From the Stowers Institute for Medical Research,
Kansas City, Missouri 64110, ¶ Harvard Microchemistry and
Proteomics Analysis Facility, Harvard University, Cambridge,
Massachusetts 02138,
Division of Biology, California Institute
of Technology, Pasadena, California 91125, ** Department of
Biochemistry and Molecular Biology, Kansas University Medical Center,
Kansas City, Kansas 66160, and
Department
of Biochemistry and Molecular Biology, University of Oklahoma Health
Sciences Center, Oklahoma City, Oklahoma 73190
Received for publication, February 5, 2003
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ABSTRACT |
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The Mediator is a multiprotein coactivator
required for activation of RNA polymerase II transcription by DNA
binding transactivators. We recently identified a mammalian homologue
of yeast Mediator subunit Med8 and partially purified a Med8-containing
Mediator complex from rat liver nuclei (Brower, C. S., Sato, S.,
Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner,
R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc.
Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis of
proteins present in the most highly purified Med8-containing fractions
by tandem mass spectrometry led to the identification of many known
mammalian Mediator subunits, as well as four potential Mediator
subunits exhibiting sequence similarity to yeast Mediator subunits
Srb5, Srb6, Med11, and Rox3. Here we present direct biochemical
evidence that these four proteins are bona fide mammalian
Mediator subunits. In addition, we identify direct pairwise binding
partners of these proteins among the known mammalian Mediator subunits.
Taken together, our findings identify a collection of novel
mammalian Mediator subunits and shed new light on the underlying
architecture of the mammalian Mediator complex.
Transcription of eukaryotic protein-coding genes by RNA polymerase
II and the general initiation factors is controlled by a large
collection of DNA binding transcriptional activators through the action
of an intermediary multiprotein coactivator referred to as Mediator.
The Mediator complex was first identified in Saccharomyces cerevisiae and found to be composed of more than 20 proteins
(designated Srb2, Srb4, Srb5, Srb6, Srb7, Srb8, Srb9, Srb10, Srb11,
Med1, Med2, Pgd1, Med4, Med6, Med7, Med8, Med11, Rox3, Cse2, Nut1,
Nut2, Gal11, Rgr1, and Sin4 (1)). Multiprotein mammalian Mediator complexes with structural and functional properties similar to S. cerevisiae Mediator were subsequently identified in several laboratories and variously designated
TRAP1 (thyroid hormone
receptor-associated proteins)/SMCC (SRB-MED-containing cofactor) (2,
3), DRIP (vitamin D receptor-interacting proteins) (4), ARC
(activator-recruited cofactor) (5), CRSP (cofactor required for Sp1
activation) (6), and mouse Mediator (7). Biochemical characterization
of these different mammalian Mediator complexes has revealed that they
are composed of many of the same proteins. Among the consensus
mammalian Mediator subunits are easily identifiable structural
homologues of several S. cerevisiae Mediator subunits,
including Srb7, Med6, Med7, Nut2, and Rgr1; in addition, recent
bioinformatic evidence suggests that, among the consensus mammalian
Mediator subunits, TRFP, TRAP80, TRAP240, TRAP230, Cdk8, Cyclin C,
TRAP220, and TRAP36 are homologous to S. cerevisiae Mediator
subunits Srb2, Srb4, Srb8, Srb9, Srb10, Srb11, Med1, and Med4,
respectively (8).
We recently identified a mammalian homologue of S. cerevisiae Mediator subunit Med8 and found that it is an Elongin
BC-interacting protein that can assemble with Elongins B and C and a
Cul2/Rbx1 module to reconstitute a potential E3 ubiquitin ligase. As
part of our effort to explore the possible relationship between
Mediator and the ubiquitin pathway, we have purified a Med8-containing Mediator complex from rat liver nuclei. Analysis of proteins present in
the most highly purified Med8-containing fractions by ion-trap MS/MS
identified many consensus mammalian Mediator subunits, as well as
additional proteins not previously recognized as subunits of mammalian
Mediator. In this report, we identify four of these proteins as
integral subunits of the mammalian Mediator complex.
Materials--
Anti-c-Myc (9E10) monoclonal antibody was
purchased from Roche Molecular Biochemicals. Rabbit anti-c-Myc antibody
(C-3956), anti-Flag (M2) monoclonal antibody, and anti-Flag M2 agarose
were obtained from Sigma. Anti-Med6 (E-20) antibody was from Santa Cruz
Biotechnology. Rabbit anti-Med8 antibody was raised against a peptide
corresponding to Med8 residues 247-268, and rabbit anti-p28b, anti-Surf5, anti-HSPC296, anti-LCMR1, and anti-FLJ23445 antibodies were
raised against full-length recombinant proteins produced in either
Escherichia coli or insect cells (Cocalico Biologicals, Inc). Rabbit anti-TRAP80 antibody was kindly provided by Dr.
R. G. Roeder.
Expression of Recombinant Proteins in Insect
Cells--
Sf21 cells were cultured at 27 °C in Sf-900 II
SFM (Invitrogen) with 10% fetal calf serum, 100 units/ml penicillin,
and 100 µg/ml streptomycin. 1 × 108 Sf21
cells in suspension (for large-scale preparations used in preparative
chromatography) or 1 × 107 Sf21 cells in
monolayer cultures (for small-scale preparations used for analytical
immunoprecipitations) were infected at a multiplicity of infection of
~5-10 with the recombinant baculoviruses indicated in the figures.
Forty-eight hours after infection, cells were collected and lysed in
ice-cold buffer containing 50 mM Hepes-NaOH (pH 7.9), 500 mM KCl, 5 mM MgCl2, 0.2% (v/v)
Triton X-100, 20% (v/v) glycerol, 0.28 µg/ml leupeptin, 1.4 µg/ml
pepstatin A, 0.17 mg/ml PMSF, and 0.33 mg/ml benzamidine. For
large-scale preparations, lysates were centrifuged at 100,000 × g for 30 min at 4 °C, and for analytical preparations,
lysates were centrifuged at 10,000 × g for 20 min at
4 °C. Where indicated in the figure legends, histidine-tagged
proteins were applied to a 1-ml HiTrap chelating column (Amersham
Biosciences) charged with nickel ions according to the
manufacturer's instructions. The column was washed with buffer I (40 mM Hepes-KOH (pH 7.6), 100 mM KCl, 20% (v/v)
glycerol, 5 mM 2-mercaptoethanol, 0.28 µg/ml leupeptin,
1.4 µg/ml pepstatin A, 0.17 mg/ml PMSF, and 0.33 mg/ml benzamidine)
containing 10 mM imidazole. The column was eluted with a
20-column volume gradient from buffer I containing 10 mM
imidazole to buffer I containing 500 mM imidazole.
We previously identified in rat liver nuclear extracts a
Med8-containing Mediator complex with an apparent native molecular mass
by SW4000 gel filtration of more than 1000 kDa (9). Although the Med8
complex appeared to fractionate chromatographically as a discrete
species, it proved to be extremely labile and refractory to complete
purification, with estimated yields as poor as 10% at each step of
purification. As a consequence, in an effort to identify proteins
present in the Med8 complex, we resorted to a proteomics approach
involving large scale enrichment of the complex by multistep
conventional chromatography and HPLC followed by exhaustive sequencing
of Med8-associated proteins by HPLC/MS/MS.
Among the proteins present in the most highly enriched Med8-containing
fractions and identified by mass spectrometry were the known mammalian
Mediator subunits TRAP230, TRAP220, TRAP80, Cdk8, Cyclin C, TRAP37,
Med6, Med7, TRFP, TRAP25, Nut2, and Soh1 (Table I in the Supplemental
Material). In addition to these known mammalian Mediator subunits were
a collection of additional proteins including the p28b, Surf5, HSPC296,
AK007855, and LCMR1 (lung cancer metastasis-related protein 1)
proteins, which were not previously recognized as consensus mammalian
Mediator subunits.
The p28b protein was previously identified by protein sequencing as a
constituent of a mammalian Mediator preparation from mouse B-cells (7),
and its apparent Drosophila melanogaster homologue was
recently identified as a component of a D. melanogaster Mediator complex (10). However, the p28b protein had not previously been identified as a subunit of other mammalian Mediator complexes, including the TRAP/SMCC, DRIP, ARC, and CRSP complexes (2-6). The
proteins encoded by the Surf5, HSPC296, and AK007855 open reading frames had not previously been identified as components of
mammalian Mediator complexes, but their apparent D. melanogaster homologues were recently identified as the Med24,
Med21, and Med23 components of a D. melanogaster Mediator
complex (10). The LCMR1 protein is defined as a metastasis-related
protein (GenBankTM accession number AAN16075).
however, no information about the function of LCMR1 has been reported,
and the LCMR1 protein had not previously been identified as a
constituent of either mammalian or D. melanogaster Mediator complexes.
On the basis of bioinformatic evidence, Bourbon and co-workers (8)
recently predicted that the p28b and Surf5 proteins are higher
eukaryotic homologues of the S. cerevisiae Mediator subunits
Srb5 and Srb6. Results of our PSI-BLAST searches and multiple sequence
alignments revealed structural similarities between LCMR1 and HSPC296
and the yeast Mediator subunits Rox3 and Med11, respectively,
suggesting that LCMR1 and HSPC296 could be mammalian homologues of
yeast Rox3 and Med11 (Fig. 1 in the Supplemental Material).
Because of their apparent structural similarities to S. cerevisiae Mediator subunits, we sought to determine whether the
p28b, Surf5, HSPC296, and LCMR1 proteins are bona fide
subunits of the mammalian Mediator complex. To begin to address this
possibility, we took advantage of a HeLa cell line that stably
expresses Mediator subunit Nut2 with an N-terminal Flag tag (f:Nut2).
This HeLa cell line has been used extensively as a source for
immunoaffinity purification of the transcriptionally active TRAP/SMCC
Mediator complex (11). Rabbit polyclonal antibodies were raised against the p28b, Surf5, HSPC296, and LCMR1 proteins and used in Western blotting experiments to assay for the presence of these proteins in
immunoaffinity-purified preparations of the HeLa cell TRAP/SMCC complex. As shown in Fig. 1, all four
proteins were readily detected by Western blotting in
immunoaffinity-purified preparations of the TRAP/SMCC complex, along
with f:Nut2 and known Mediator subunits TRAP80, Med6, and Med8. A
protein encoded by the FLJ23445 open reading frame, which was also
identified by mass spectrometry in the most highly enriched
Med8-containing fractions, was not detected by Western blotting in
purified preparations of the TRAP/SMCC complex (Fig. 1). Thus, although
the p28b, Surf5, HSPC296, and LCMR1 proteins were not previously
identified in the TRAP/SMCC complex by protein sequencing, they were
all detected by Western blotting in purified preparations of the
complex, suggesting that they are subunits of the complex.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
The p28b, Surf5, HSPC296, and LCMR1 proteins
are present in the purified TRAP/SMCC mammalian mediator complex.
Immunoaffinity purification of the TRAP/SMCC Mediator complex was
carried out essentially as described (11). Six ml of undialyzed nuclear
extract (12) (~30 mg protein) from either parental HeLa cells (HeLa)
or HeLa M10 cells (11) stably expressing Flag-tagged mammalian Mediator
subunit Nut2 (f:Nut2) were incubated with 100 µl of
anti-Flag M2 agarose in buffer A (10 mM Hepes-NaOH, pH7.9,
10 mM KCl, 1 mM MgCl2, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1%
Triton X-100) containing 0.3 M KCl for at least 4 h at
4 °C. Beads were washed five times with 5 ml of buffer A containing
0.3 M NaCl and once with buffer A containing 0.1 M NaCl. Bound proteins were eluted by incubating beads
twice with 100 µl of buffer A containing 0.1 M NaCl and
0.2 mg/ml Flag peptide and analyzed by Western blotting with the
antibodies indicated in the figure. Western blots were developed
using horseradish peroxidase-labeled secondary antibodies and either
SuperSignal West Dura extended duration substrate or SuperSignal West
Femto maximum sensitivity substrate (Pierce).
To obtain additional evidence supporting assignment of the p28b, Surf5,
HSPC296, and LCMR1 proteins as mammalian Mediator subunits, we sought
to identify pairwise binding partners of these proteins among the known
mammalian Mediator subunits. To this end, we used a convenient and
scalable screen to assess the ability of these proteins to interact
with known mammalian Mediator subunits prepared by in vitro
translation in rabbit reticulocyte lysates. pcDNA3.1 expression
vectors encoding the known mammalian Mediator subunits indicated in
Fig. 2 were constructed and used to
program rabbit reticulocyte lysates for translation of
35S-labeled Mediator proteins. Binding of
35S-labeled Mediator proteins to purified recombinant
Flag-p28b, Flag-TRFP, GST-Surf5, GST-TRAP25, and GST-LCMR1 fusion
proteins was assayed in pull-down experiments using anti-Flag-agarose
or glutathione-agarose beads, and bound proteins were visualized by
autoradiography. The results of these experiments revealed significant
and reproducible binding (i) of p28b to Mediator subunit TRFP, (ii) of
TRAP25 to itself and to Mediator subunits TRAP36 and Surf5, (iii) of
Surf5 to both Mediator subunit TRAP25 and HSPC296, and (iv) of LCMR1 to
Mediator subunit Soh1 (Fig. 3). Additionally, GST-TRAP25, GST-Surf5, and GST-LCMR1 all bound to TRAP80
and TRAP37 in these experiments (Fig. 2).
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To confirm and extend these findings, we sought to reconstitute the protein-protein interactions observed in in vitro translation experiments in transfected 293T cells or with recombinant proteins expressed in Sf21 insect cells or in E. coli. Consistent with the results of our initial screening experiments, p28b could be coimmunoprecipitated from 293T cell lysates with mammalian Mediator subunit TRFP, Surf5 could be coimmunoprecipitated with both HSPC296 and Mediator subunit TRAP25, and LCMR1 could be coimmunoprecipitated with Mediator subunit Soh1 (Fig. 3).
As shown in Fig. 4A,
recombinant TRFP purified from E. coli, but not bacterially
expressed FLJ23445, bound specifically to purified GST-p28b. In control
experiments, TRFP did not bind to GST-Med8. Consistent with these
observations, a TRFP-p28b heterodimer could be reconstituted with
His-TRFP and His-p28b that had been purified from baculovirus-infected
insect cells by nickel chromatography. TheTRFP-p28b heterodimer was
purified to near homogeneity by TSK DEAE-NPR HPLC. After mixing
recombinant TRFP and p28b, both proteins bound the TSK DEAE-NPR column
and were eluted with ~130 mM KCl. In control experiments
p28b flowed through the TSK DEAE-NPR column when TRFP was not present
(Fig. 4B). Similarly, an HSPC296-Surf5 heterodimer could be
purified to near homogeneity by anti-Flag immunoaffinity chromatography
of Flag-tagged HSPC296 and C-Myc-tagged Surf5 that had been coexpressed
in Sf21 cells (Fig.
5A).
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To determine whether Surf5 can bind directly to TRAP25, either GST-TRAP25 and Surf5 or GST and Surf5 were coexpressed in E. coli, and cell lysates were subjected to glutathione-agarose chromatography. Because GST and Surf5 coelectrophorese on SDS-polyacrylamide gels, the presence or absence of Surf5 in column eluates was detected by Western blotting with anti-Surf5 antibodies. As shown in Fig. 5B, bacterially expressed Surf5 bound specifically to bacterially expressed GST-TRAP25 but not to GST.
Finally, binding of LCMR1 to Soh1 was detected following pairwise
coexpression of these proteins in Sf21 cells. As shown in Fig.
6, lanes 1-4, LCMR1 and Soh1
could be coimmunoprecipitated efficiently with anti-LCMR1 antibodies
from lysates of cells expressing both LCMR1 and Soh1, whereas only a
small amount of Soh1 was detected in immunoprecipitates from cells
expressing Soh1 alone. To determine whether LCMR1 can bind directly to
Soh1, thioredoxin-LCMR1, which had been expressed in and purified from
E. coli, was mixed with either GST-Soh1 or GST and subjected
to glutathione-agarose chromatography. Full-length Trx-LCMR1 and
GST-Soh1 coelectrophorese on SDS-polyacrylamide gels (data not shown).
Therefore, Trx-LCMR1 was detected by Western blotting with
anti-LCMR1 antibodies. As shown in Fig. 6, lanes 8-10,
Trx-LCMR1 bound GST-Soh1 but not GST. Further supporting the
specificity of this interaction, a large fraction of Trx-LCMR1 in
binding reactions had been proteolyzed during its expression and/or
purification (lanes 5-7); however, the full-length protein bound most efficiently to GST-Soh1.
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In summary, in this report we present direct biochemical evidence that
the mammalian p28b, Surf5, HSPC296, and LCMR1 proteins are bona
fide subunits of the mammalian Mediator complex. Taken together
with the bioinformatic evidence of Bourbon and co-workers (8), which
suggests that the p28b and Surf5 proteins may be homologues of S. cerevisiae Mediator subunits Srb5 and Srb6, our finding that the
HSPC296 and LCMR1 proteins share sequence similarity with S. cerevisiae Mediator subunits Med11 and Rox3 suggests that yeast
and higher eukaryotic Mediator complexes may be more highly conserved
throughout evolution than previously thought. Ultimately, however,
experiments that directly explore the structural and functional
relationships between apparent homologues of yeast and higher
eukaryotic Mediator subunits will be required to determine the extent
of evolutionary conservation between yeast and higher eukaryotic
Mediator complexes.
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ACKNOWLEDGEMENTS |
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We thank R. Roeder and S. Malik for the HeLa cell line expressing FLAG-Nut2.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R37 GM41628.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.
The on-line version of this article (available at
http://www.jbc.org) includes as Supplemental Material (i) a sequence
comparison of yeast and mammalian Mediator subunits and (ii) peptide
sequences obtained from the purified rat liver Mediator complex.
§ These authors contributed equally to this work.
§§ To whom correspondence should be addressed. Tel.: 816-926-4091; Fax: 816-926-2093; E-mail: jlc@stowers-institute.org.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.C300054200
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ABBREVIATIONS |
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The abbreviations used are: TRAP, thyroid hormone receptor-associated protein; Med, Mediator; Srb, suppressor of RNA polymerase B; SMCC, Srb-Med-containing cofactor; DRIP, vitamin D receptor-interacting protein; ARC activator-recruited cofactor, CRSP, cofactor required for Sp1 activation; MS/MS, tandem mass spectrometry; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; PMSF, phenylmethylsulfonyl fluoride; TRFP, TATA-binding protein related factor-proximal protein.
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