatory subunit of the broad specificity,
energy-dependent protease ClpP. In E. coli, ClpX
and ClpP are translated from a single heat-shock-inducible transcript
in accordance with their involvement in stress tolerance (7-9). In
this two-component chaperone-protease system, ClpX does not refold
proteins to mediate functional reactivation but rather utilizes its
chaperone activity to selectively target specific substrates for
degradation by channeling them into the proteolytic chamber of the
two-ring ClpP tetradecamer. Negative staining electron microscopy (14)
and crystallographic analysis of ClpP (15) reveal a structural
organization that is homologous to that of the eukaryotic 26 S
proteasome. Specific targets of ClpX/P include While prokaryotes possess pan-cellular chaperone distribution,
eukaryotes require compartment-specific chaperones to negotiate and
maintain polypeptide chains within the appropriate tertiary structure
and to facilitate the degradation of unsalvageable or transient protein
molecules and complexes (25, 26). As expected from the proposed
endosymbiotic origins of the mitochondria (27, 28), the molecular
chaperones that direct mitochondrial function display significant
sequence homolog to bacterial counterparts. In particular,
mitochondrial proteins Hsp70, chaperonins Hsp60/Hsp10 and Hsp78 are
homologous to E. coli DnaK, GroEL/GroES, and the class I
Hsp100/Clp family, respectively. The conspicuous absence of a
eukaryotic homolog of the ClpX class II Hsp/Clp subfamily was remedied
following the sequencing of the complete genome of Saccharomyces
cerevisiae (29, 56) and the subsequent demonstration that the gene
product termed Mcx1p partitioned to the mitochondria (30). However, a
mammalian homolog had remained until this point unidentified. The
existence of such a member has been strongly implied by the cloning of
a human homolog of ClpP that sorts to the mitochondrial matrix (31,
32).
In this report, we describe the identification and initial
characterization of murine ClpX, a novel mammalian member of the Hsp100/Clp family of molecular chaperones that displays distinct sequence similarity with its E. coli counterpart. We
demonstrate that murine ClpX is directed to the mitochondria by an
N-terminal targeting peptide. In line with its likely role as a
mitochondrial molecular chaperone, we show that ClpX possesses an
intrinsic ATPase activity that is resilient in vitro to
fluctuations in reaction conditions reflecting environmental stress.
Its capacity to interact with mouse ClpP in mammalian overexpression
experiments suggests that mouse ClpX/P may represent a novel system for
the regulation of mitochondrial protein homeostasis.
Cloning of the Murine Homolog of Bacterial ClpX, Chromosomal
Localization, and Tissue Expression--
Blast searches of the data
base of expressed sequence tags (dBEST) (33) using E. coli
ClpX revealed the partial murine ClpX EST clone Aa013832 (Image
Consortium clone 441677). This ClpX fragment was random prime
[ Construction of ClpX in Vitro Transcription-Translation and
Mammalian Expression Vectors--
To create serial N-terminal
deletions of ClpX in the Sp6 promoter driven vector pCEV27, PCR
products bearing optimal Kozak translation start sequences were cloned
between BamHI of the polylinker and an AvaI site
internal to ClpX (pCEV27ClpXM1, pCEV27ClpXM2, pCEV27ClpX Cell Culture and Recombinant Eukaryotic Protein
Expression--
The human embryonic kidney fibroblast line, 293T, was
grown in Dulbecco's modified Eagle's medium containing 5% fetal
bovine serum at 37 °C in a 5% CO2 atmosphere. GST
fusion proteins of ClpX were overexpressed from the pEBG vector (see
above) by transient transfection of 293T cells at 25% confluence using
the calcium phosphate precipitation method. Cells were harvested
48 h post-transfection and processed as described previously (38)
and dialyzed in 25 mM Tris, pH 8.0, 200 mM
NaCl, 2 mM EDTA, 10 mM ATPase and Nucleotide Binding Assays--
Indicated
concentrations of recombinant GSTClpX wild-type and mutant proteins
were incubated in a 20-µl reaction with 25 mM Tris, pH
7.4, 10 mM MgCl2, 1 mM
dithiothreitol, BSA 0.1 µg/µl, 1-500 µM ATP, and 0.5 µl of [ ClpX/ClpP Protein Interaction--
Recombinant proteins, GSTClpX
full-length or N-terminal truncations in pEBG, and 3'-HA-tagged ClpP in
pEBB were transiently overexpressed in immunoprecipitation cells using
calcium phosphate precipitation. Cells were harvested in phosphate
buffered saline (PBS) 48 h post-transfection and freezed-thawed
once at room temperature. Cells were resuspended in immunoprecipitation
lysis buffer containing 20 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM phenylmethysulfonyl fluoride, 0.1 µM
aprotinin, 1 µM leupeptin, and 1 µl of pepstatin and
sonicated extensively. The lysate was cleared in a microcentrifuge for
15 min at 14,000 rpm and 4 OC. An aliquot was removed for
protein expression evaluation, and the remainder of the lysate was
incubated on glutathione beads for 2 h at 4 °C with gentle
rocking. The beads were washed five times with mild vortexing in
immunoprecipitation lysis buffer and finally resuspended in 2 × SDS-PAGE loading buffer. The interaction was subsequently evaluated
with Western analysis.
Cellular Localization of Recombinant ClpX--
10 µg of
pEGFPN3, pClpXM1EGFP, pClpXM2EGFP, or
pClpX Isolation of a Murine Homolog of Bacterial ClpX and Evolutionary
Conservation of Functional Domains--
Due to our laboratory's
interest in the remodeling of higher order nucleoprotein complexes in
mammalian transposition and recombination reactions, we screened the
data base of expressed sequenced tags (dBEST) (33) for a mouse homolog
of the E. coli ATP-dependent remodeling factor
ClpX. Identification of EST clone Aa013832 permitted isolation of a
full-length cDNA of 2847 base pairs encoding mouse ClpX (Fig.
1A) from a keratinocyte library. The
potential open reading frame is demarcated by two stop codons, a 5' TGA
(nt 128-130) and a 3' TAA (nt 2075-2077), which contain two possible
sites of translation initiation, ATG1 (nt 179-181: M1) and
ATG2 (nt 287-289: M2). From ATG1, the cDNA comprises
an open reading frame of 1896 base pairs and encodes a predicted protein of 632 amino acids with a molecular mass of 69,174 Da and a
theoretical isoelectric point of 7.76. The mouse ClpX as compared with
Caenorhabditis elegans, E. coli, and S. cerevisiae ClpX demonstrates 40, 38, and 32% amino acid identity
and 59, 46, and 48% similarity, respectively (Fig. 1B). A
consensus polyadenylation site (AATAAA) is located 14 base pairs before
the 3' poly(A) start (Fig. 1A).
Functional analysis of mouse ClpX is dependent upon an understanding of
its component motifs. We thereby note that the primary structure of the
69-kDa mouse ClpX polypeptide displays three discernable domains that
are evolutionary conserved through E. coli (Fig. 1,
A and B). First, along with the C. elegans and E. coli homologs, murine ClpX possesses
toward the N terminus a C4-type zinc finger (amino acids 106-131) of
unknown function. Second, ClpX bears a characteristic ATPase motif with
a classic P-loop (amino acids 290-305) and Walker B Mg2+
binding pocket (amino acids 355-362) with distinct similarity to those
of the F1-ATPase and P-type transporters (40). Third, the C
terminus contains two tandem PDZ-like domains, which display 44, 31, and 41% identity and 65, 58, and 58% similarity with the PDZ-like
domains of C. elegans, S. cerevisiae, and
E. coli, respectively. In E. coli, these domains
mediate specific substrate recognition (3). In addition to these three
highly conserved motifs, mouse ClpX possesses at the N terminus an
apparent mitochondrial targeting sequence (41-43) spanning as much as
the first 65 amino acids of the protein (Fig. 1A). This
region is characterized by the predicted capacity to form an
amphiphilic helix coupled with a biased distribution of positively
charged amino acids (7 arginines), an abundance of hydroxylated
residues (13 serines or threonines), and a paucity of negatively
charged amino acids (1 aspartate and 1 glutamate). Maturation of the
ClpX preprotein can be predicted to result following cleavage by the
mitochondrial processing peptidase at three putative sites, one site
within an R-2 motif and another two sites within an R-10 motif (Fig.
1A) (42, 43).
To determine whether translation preferentially initiates at AUG 1 or
2, the migration was compared between full-length ClpX cDNA
in vitro translation products and the products from
truncated forms of ClpX starting from either ATG1 (ClpXM1),
ATG2 (ClpXM2), or an N-terminal deletion of the first 65 amino acids (ClpX Tissue-specific Expression and Assignment of Chromosomal
Localization--
Northern blot analysis was used to study the tissue
distribution of ClpX encoding mRNA. Samples of poly(A)+
RNA from eight different BALB/c mouse tissues were hybridized with
labeled probe specific for ClpX (Fig. 2A). The relative
expression level is highly variable between different tissues. ClpX was
expressed predominantly in the liver as a single transcript of ~2.9
kb and in the testes as two transcripts of ~2.6 and ~2.9 kb. The
length of the 2.9-kb mRNA corresponds to the size of the
full-length cDNA (Fig. 1A). The two mRNAs in the
testes may result from alternative RNA splicing. Lower expression of
the 2.9-kb ClpX transcript was detected in the heart and kidney. Very
low levels were observed in skeletal muscle (upon overexposure) with no
apparent expression detectable in the brain, spleen, and lung.
Human chromosomal localization of ClpX was assigned using the Gene Map
of the Human Genome, which is a physical map of cDNA-based sequence-tagged sites (35, 36) available on the World Wide Web. This
resource of currently 30,181 genes utilizes the unique 3'-untranslated
region of a cDNA to position ESTs relative to microsatellite
markers by radiation hybrid mapping with an error rate of 1.08%. ClpX
was typed on a Genebridge4 radiation hybrid panel using PCR primers
designed to the 3'-untranslated region of the human gene and determined
to map to chromosome 15q22.2-22.3 between fixed reference markers
D15S125 and D15S216.
ATPase Activity of Mouse ClpX--
The presence of a consensus
ATPase motif (Fig. 1A) led us to analyze the kinetics of ATP
hydrolysis by ClpX. Overexpressed recombinant ClpX was purified from a
human kidney fibroblast cell line, 293T, as a GST fusion protein (Fig.
3A). Titration of GSTClpX
To demonstrate that the ATPase activity is intrinsic to ClpX, lysine
300 of the P-loop motif was replaced by alanine (K300A) (Fig.
4A, lane 4).
Consonant with the role of this lysine in other ATPases as an integral
structural component of the P-loop and a point of coordination for the
Both of the two previously cloned members of the Hsp100/Clp family in
S. cerevisiae, Hsp104 and Hsp78, mediate stress tolerance under conditions of extreme temperature (6, 45), and Hsp104 is also of
critical importance for tolerance to ethanol (46). Hence, we analyzed
the ATPase profile of ClpX under various conditions reflecting
environmental stress (4). Interestingly, ClpX is resilient to varied
alterations in reaction conditions. ATP hydrolysis was essentially
unaltered over a pH range of 6.8-8.8 (Fig.
5A) and decreased only 15%
when the salt range was varied from the physiological value of 150-450
mM NaCl (Fig. 5B). In addition, ClpX retained
75% of wild-type activity in 20% ethanol (Fig. 5C) and
90% of wild-type activity at 55 °C (Fig. 5D). The
ability to function across a broad scope of reaction parameters
suggests that ClpX may function along with the other Hsp100/Clp family members in response to cellular stress.
ClpX Interacts with ClpP--
The absence of an open reading frame
in the complete yeast genome demonstrating distinct homology to
bacterial ClpP (29, 56) suggests the possibility that ClpX may have
evolved in eukaryotes to function independently of ClpP. To address
whether mouse ClpX could still associate to form a complex with mouse
ClpP, we performed co-precipitation assays between N-terminal serial
deletions of ClpX fused in frame with the C terminus of glutathione
S-transferase and ClpP (amino acids 56-272) with a
C-terminal HA tag. Recombinant proteins were overexpressed in 293T
cells and interaction was evaluated by affinity precipitation of
complexes on glutathione beads. GSTClpXM1 does not interact
nonspecifically with any anti-HA cross-reactive species in 293T cells
not transfected with ClpP3'HA (Fig. 6,
lane 1 in the middle panel). GST alone did not
interact with ClpP3'HA (Fig. 6, lane 2 in the middle
panel). GSTClpXM1, GSTClpXM2, and
GSTClpX Subcellular Sorting of Mouse ClpX-Green Fluorescent Protein
Fusions--
The presence of an apparent mitochondrial targeting
sequence on the N terminus of ClpX strongly suggested that ClpX would localize to the mitochondria along with its interacting partner protein
ClpP (31). To explore the intracellular distribution of ClpX, we fused
the C terminus of the full-length ClpXM1 preprotein with
the N terminus of a red-shifted variant of green fluorescent protein
(EGFP). In addition, the possibility that use of the second ATG
(M2) might direct differential compartmentalization of this in vitro minor translation species (Fig. 2B) was
explored using a ClpXM2-EGFP fusion initiated directly
from the second methionine. The fusions were overexpressed in 293T
cells and analyzed using confocal laser scanning microscopy. With EGFP
alone, fluorescence was observed throughout the nucleus and cytoplasm
(Fig. 7A). On the other hand,
both the ClpXM1-EGFP and ClpXM2-EGFP fusions
generated fluorescence in the form of discrete cytoplasmic rod-like
elements, suggesting distribution of ClpX to the paracrystalline
structures of the mitochondria (Fig. 7, B and C).
To verify that the punctate staining suggestive of mitochondrial
localization was dependent on the integrity of the N-terminal 65-amino
acid targeting peptide, we analyzed distribution of ClpX The current study presents the cloning and initial
characterization of murine ClpX, a novel member of the Hsp100/Clp
family of molecular chaperones and energy-dependent
protease regulators. The encoded protein of 632 amino acids is a class
II member as characterized by its general structural organization as
well as the presence of only one and not two nucleotide binding domains (1). Sequence homology exhibited between the E. coli,
S. cerevisiae, C. elegans, and mouse members of
this chaperone subfamily is concentrated toward the central and
C-terminal regions of the molecules corresponding to the ATPase and the
PDZ-like substrate recognition domains. Significant amino acid sequence
divergence is observed at the N terminus due to the nonconserved nature
of mitochondrial targeting peptides and the obvious absence of the
peptide in E. coli. An additional N-terminal difference is
that only three of the homologs contain a C4-zinc finger, with its
noticeable absence in S. cerevisiae (Fig. 1B).
Since a ClpP homolog is also not evident in S. cerevisiae (29, 56) the C4-zinc finger of E. coli, C. elegans, and mouse ClpX is a potential candidate for mediating
recruitment of ClpP. However, as is the case with DNA J, the C4-zinc
binding domain may also direct recognition and binding of denatured
protein substrates (47). It is interesting to speculate that ClpX may
possess a bimodal capacity for substrate recognition with the
N-terminal C4-zinc finger nonspecifically contacting denatured
polypetides and the C-terminal PDZ domains directing specific
interactions with native proteins.
Since the molecular chaperone activity of Hsp100/Clp family members has
been directly linked to their ability to hydrolyze ATP (13, 48, 49), it
is noteworthy that the Km of basal ATPase hydrolysis
by ClpX is ~25 µM. This value is over 20- (low salt) to
200-fold (high salt) lower than that obtained for Hsp104 (4), 20-fold
lower than that for E. coli ClpX (13), 8-fold lower than
that for E. coli ClpA (48), and over 40-fold lower than the
value for ClpB (50). While the basis of these differences is presently
unclear, the resiliency of ClpX ATPase activity under conditions
mimicking cellular stress (4) supports its membership in a Hsp100 class
of stress tolerance proteins (6-9, 30, 45, 51).
The absence of multiple transcripts in all tissues but the testes and
the direct correspondence of the ~2.9-kb cDNA, which we have
cloned with the single ~2.9-kb transcript identified by Northern
analysis, suggest the existence of a single form of ClpX in most
tissues. Within this single transcript, the identification of two
alternative start ATGs (M1 and M2) by in
vitro transcription/translation experiments initially posed the
exciting possibility that both mitochondrial and cytosolic forms of
ClpX could be generated from the same transcript as has been documented
for other proteins (52, 53). Such a mechanism for subcellular
compartmentalization would be both biologically economical as well as
resourceful, since it could provide a means of controlling differential
compartmentalization in response to cellular metabolic status. However,
GFP fusions with ClpXM1 and ClpXM2 both
localized to the mitochondria and suggest that mice, like yeast, do not
possess a cytosolic form of ClpX. Indeed, the truncated second form
observed in the transcription/translation reactions may simply
represent an aberrant product of the in vitro reaction.
The tissue-specific pattern of ClpX expression is inconsistent with a
role as a constitutive chaperone and suggests that ClpX has acquired
tissue-specific mitochondrial functions. The lack of an essential
requirement of eukaryotic mitochondrial ClpX for general cell viability
under normal growth conditions has been supported by the absence of an
obvious phenotypic effect following disruption of the ClpX homolog in
yeast (30). It is particularly interesting that mouse ClpX is most
highly expressed in the liver where the mitochondria participate in
numerous cell type-specific functions. These liver-specific
mitochondrial processes include the oxidation of drugs and other toxic
compounds, the formation of ketone bodies, the synthesis of components
of fatty acid precursors, and the generation of critical components of
the nitrogen metabolic pathway. Elucidation of the cis-acting elements
responsible for the varied pattern of basal tissue transcription and
the characterization of potential stress response elements including
the heat shock promoter element binding sites for heat shock
transcription factors (54) awaits upcoming promoter analysis.
Interestingly, the common regulatory mechanism governing expression of
bacterial ClpX and ClpP (7), which are translated from a single
transcript appears to be lost in humans where ClpX localizes to
chromosome 15q22.2-22.3 and ClpP to chromosome 19q13. The divergent
regulatory control is supported by the highly distinct pattern of ClpX
and ClpP transcripts found in analogous tissues (this study and Ref.
30). Such divergence suggests that ClpX and ClpP may have acquired some
independent cellular functions. However, the ability of ClpX and ClpP
to function as part of a chaperone-protease system is still implied by
their capacity to form a stable complex in vivo.
Identification of the eukaryotic targets of mouse ClpX may be informed
by parallels with the bacterial system. E. coli ClpX binds
to the C-terminal 7-11 amino acids of the Mu transposase (22), of the
Mu repressor (24), of the SsrA C-terminal peptide tails (18), and of
the C. crescentus cell-cycle regulator CtrA (21) through a
direct interaction with the ClpX C-terminal PDZ domains (3). Although
not highly conserved in amino acid sequence, this C-terminal targeting
sequence (CTS) is characterized by a central charged core flanked by
hydrophobic amino acids. Transfer of the CTS to heterologous proteins
transforms them into substrates for E. coli ClpX function
(22). Since the PDZ domains of mouse are approximately 60% similar to
those of C. elegans, S. cerevisiae, and E. coli, it is highly plausible that the mode and targets of
substrate recognition by ClpX will also be evolutionarily conserved. To
identify eukaryotic mitochondrial proteins possessing a CTS homologous
to that of MuA, the Mu repressor, SsrA proteins, or CtrA, we analyzed
the mitochondrial subcategory of the Yeast Protein Data base
(http://www.proteome.com/YPDhome.html) (55). Since this resource is a
compilation of nearly all yeast mitochondrial genes as identified from
the complete sequence of the yeast genome (29, 56), it provides a
thorough representation of the entire range of potential ClpX
substrates. Interestingly, of the 293 mitochondrial proteins in the
data base, only nine fulfilled our criteria (see "Experimental
Procedures") for homology to the CTS within the last 11 amino acids:
electron transferring flavoprotein, 1
µg
1, which is active over a broad range of pH,
temperature, ethanol, and salt parameters. Substitution of lysine 300 with alanine in the ATPase domain P-loop abolishes both ATP hydrolysis
and binding. Recombinant ClpX can also interact with its putative
partner protease subunit ClpP in overexpression experiments in 293T
cells. Subcellular studies by confocal laser scanning microscopy
localized murine ClpX green fluorescent protein fusions to the
mitochondria. Deletion of the N-terminal mitochondrial targeting
sequence abolished mitochondrial compartmentalization. Our results thus
suggest that murine ClpX acts as a tissue-specific mammalian
mitochondrial chaperone that may play a role in mitochondrial protein homeostasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
O replication protein, to dissociate
preformed
O aggregates, and to stimulate the binding of
O to
ori
DNA (13).
O (7, 16), starvation
sigma factor (
S) (17), SsrA-tagged proteins generated
from defective mRNAs (18), the Phd protein of plasmid prophage P1
(19), the Caulobacter crescentus cell cycle regulator CtrA
protein (20, 21), MuA (22), and the Mu repressor protein (23, 24). Many
of these substrates possess 7-11 amino acid C-terminal peptides that
are required for recognition by the PDZ-like domains of ClpX (3).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP-labeled with Klenow fragment and used to
screen a mouse keratinocyte library BALB/MK (34). Full-length murine
ClpX in pCEV27 was identified (pM11/18) and nucleotide sequence was
obtained by automated sequencing of both strands (Utah State University Biotechnology Center). A human chromosomal location of ClpX was assigned by analyzing the physical map of the human genome available at
http://www.ncbi.nlm.nih.gov/genemap/ (35, 36). A mouse Multiple Tissue Northern (CLONTECH) was hybridized
as recommended by the manufacturer with an [
-32P]dCTP
body-labeled PCR1 fragment
generated using primers 467 (5'-ATGTTAGGAAGACTGGGGACG-3') and 238 (5'-TTATAATGATTATACACGGC-3'). The C terminus of proteins were
considered homologous to the C-terminal targeting sequence of bacterial
ClpX substrates if all four of the following criteria were fulfilled:
1) the final two amino acids were nonpolar, 2) the preceding 7 amino
acids were preferentially polar or charged, 3) at least 6 of the 11 amino acids were similar to the final amino acids of the SsrA tag (18),
and 4) at least 1 of the first 2 amino acids was nonpolar.
65). To generate fusions to the C terminus of glutathione S-tranferase of the N-terminal serial deletions,
BamHI fragments from the start ATG to internal to the
3'-untranslated region (nt 2439) were subcloned from the pCEV27 vectors
into the eIF2 promoter driven mammalian expression vector pEBG (37)
(pEBGClpXM1, pEBGClpXM2, and pEBGClpX
65).
The mutation of K300A was introduced with a mutated PCR oligonucleotide
and the fragment was subcloned between AvaI (nt. 628) and
XbaI (nt 1341) (pEBGClpXM2K300A). Red-shifted green fluorescent protein fusions in the mammalian expression vector
pEGFPN3 (CLONTECH) were generated by PCR
amplification from the ApaLI site (nt 1853) to the C
terminus, which was fused in frame with the N terminus of EGFP using
the KpnI site. The N terminus of ClpX was inserted
simultaneously as a BamHI-ApaLI fragment using
the 5' polylinker site BglII (pClpXEGFPM1,
pClpXEGFPM2, and pClpXEGFP
65). The mouse homolog of ClpP
(GenBankTM accession number AJ005253) was amplified by PCR
from the mouse keratinocyte library BALB/MK. The fragment spanning the
nucleotides coding for amino acids 56-272 (with a deletion of the
first 55 N-terminal amino acids, which are proteolytically removed in
mature ClpP) was fused in frame with a C-terminal HA tag (amino
acids:YPYDVPDYA) in the eIF2 promoter vector pEBB (pEBBClpP
55
3'-HA). All PCR-generated constructs were confirmed by sequencing.
-mercaptoethanol, and
20% glycerol. Protein quantitation was conducted following SDS-PAGE
and Coomassie staining using dilutions of bovine serum albumin as a standard.
-32P]ATP (3.3 µM; 3000 Ci/mM, Amersham Pharmacia Biotech) at 37 °C for
indicated times. Reactions were stopped by the addition of 25 mM EDTA and freezing at
70OC.
th
of the reaction volume was spotted onto thin layer chromatography (TLC)
plates (polyethyleneimine cellulose F; EM Science), and separation of
Pi from ATP was attained in 1.0 M formic acid
and 0.5 M LiCl2. Following autoradiography, quantitation of hydrolyzed ATP was determined by Molecular Analyst phosphoimaging (Bio-Rad). Nucleotide binding studies (39) were performed in 20-µl binding reactions with 25 mM Tris, pH
7.4, 10 mM MgCl2, 1 mM
dithiothreitol, 0.1 µg/µl BSA, 0.33 µM
[
-32P]ATP (3,000 Ci/mM and 3.3 µM) and 0.1 µM wild-type or mutant GSTClpX
protein for 10 min at 4OC. Free nucleotides were removed
with G-50 Sephadex spin columns (Roche Molecular Biochemicals),
collected in tubes containing 5 µl of 500 mM EDTA, pH
7.5, and placed immediately on dry ice to stop the reaction. Bound
nucleotides in the spin-through were quantitated on a Beckman LS 6500 scintillation system and the ratio of ADP/ATP in the flow-through was
determined following separation of an aliquot of the spin-through by
TLC. Following autoradiography, values were derived by phosphoimaging.
65EGFP were transiently transfected in 293T cells as described
above. Thirty-six hours post-transfection, the cells were incubated
with 1 µM Mitotracker® Red CMXros (Molecular
Probes) for 2 h at 37 OC. Cells were rinsed once in
PBS prewarmed to 37 OC, incubated for 20 min in 4%
paraformaldehyde, rinsed three times in room temperature PBS, fixed for
15 min in cold methanol/acetone (1:1), and rehydrated in PBS for 10 min. Following three washes in PBS + 1% BSA, the cells were incubated
for 10 min at room temperature with 5 µg/ml
4',6'-diaminidino-2-phenylindole (Sigma) in PBS + 1% BSA. Following
three washes in PBS + 1% BSA, the coverslips were mounted. Images were
acquired using confocal laser microscopy.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
cDNA and deduced amino acid sequence of
murine ClpX indicating evolutionary conservation of protein
domains. A, the cDNA and protein sequences of
murine ClpX are displayed. Putative translation initiation and stop
codons are emphasized in uppercase letters. Two possible
start methionines are indicated (M1 and M2) in
bold and the three consensus cleavage sites (/1
of the R-2 motif and /2 and /3 of the R-10
motif) (42) for the matrix processing peptidase are marked. The
predicted C4-zinc finger is underlined by dots with the
putative Zn2+ coordinating cysteines highlighted in
bold. The P-loop and Walker B sites of the ATPase motif are
single underlined with lysine 300 of the GKT motif indicated
in bold. The region displaying homology to PDZ domains is
double underlined with the boundary between PDZ domain A and
B demarcated by a //. The polyadenylation consensus is single
underlined and in small caps. B, protein
sequence alignment of mouse, C. elegans
(GenBankTM accession number Z73906), E. coli
(GenBankTM accession number Z23278), and S. cerevisiae (GenBankTM accession number Z36096) ClpX
with areas of similarity shaded. Percentage amino acid
identity and similarity reported in the text were calculated using the
putative mature form of mouse ClpX cleaved after the first 65 amino
acids.
65) (Fig.
2B). While over 98% of the
translation products from the full-length cDNA (Fig. 2B,
arrow a in lane 1) co-migrated with ClpXM1 (lane 2), a minor species was produced
(arrow b in lane 1) that co-migrated with the
ClpXM2 product (lane 3). The preponderance of
initiation at AUG1 suggests that this is the biologically relevant start site. However, while translation initiation at AUG2 may be
specific to the in vitro transcription/translation system
and may not occur in vivo, the possibility that the two
translation isoforms are targeted differentially to separate
subcellular compartments lead us to characterize both start forms in
subsequent studies.
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Fig. 2.
Northern blot analysis of mouse
poly(A)+ RNA from a multiple tissue Northern blot and
initiation codon usage analysis by in vitro
transcription-translation. A, a multiple tissue
Northern blot (CLONTECH) of poly(A)+
RNA from BALB/c mouse heart (lane 1), brain (lane
2), spleen (lane 3), lung (lane 4), liver
(lane 5), skeletal muscle (lane 6), kidney
(lane 7), and testis (lane 8) was probed with a
32P-body-labeled PCR product spanning nucleotides 287-777.
Numbers on the left indicate sizes in kilobases.
B, [35S]methionine-labeled forms of ClpX were
generated using a coupled in vitro transcription-translation
rabbit reticulocyte lysate system and resolved by SDS-PAGE. Lane
1 contains translation products (arrows a and
b) from the ClpX full-length cDNA (pM11/18) with the
entire reported 5'-untranslated region and both initiation codons ATG1
(M1) and ATG2 (M2) intact. Migration can be
compared with products initiated directlyfrom either ATG1 in the
absence of a 5'-untranslated region (pCEV27ClpXM1)
(lane 2), from ATG2 (pCEV27ClpXM2) (lane
3), or from a deletion of the first 65 amino acids spanning the
mitochondrial targeting peptide (pCEV27ClpX 65) (lane
4).
65 (Fig.
3B) and a time course of Pi release (Fig.
3C) revealed linear kinetics in the presence of 0.05 µM ClpX over an incubation of 5-10 min. These conditions
were used for all subsequent assays. ClpX hydrolyzed ATP with a
Km of ~25 µM and a
Vmax of ~660 pmol min
1
µg
1, which corresponds to the hydrolysis of ~1
molecule of ATP/molecule of ClpX/s (Fig. 3D). All three
fusions shown in Fig. 3A display equivalent reaction
kinetics. When the ATPase activity of ClpX was tested at 150 mM NaCl to more closely resemble physiological ionic
strength, the Km was essentially unchanged (data not
shown). Either Mg2+ or Mn2+ was required for
efficient ClpX ATPase activity. Like ClpA and ClpB, ClpX retained
partial ATPase activity in Ca2+. Unlike Hsp104, which
hydrolyzes ATP in Co2+ and Ni2+ (4), ClpX
demonstrated negligible activity using these divalent cations (Table
I).
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Fig. 3.
Characterization of the ATPase activity of
murine ClpX. A, SDS-PAGE analysis of
GSTClpXM1 (lane 1), GSTClpXM2
(lane 2), and GSTClpX 65 (lane 3) purified
following transient overexpression in 293T cells. B,
picomoles of ATP hydrolyzed versus concentration of ClpX.
The amount of ATP hydrolyzed in 6 min at 37 °C by increasing amounts
of GSTClpX
65 (0.006-0.2 µM) in reactions containing
1000 pmol ATP (50 µM final ATP concentration) was
determined. Pi was separated from unhydrolyzed ATP by thin
layer chromatography, and amounts were quantitated following
autoradiography. C, time course of ATPase activity;
picomoles of ATP hydrolyzed at 37 °C over time (minutes) by 0.05 and
0.025 µM GSTClpX
65 with 1000 pmol ATP (50 µM) as the initial substrate amount. D, rate
of hydrolysis was determined from the picomoles of Pi
released over 6 min by 100 ng of ClpX (0.05 µM).
Km and Vmax values were also
derived from a Lineweaver-Burk plot of the same values (graph not
shown). Reaction rates were calculated using 0.05 µM
GSTClpX
65 at initial ATP concentration values of 5, 10, 15, 25, 50, 100, 200, and 250 µM ATP. GSTClpXM1 and
GSTClpXM2 yielded similar Km and
Vmax values.
ATPase activity of ClpX in the presence of various divalent cations
65
at 37 °C for 6 min with 50 µM initial ATP (1000 pmol)
and 10 mM amounts of the indicated divalent cation. No
activity was noted in the presence of 10 mM EDTA. The
percent activity reported is standardized with hydrolysis in
Mg2+ as 100%. The divalent ion dependence of other Hsp100/Clp
family members has been compiled previously by Schirmer et
al. (4).
- and
-phosphates of bound nucleotides (44), the mutation
entirely abolished detectable ClpX mediated ATPase activity (Fig.
4B). Next, the effect of the mutation on nucleotide binding
was evaluated to determine whether the deficiency in hydrolysis
resulted from inability to recruit ATP. At 4 OC, wild-type
GSTClpXM1, GSTClpXM2, and GSTClpX
65
associated with both ATP and ADP (Fig. 4C). The ratio of
bound ATP to bound ADP was about 1:5. This association required the
presence of Mg2+ (data not shown). In contrast,
GSTClpX
65K300A binding to ATP at 4 °C was negligible (Fig.
4C), suggesting that Lys300 is critical for both
ATP binding and hydrolysis.
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Fig. 4.
ATP hydrolysis and nucleotide binding of
P-loop mutation K300A. A, SDS-PAGE analysis of GST,
wild-type GSTClpX 65, and the P-loop substitution GSTClpX
65K300A.
B, time courses of ATP hydrolysis for reactions containing
0.05 µM GSTClpX
65 or 0.05 µM
GSTClpX
65K300A at 37 °C with 1000 pmol as the initial amount of
ATP. C, nucleotide binding was assayed by incubating ATPase
assay reactions containing 0.05 µM protein at 4 °C for
10 min and removing unincorporated nucleotides with G-50
Sephadex® spin columns. The ADP/ATP ratio of the bound
nucleotides was determined following separation by thin layer
chromatography for reactions with no protein, GST alone,
GSTClpX
65K300A, GSTClpXM1, GSTClpXM2, and
GSTClpX
65. Total bound nucleotides, bound ADP, and bound ATP for
each protein reaction are reported as counts per minute. Experiments
were repeated three times with deviations from the mean <10%.
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Fig. 5.
ClpX ATPase activity under stress
conditions. The ATPase activity of GSTClpX 65 under conditions
of varied pH (A), salt (B), ethanol
(C), and temperature (D). Reactions were
conducted at 37 °C for 6 min using 0.05 µM
GSTClpX
65 and 1000 pmol of ATP.
65 all co-precipitated ClpP3'HA with approximately equal
efficiency (Fig. 6, lanes 3-5 in the middle
panel). Precipitated levels of the GST and GSTClpX proteins were
evaluated by Western analysis (Fig. 6, upper panel).
Expression levels of ClpP3'HA were assessed by Western analysis of
whole cell extracts from the transfected cells (Fig. 6, lower
panel). As a control, GSTClpX proteins did not interact with
various HA-tagged forms of Rag 1 and 2 proteins (data not shown). In
all, the data demonstrate the capacity of mouse ClpX/ClpP to form a
stable complex. This interaction suggests that mouse ClpX may function
analogously to E. coli ClpX as an
energy-dependent regulator of ClpP function.
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Fig. 6.
Interaction of ClpX and ClpP.
Co-precipitation assays were used to analyze the interaction between
wild-type or mutant recombinant GSTClpX proteins transiently expressed
in 293T cells with a N-terminally truncated mouse ClpP ( 55) tagged
at the C terminus with an HA peptide. Interaction with ClpP(3'HA) was
assayed with GST alone, GSTClpXM1, GSTClpXM2,
and GSTClpX
65 by co-precipitation with glutathione beads followed by
Western analysis. The upper panel reveals the amount of GST
or GSTClpX fusions precipitated in each lane as monitored by a
monoclonal anti-GST antibody. The middle panel represents
interacting ClpP(3'HA) as detected by a monoclonal anti-HA antibody
12CA5. Expression levels of ClpP(3'HA) are depicted in the lower
panel by an anti-HA blot of total cellular lysates.
65-EGFP
lacking the targeting sequence. Consistent with the existence of an
N-terminal mitochondrial targeting peptide, this deletion ablated the
punctate fluorescence and, in turn, generated homogenous cytoplasmic
staining (Fig. 7D). Subcellular compartmentalization of
ClpXM1-EGFP to the mitochondria was further supported by
co-localization studies using a mitochondrion-selective dye,
Mitotracker® Red (Fig. 7, E-G), in which
co-segregation of the EGFP fluorescence (Fig. 7E) with the
rhodamine emission of Mitotracker® (Fig. 7F)
was observed through numerous confocal sections (Fig. 7G).
Similar co-localization was observed for ClpXM2-EGFP (data not shown).
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Fig. 7.
Subcellular localization of murine ClpX in
transfected 293T cells using green fluorescent protein. Serial
N-terminal deletions of ClpX were overexpressed as fusions to the N
terminus of enhanced green fluorescent protein (EGFP). The 293T cells
were fixed and processed 24-36 h post-transfection and visualized by
confocal laser-scanning microscopy. The nuclei in all panels are
visualized by 4',6'-diaminidino-2-phenylindole autofluorescence. Cells
in A were transfected with EGFP alone. In B and
C, ClpX-EGFP fusion proteins were initiated from either ATG
1 (pClpXM1EGFP) or directly from ATG 2 (pClpXM2EGFP), respectively. In D, the 65 N-terminal amino acids from the full-length preprotein were deleted,
and translation was initiated by replacing the codon for alanine 66 with an ATG (pClpX 65EGFP). In E-G,
pClpXEGFPM1 was expressed in cells stained with the
mitochondrial specific dye MitoTracker® Red CMXros. In
E, the GFP fluorescence of ClpXM1EGFP is
demonstrated. The same field revealing MitoTracker staining is
displayed in F, and co-localization of the two staining
patterns is revealed in G.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain (GenBankTM
accession number 1323371), citrate transport protein 1 (GenBankTM accession number 536746), import receptors of
the outer membrane TOM70 and TOM72 (GenBankTM accession
numbers 1302050 and 529136), RIP1 component of the cytochrome
bc1 complex (GenBankTM accession number
602391), NADH-ubiquinone oxidoreductase (GenBankTM
accession number 805022), MSH1, the yeast homolog of E. coli MutS (GenBankTM accession number 529134), proline oxidase
(GenBankTM accession number 1360564), and the E2 component
of the pyruvate dehydrogenase complex (GenBankTM accession
number 1301955). The first six potential substrates are in agreement
with the tight inner membrane association observed for S. cerevisiae ClpX (30). This information, which suggests a possible
role of ClpX in modulation of molecular import, electron transport,
biochemical pathways and cell viability during normal or stressed
cellular conditions, may ultimately prove valuable in elucidating its
role in mammalian mitochondrial protein homeostasis.
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ACKNOWLEDGEMENTS |
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We thank Zhen-Qiang Pan, Stuart Aaronson, and Patricia Cortes for their kind and generous guidance and support. We also thank Adolfo Garcia-Sastre, Ulrich Hermanto, Vassilis Aidinis, and Jose Trincao for their generous help and Larry Shapiro for critical review of the manuscript.
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Addendum |
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A human homolog has been reported recently (GenBankTM accession number AJ006267 (C. Jespersgaard, P. Bross, T. J. Corydon, B. S. Andresen, T. Kruse, L. Bolund, and N. Gregersen, unpublished data)).
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FOOTNOTES |
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* This work was supported by Department of the Army/Department of Defense Breast Cancer Predoctoral Training Grant DAMD17-94-J-4111 (to S. S.) and National Institutes of Health Grant 1RO1 AI40191-02 (to E. S.). Confocal laser scanning microscopy was performed with the guidance of Scott Henderson at Mount Sinai School of Medicine Confocal Laser Scanning Microscopy core facility, supported with funding from National Institutes of Health Shared Instrumentation Grant 1 S10 RR0 9145-01 and National Science Foundation Major Research Instrumentation Grant DBI-9724504.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to the memory of Andrew Hodtsev and Eugenia Spanopoulou.
Our mentors, Andrew Hodtsev and Eugenia Spanopoulou, who were the
motivating inspiration behind this work, were tragically killed during
the crash of Swiss Air Flight 111 on September 2, 1998.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF134983.
§ To whom correspondence should be addressed: Mount Sinai School of Medicine, Ruttenberg Cancer Center, 1425 Madison Ave., East Bldg., Box 1130, New York, NY 10029. Tel.: 212-659-5525; Fax: 212-849-2446; E-mail: santas01{at}doc.mssm.edu.
Assistant investigator in the Howard Hughes Medical Institute
and a Cancer Research Institute Clinical Investigator.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; nt, nucleotide(s); HA, hemagglutinin; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PBS, phosphate-buffered saline; EST, expressed sequence tag; GFP, green fluorescent protein; EGFP, enhanced GFP; CTS, C-terminal targeting sequence.
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REFERENCES |
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