From the Program in Molecular and Cell Biology,
Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, the ¶ Howard Hughes Medical Institute, Oklahoma City, Oklahoma
73104, the ** Department of Biochemistry and Molecular Biology,
University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
73190, and the
Edward A. Doisy Department of Biochemistry, St.
Louis University School of Medicine, Saint Louis, Missouri 63104
Received for publication, November 7, 2000, and in revised form, February 3, 2001
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ABSTRACT |
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RNA polymerase II elongation factor ELL was
recently purified from rat liver as a component of a multiprotein
complex containing ELL and three ELL-associated
proteins (EAPs) of ~45 (EAP45), ~30 (EAP30), and ~20
(EAP20) kDa (Shilatifard, A. (1998) J. Biol. Chem.
273, 11212-11217). Cloning of cDNA encoding the EAP30 protein revealed that it shares significant sequence similarity with the product of the Saccharomyces cerevisiae SNF8 gene (Schmidt,
A. E., Miller, T., Schmidt, S. L., Shiekhattar, R., and
Shilatifard, A. (1999) J. Biol. Chem. 274, 21981-21985), which is required for efficient derepression of
glucose-repressed genes. Here we report the cloning of cDNAs
encoding the EAP45 and EAP20 proteins. In addition, we identify the
S. cerevisiae VPS36 and YJR102c genes as
potential orthologs of EAP45 and EAP20 and show
that they are previously uncharacterized SNF genes with
properties very similar to SNF8.
RNA polymerase II elongation factor ELL (for 11-19 lysine-rich in
leukemia) was originally identified in rat liver nuclear extracts and
purified by its ability to increase the overall rate of elongation by
RNA polymerase II in vitro by suppressing transient pausing
by polymerase at many sites along the DNA (1). The human ELL
gene was also identified as a gene involved in t(11:19)(q23:p13.1) translocations with the MLL gene in acute myeloid leukemia
(2, 3).
Biochemical studies have revealed that ELL has multiple transcriptional
activities (4). In addition to its ability to stimulate the rate of
elongation by RNA polymerase II, ELL is capable of inhibiting
promoter-specific transcription initiation by RNA polymerase II
in vitro by binding polymerase and preventing it from
entering the preinitiation complex (4).
Recently, ~40% of the detectable ELL protein in rat liver whole cell
extracts was found to be in a high molecular mass, multiprotein complex. Purification of this complex to near homogeneity revealed that, in addition to ELL, it includes three
ELL-associated proteins (EAPs)1 of ~45, ~30, and
~20 kDa (5). The ELL-EAP complex was found to possess elongation
stimulatory activity indistinguishable from that of the individual ELL
protein, but to lack transcription initiation inhibitory activity,
suggesting that the EAPs blocked this ELL activity (5). This hypothesis
was confirmed when EAP30 was cloned and shown to interact directly with
ELL in vitro and to prevent it from inhibiting transcription
initiation (6).
Cloning of the cDNA encoding EAP30 revealed that it is a potential
ortholog of the product of the Saccharomyces cerevisiae SNF8
gene (6). The SNF8 gene was originally identified by Carlson and co-workers (7) in a genetic screen for yeast genes essential for
growth on sucrose. Yeast snf8 mutants are defective in
derepression of glucose-repressed genes such as SUC2, which
encodes the enzyme invertase that hydrolyzes the non-fermentable carbon
sources sucrose and raffinose. Yeast bearing snf8 mutations
are able to repress SUC2 expression in the presence of
glucose, but do not support maximal SUC2 derepression when
switched to low glucose-containing media (7).
In this report, we present identification, cloning, and
characterization of the remaining two EAPs, EAP45 and EAP20. In
addition, we identify the S. cerevisiae VPS36 and
YJR102c genes as potential orthologs of EAP45 and
EAP20 and show that yeast containing mutations in
VPS36 and YJR102c exhibit phenotypes very similar
to those of snf8 mutants.
Materials--
Anti-T7 antibody was purchased from Novagen.
Anti-HA (12CA5) and anti-Myc (9E10) antibodies were obtained from Roche
Molecular Biochemicals. Protein A/G-Sepharose was from Amersham
Pharmacia Biotech. 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) was purchased from Sigma. Glucose oxidase and horseradish
peroxidase were obtained from Roche Molecular Biochemicals. Hybond-P
PVDF membranes were from Amersham Pharmacia Biotech. Chemiluminescence reagent (Super Signal West Dura Extend) was purchased from Pierce.
Identification of cDNAs Encoding the EAP45 and EAP20
Proteins--
The ELL-EAP complex was purified as described (5). The
EAP45 and EAP20 proteins were resolved by SDS-polyacrylamide gel electrophoresis. Approximately 25 pmol of the EAP45 and EAP20 proteins
were reduced, S-carboxyamidomethylated, and digested with
trypsin in gel slices. An aliquot of the tryptic digests was analyzed
by matrix-assisted laser desorption ionization mass spectrometry.
Another aliquot of the tryptic digests was fractionated by microbore
high performance liquid chromatography, and isolated peptides were
sequenced by automated Edman degradation. ESTs encoding EAP45 and EAP20
peptide sequences were identified through searches of the GenBank EST
data base. ESTs encoding mouse EAP45 (AA274602 and AA086895) and mouse
EAP20 (AA032693) were obtained from Research Genetics (IMAGE
consortium) and sequenced on both strands.
Expression of Recombinant EAP45, EAP30, and EAP20 in Insect
Cells--
Mouse EAP45 containing an N-terminal Myc tag, mouse EAP30
containing an N-terminal HA tag, and mouse EAP20 containing an
N-terminal T7 tag were subcloned into pBacPAK8, and recombinant
baculoviruses were generated by the BacPAK baculovirus expression
system (CLONTECH). Sf21 cells were cultured
in Sf-900 II SFM with 5% fetal calf serum at 27 °C and infected
with appropriate recombinant viruses. Sixty hours after infection,
cells were collected and lysed by gentle vortexing in ice-cold buffer
containing 40 mM Hepes-NaOH (pH 7.9), 150 mM
NaCl, 1 mM DTT, 0.5% Triton X-100, 10% glycerol, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml pepstatin A, and 5 µg/ml aprotinin. Lysates were centrifuged at 10,000 × g for 20 min at 4 °C. The supernatants were used for
Western blotting and immunoprecipitations.
Expression of Recombinant EAP45, EAP30, and EAP20 in Mammalian
Cells--
Mouse EAP45 containing an N-terminal Myc tag was subcloned
into pCDNA3.1 (Invitrogen), and mouse EAP30 containing an
N-terminal HA tag, and mouse EAP20 containing an N-terminal T7 tag were
subcloned into the pCI-neo expression vector (Promega). 48 h after
transfection of 293T cells using Fugene 6, cells were collected and
lysed in ice-cold buffer containing 40 mM Hepes-NaOH (pH
7.9), 150 mM NaCl, 1 mM DTT, 0.5% Triton
X-100, 10% glycerol, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml pepstatin A, and 5 µg/ml aprotinin. Lysates were centrifuged
at 10,000 × g for 20 min at 4 °C. The supernatants
were used for Western blotting and immunoprecipitations.
Immunoprecipitations and Western Blotting--
To measure
interactions among the EAP proteins, baculovirus-infected insect cell
lysates or transfected mammalian cell lysates were incubated with the
indicated antibodies for 1 h at 4 °C and then with protein
A/G-Sepharose for 1 h at 4 °C. Protein A/G-Sepharose was washed
three times in buffer containing 40 mM Hepes-NaOH (pH 7.9),
250 mM NaCl, 1 mM DTT, and 0.5% Triton X-100.
Immunoprecipitated proteins were analyzed by electrophoresis through
SDS-polyacrylamide gels. Proteins were transferred to Hybond-P PVDF
membranes, probed with appropriate antibodies, and visualized using the
Super Signal West Dura Extend chemiluminescence reagent.
Yeast Deletion Strains--
To generate the
yjr102c
Cell growth was analyzed under non-fermenting conditions by growing
wild type BY4733 cells and isogenic strains containing the respective
knockouts on plates containing 1% yeast extract, 2% bacto-peptone and
either 2% glucose (YPD) or 2% raffinose plus 1 µg/ml antimycin A
(7). Temperature sensitivity was assessed by growth of yeast on YPD at
39 °C.
Assay of Invertase--
Yeast were grown in log phase for at
least three generations to an A600 of 1 in YPD
with 4% glucose. Cells to be derepressed were washed twice in 1%
yeast extract, 2% bacto-peptone, and 0.05% glucose and then grown in
the same medium plus 5% glycerol for 2.5 h. One
A600 unit of cells was washed in cold 10%
sodium azide. Glucose repressed cells were resuspended in 50 µl of
10% sodium azide, and derepressed cells were resuspended in 200 µl of 10% sodium azide. Prior to assay, the derepressed cell
suspension was diluted 10-fold in water.
To assay invertase activity, 50 µl of cell suspension were added to
100 µl of 0.2 M sodium acetate (pH 5.1). 50 µl of 0.5 M sucrose was then added, and the cell suspension was
allowed to incubate for 20 min at 37 °C. Reactions were stopped by
addition of 300 µl of 0.2 M
K2HPO4. 100 µl of the resulting cell
suspension was added to an additional 400 µl of 0.2 M
K2HPO4 and boiled for 3 min. Two ml of
glucostat reagent containing 0.2 mM potassium phosphate (pH
7.0), 40 µg of glucose oxidase (Roche Molecular Biochemicals), 5 µg
of horseradish peroxidase, and 0.53 mg of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) were added, the
reaction was incubated at room temperature for at least 30 min, and the
A420 of the samples was determined. Invertase
units (µmol glucose released/min/100 mg dry weight of yeast) were
calculated by correcting for initial dilution of cell volumes and by
comparison to standard curves performed in parallel with known
concentrations of glucose.
Measurement of Invertase (SUC2) RNA--
Yeast cells were grown
under glucose repressed or derepressed conditions as described above.
Total RNA was isolated from 2 A600 units of
cells using the Qiagen RNeasy Mini-kit. Reverse-transcription was
performed in 100-µl reactions using the Taq-Man reverse
transcription kit (PerkinElmer Life Sciences) with 2 µg of total RNA.
Reverse transcriptase reactions were primed with either a
SUC2 antisense primer (5'-TTG GTT CGG CTT TCA AAT TGA-3') or
an ACT1 antisense primer (5'-GAG CAG CGG TTT GCA CT-3').
Real time quantitative PCR reactions were performed in a LightCycler
(Roche) using SUC2 antisense and sense (5'-CCC ATG GAG ATC
ATC CAT GTC T-3') or ACT1 antisense and sense (5'-CCC ATG
GAG ATC ATC CAT GTC-3') primers; the SUC2 and
ACT1 PCR products were both ~100 base pairs long. PCR
reactions were performed according to the manufacturer's instructions with the Light- Cycler-FastStart DNA Master SYBR Green I kit (Roche) and contained 5 mM MgCl2, 0.5 µM
of each primer, and 2 µl (2%) of the cDNA products of reverse
transcription reactions. Reactions were initiated by heating at
95 °C for 10 min, followed by 45 cycles programmed as follows:
95 °C for 0 s, 60 °C for 5 s, and 72 °C for 6 s. To prepare the standard curve, 1, 0.1, 0.01, and 0.001 pg of
purified SUC2 or ACT1 PCR product was used in
identical PCR reactions.
Identification of cDNAs Encoding EAP45 and EAP20--
The
ELL-EAP complex, which is composed of the ELL, EAP45, EAP30, and EAP20
proteins, was purified to near homogeneity from rat liver whole cell
extracts as described previously (5). The sequences of peptides derived
from EAP45 and EAP20 were determined by Edman degradation or
matrix-assisted laser desorption ionization mass spectrometry (Fig.
1). No matches to peptides from EAP45 or
EAP20 were identified in searches of the GenBankTM non-redundant data
base, indicating that EAP45 and EAP20 are previously uncharacterized mammalian proteins. A search of the GenBankTM EST data base, however, identified mouse and human ESTs encoding the sequenced peptides and
containing potential full-length EAP45 and EAP20 ORFs. The mouse EAP45
ORF encodes a 387-amino acid protein with a calculated molecular mass
of 43,844 Da and a pI of 6.0 (Fig. 2).
Mouse EAP45 is 97% identical in amino acid sequence to human EAP45.
The mouse and human EAP20 ORFs encode identical 176-amino acid
proteins with calculated molecular masses of 20,971 Da and pIs of 6.8 (Fig. 3).
Reconstitution of the EAP Complex--
Bacterially expressed EAP30
was previously shown to bind directly to ELL in vitro and to
block its ability to inhibit promoter-specific transcription initiation
by RNA polymerase II (6). To investigate interactions among the EAP
proteins, 293T cells were transfected with various combinations of
pCI-neo expression vectors encoding epitope tagged EAP45, EAP30, and
EAP20. As shown in Fig. 4A,
when all three EAP proteins were coexpressed in 293T cells, all three proteins could be coimmunoprecipitated with antibodies specific for the
epitope tags on either EAP45 or EAP20. In addition, EAP30 could be
coimmunoprecipitated with EAP20 in the absence of exogenously expressed
EAP45, and EAP20 could be coimmunoprecipitated with EAP45 in the
absence of exogenously expressed EAP30. Because EAP45 was only
expressed to detectable levels when coexpressed in 293T cells with
EAP20, it was not possible to assess the ability of EAP30 to interact
with EAP45 in the absence of exogenously expressed EAP20. We therefore
investigated interactions among the EAP proteins in Sf21 insect
cells.
In these experiments, Sf21 cells were infected with various
combinations of baculoviruses encoding epitope-tagged EAP45, EAP30, and
EAP20. Consistent with the results of experiments carried out with 293T
cells, when coexpressed in Sf21 cells, all three EAP proteins
could be coimmunoprecipitated with antibodies specific for the epitope
tags on either EAP45 or EAP20 (Fig. 4B). In addition, EAP30
could be coimmunoprecipitated with EAP20 in the absence of exogenously
expressed EAP45, and EAP20 could be coimmunoprecipitated with EAP45 in
the absence of exogenously expressed EAP30. Only a very small fraction
of EAP30, however, was coimmunoprecipitated with EAP45 in the absence
of exogenously expressed EAP20. Taken together, the results of
experiments carried out with both mammalian and insect cells suggest
that EAP20 bridges EAP30 and EAP45 and thereby nucleates assembly of
the EAP complex.
The S. cerevisiae VPS36 and YJR102c Genes Encode Two Previously
Uncharacterized SNF Proteins--
The S. cerevisiae snf8
protein was previously identified as a potential ortholog of mammalian
EAP30 (6). The SNF8 gene was originally isolated by Carlson
and co-workers (7) in a screen for yeast mutants defective in
derepressing expression of glucose-repressed genes such as
SUC2, which encodes the sucrose and raffinose hydrolyzing
enzyme invertase. How Snf8 participates in regulation of gene
expression by glucose is not yet known.
GenBankTM data base searches identified the S. cerevisiae
Vps36 protein and the product of S. cerevisiae ORF YJR102c
are potential orthologs of mammalian EAP45 and EAP20, respectively. The
S. cerevisiae VPS36 gene was previously identified in a
genetic screen for yeast defective in a variety of protein trafficking
pathways (10, 11). In addition, data base searches identified the
products of Drosophila melanogaster ORF CG10711,
Caenorhabditis elegans ORF F17C11.8, and
Schizosaccharomyces pombe ORF SPBC3B9.09 as potential
orthologs of EAP45 (Fig. 2) and D. melanogaster ORF CG14750,
C. elegans ORF W02A11.2, and S. pombe ORF
SPBC4B4.06 as potential orthologs of EAP20 (Fig. 3).
In light of the amino acid sequence similarities between EAP30 and
Snf8, EAP45 and Vps36, and EAP20 and Yjr102c, we speculated that, like
Snf8, Vps36 and Yjr102c could have roles in regulation of glucose
repressed genes. To address this possibility, S. cerevisiae strains lacking the SNF8, VPS36, or
YJR102c genes were constructed and their phenotypes
compared. Carlson and co-workers (7, 12) previously demonstrated that
yeast lacking the SNF8 gene are viable, but exhibit growth
defects on raffinose, temperature sensitivity, and partial loss of the
ability to derepress SUC2 expression. Like SNF8,
neither VPS36 nor YJR102c is an essential gene.
As described previously for the snf8 deletion,
vps36 and yjr102c deletions result in a severe
growth defect on raffinose and temperature-sensitive growth on glucose
(Fig. 5).
To compare the effects of snf8, vps36, and
yjr102c deletions on SUC2 derepression, wild type
and deletion strains were grown for several generations in medium
containing 4% glucose to repress SUC2 and then transferred
to medium containing 0.05% glucose and 5% glycerol as carbon sources.
As shown previously (7, 12) and in Fig.
6A, the snf8
deletion strain was partially defective in derepression of invertase
activity after cells were transferred out of high glucose medium;
maximum invertase activity obtained under derepressing conditions was
~4-fold lower with the snf8 deletion than with the
isogenic wild type yeast strain. Maximum invertase activity under
derepressing conditions was similarly reduced in the vps36
and yjr102c deletion strains. To determine whether the
partial defect in invertase derepression in the three deletion strains
is accompanied by decreases in SUC2 RNA levels, total RNA
was prepared from wild type and mutant yeast strains grown under
repressing and derepressing conditions, and SUC2 RNA levels
were determined by real time quantitative reverse transcription-PCR. As
shown in Fig. 6B, similar to invertase activity,
SUC2 RNA was only partially derepressed in
snf8, vps36, and yjr102c deletion strains, indicating that mutations in these genes affect regulation of
SUC2 RNA synthesis and/or stability.
RNA polymerase II elongation factor ELL was recently purified from
rat liver as a component of a multiprotein complex containing ELL and
three additional proteins designated EAPs ~45 (EAP45), ~30 (EAP30),
and ~20 (EAP20) kDa; ~40% of the detectable ELL in rat liver whole
cell extracts is present in the ELL-EAP complex (5). In a previous
study, the ELL-EAP complex was found to possess elongation stimulatory
activity indistinguishable from that of the individual ELL protein. In
contrast to free ELL, however, the ELL-EAP complex lacked transcription
initiation inhibitory activity, arguing that association of the EAPs
with ELL blocked this activity (5). Cloning of the cDNA encoding the
EAP30 protein and characterization of its interaction with ELL revealed
that EAP30 alone is capable of interacting directly with ELL and
blocking ELL transcription initiation inhibitory activity (6). In
addition, comparison of the EAP30 amino acid sequence with known
protein sequences identified the product of the S. cerevisiae
SNF8 gene as a potential ortholog.
The yeast SNF8 gene was originally identified by Carlson and
co-workers (7) in a genetic screen for yeast defective in derepression
of expression of glucose-repressed genes like SUC2, which
encodes the sucrose-metabolizing enzyme invertase. Carlson and
co-workers found that yeast lacking the SNF8 gene repress SUC2 expression properly when grown in glucose-containing
media, but fail to support maximal derepression of SUC2 gene
expression when switched to low glucose-containing media.
In this report, we describe cloning of cDNAs encoding the EAP45 and
EAP20 proteins, which, together with EAP30, reconstitute the three
subunit EAP complex. In addition, we identify the S. cerevisiae
VPS36 and YJR102c genes as potential orthologs of
EAP45 and EAP20 and show that they are previously
uncharacterized SNF genes.
Taken together with our evidence that mutation of the VPS36
and YJR102c genes results in similar SNF phenotypes in
yeast, our finding that the mammalian EAP proteins function together in
a multiprotein complex raises the possibility that the yeast Snf8,
YJR102c, and Vps36 proteins may also function together in a complex in
yeast. Indeed, in an independent line of research, Emr and
co-workers2 have identified
the S. cerevisiae SNF8 and YJR102c genes in a genetic screen for yeast defective in intracellular protein trafficking and have shown that the Snf8, YJR102c, and Vps36 proteins are present
in yeast in a multiprotein complex.
Finally, the relationship between the functions of S. cerevisiae Snf8, Yjr102c, and Vps36 proteins in derepression of
glucose repressed genes and protein trafficking is unknown. In light of previous evidence that derepression of the SUC2 gene is
accompanied by increases in both transcription and stability of
SUC2 mRNA, our observation that deletions of the
SNF8, VPS36, and YJR102c genes results
in reduced levels of derepressed SUC2 mRNA expression indicates that the Snf8, Yjr102c, and Vps36 proteins participate in
some way in promoting transcription and/or mRNA stability during derepression. In this regard, it is noteworthy that the protein encoded
by the human tumor susceptibility 101 (TSG101)
gene, which has been implicated in transcriptional regulation, has
recently been shown to be an ortholog of the S. cerevisiae
Vps23 protein (13, 14). Mutations in the VPS23 and
TSG101 genes result in intracellular protein trafficking
defects in yeast and mammalian cells, respectively. In addition to its
role in protein trafficking, however, the TSG101 protein has been
reported both to function as a general transcriptional corepressor (15)
and to interact specifically with the AF-1 activation domain of the
glucocortoid receptor and inhibit glucocorticoid receptor-, androgen
receptor-, and estrogen receptor-dependent gene expression
in cells (15-17). In light of these observations, future studies
investigating the functions of the EAP proteins in mammalian cells and
in yeast should provide valuable insights into the intriguing
relationship between the transcriptional regulatory and protein
trafficking pathways in eukaryotic cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, snf8
, or vps36
yeast
strains, the YJR102c, SNF8, or VPS36
genes were deleted in S. cerevisiae strain BY4733
(Mata, his3-200, leu2-0, met15-0, trp1-63, ura3-0) (8) by replacing their complete ORFs with the
HIS3 gene (9). Deletions were confirmed by PCR analysis of
single colonies that grew on histidine(
) plates.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of the EAP45 and EAP20
subunits of the EAP complex. SDS-polyacrylamide gel of the sample
used for peptide sequencing is shown. Peptide sequences derived from
the individual EAP proteins are indicated.
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Fig. 2.
Alignment of mammalian EAP45 with potential
D. melanogaster, C. elegans, and
yeast orthologs. The best alignment of human (H;
GenBankTM accession no. AF151903) and mouse (M)
EAP45 with potential orthologs from D. melanogaster
(DM; GenBankTM accession no. AAF49802), C. elegans (CE; GenBankTM accession no. T21060), S. pombe (SP; GenBankTM accession no. T40348), and
S. cerevisiae (SC; GenBankTM accession no. NP
013521) was determined in part by the MACAW program (18) using the
BLOSUM 80 score table (19) and by inspection. Residues identical to
mammalian EAP45 are indicated by white letters on
black background. Residues similar to mammalian
EAP45 are indicated by black letters on
gray background. Residues considered similar are
I, L, M, and V; E, D, N, and Q; H, K, and R; F, W, and Y; and A, P,
S, and T.
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Fig. 3.
Alignment of mammalian EAP20 with potential
D. melanogaster, C. elegans, and
yeast orthologs. The best alignment of human (H;
GenBankTM accession no. BE386260) and mouse (M) EAP20 with
potential orthologs from D. melanogaster (DM;
GenBankTM accession no. AAF59066), C. elegans
(CE; GenBankTM accession no. T26073), S. pombe
(SP; GenBankTM accession no. T40478), and S. cerevisiae (SC; GenBankTM accession no. CAA89632) was
determined in part by the MACAW program (18) using the BLOSUM 80 score
table (19) and by inspection. Residues identical to mammalian EAP20 are
indicated by white letters on black
background. Residues similar to mammalian EAP20 are
indicated by black letters on gray
background. Residues considered similar are I, L, M, and V;
E, D, N, and Q; H, K, and R; F, W, and Y; and A, P, S, and T.
View larger version (39K):
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Fig. 4.
Reconstitution of the EAP complex in insect
and mammalian cells. Panel A, interactions among the
EAP proteins in mammalian 293T cells. 293T cells were transiently
transfected with vectors encoding the indicated proteins. Total cell
lysates (upper panel) and anti-T7
(middle panel) or anti-Myc (lower
panel) immunoprecipitates were subjected to 11%
SDS-polyacrylamide gel electrophoresis, followed by Western blotting
with antibodies against epitopes on the indicated proteins (T7 epitope
on EAP20; HA epitope on EAP30, and c-Myc epitope on EAP45).
HC, heavy chain. Panel B, interactions among the
EAP proteins in Sf21 insect cells. Sf21 cells were
infected with baculoviruses encoding the indicated proteins. Total cell
lysates (upper panel) and anti-T7
(middle panel) or anti-Myc (lower
panel) immunoprecipitates were subjected to
SDS-polyacrylamide gel electrophoresis, followed by Western blotting
with antibodies against epitopes on the indicated proteins.
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Fig. 5.
S. cerevisiae vps36 and
yjr102c Mutants exhibit growth phenotypes similar to
those of snf8 mutants. From a starting culture
density of 1 A600, 10-fold serial dilutions of
cultures of each strain were plated and grown under the indicated
conditions.
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Fig. 6.
S. cerevisiae VPS36 and
YJR102c genes encode previously uncharacterized SNF
proteins. Panel A, invertase activity is not fully
de-repressed in yjr102c, snf8, and
vps36 deletion strains. Invertase activity was measured as
described under "Experimental Procedures" following growth of yeast
in high glucose (repressed conditions) or low glucose (derepressed
condition) media. The data presented are derived from three independent
experiments. Panel B, the partial defect in invertase
derepression in the yjr102c, snf8, and
vps36 deletion strains is accompanied by decreases in
SUC2 (invertase) RNA levels. SUC2 RNA levels were
determined by real-time quantitative reverse transcription-PCR as
described under "Experimental Procedures" and are expressed
relative to the amount of SUC2 RNA from wild type cells
grown under de-repressed conditions. The data presented are derived
from three independent experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank M. Babst and S. Emr (Howard Hughes Medical Institute, University of California, San Diego, CA) for communicating unpublished results, M. Johnston (Washington University, St. Louis, MO) for the invertase assay protocol, and K. Jackson (Molecular Biology Resource Center, Oklahoma Center for Molecular Medicine, Oklahoma City, OK) for oligonucleotide synthesis.
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FOOTNOTES |
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* This work was supported in part by American Cancer Society Grant RP69921801 (to A. S.) and by National Institutes of Health Grant R37-GM41628 and a grant to the Oklahoma Medical Research Foundation by the H. A. and Mary K. Chapman Charitable Trust (to R. C. C. and J. W. C.).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.
§ These authors contributed equally to this work.
Associate investigator of the Howard Hughes Medical Institute.
§§ An Edward Mallinckrodt, Jr., Young Investigator. To whom correspondence should be addressed. Tel.: 314-577-8137; Fax: 314-268-5737; E-mail: shilatia@slu.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M010142200
2 M. Babst and S. Emr, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: EAP, ELL-associated protein; SNF, sucrose nonfermenting; ORF, open reading frame; VPS, vacuolar protein sorting; PVDF, polyvinylidene difluoride; EST, expressed sequence tag; DTT, dithiothreitol; HA, hemagglutinin; ORF, open reading frame; PCR, polymerase chain reaction.
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REFERENCES |
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1. | Shilatifard, A., Lane, W. S., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1996) Science 271, 1873-1876[Abstract] |
2. |
Thirman, M. J.,
Levitan, D. A.,
Kobayashi, H.,
Simon, M. C.,
and Rowley, J. D.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12110-12114 |
3. |
Mitani, K.,
Kanda, Y.,
Ogawa, S.,
Tanaka, T.,
Inazawa, J.,
Yazaki, Y.,
and Hirai, H.
(1995)
Blood
85,
2017-2024 |
4. |
Shilatifard, A.,
Haque, D.,
Conaway, R. C.,
and Conaway, J. W.
(1997)
J. Biol. Chem.
272,
22355-22363 |
5. |
Shilatifard, A.
(1998)
J. Biol. Chem.
273,
11212-11217 |
6. |
Schmidt, A. E.,
Miller, T.,
Schmidt, S. L.,
Shiekhattar, R.,
and Shilatifard, A.
(1999)
J. Biol. Chem.
274,
21981-21985 |
7. | Yeghiayan, P., Tu, J., Vallier, L. G., and Carlson, M. (1995) Yeast 11, 219-224[Medline] [Order article via Infotrieve] |
8. | Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Yeast 14, 115-132[CrossRef][Medline] [Order article via Infotrieve] |
9. | Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993) Nucleic Acids Res. 21, 3329-3330[Medline] [Order article via Infotrieve] |
10. | Nothwehr, S. F., Bryant, N. J., and Stevens, T. H. (1996) Mol. Cell. Biol. 16, 2700-2707[Abstract] |
11. |
Luo, W. J.,
and Chang, A.
(1997)
J. Cell. Biol.
138,
731-746 |
12. |
Vallier, L. G.,
and Carlson, M.
(1991)
Genetics
129,
675-684 |
13. | Babst, M., Odorizzi, G., Estepa, E. J., and Emr, S. D. (2000) Traffic 1, 248-258[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Li, Y.,
Kane, T.,
Tipper, C.,
Spatrick, P.,
and Jenness, D. D.
(1999)
Mol. Cell. Biol.
19,
3588-3599 |
15. | Watanabe, M., Yanagi, Y., Masuhiro, Y., Yano, T., Yoshikawa, H., Yanagisawa, J., and Kato, S. (1998) Biochem. Biophys. Res. Commun. 245, 900-905[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Hittelman, A. B.,
Burakov, D.,
Iniguez-Lluhi, J. A.,
Freedman, L. P.,
and Garabedian, M. J.
(1999)
EMBO J.
18,
5380-5388 |
17. | Sun, Z., Pan, J., Hope, W. X., Cohen, S. N., and Balk, S. P. (1999) Cancer 86, 689-696[CrossRef][Medline] [Order article via Infotrieve] |
18. | Schuler, G. D., Altschul, S. F., and Lipman, D. J. (1991) Proteins Struct. Funct. Genet. 9, 180-190[Medline] [Order article via Infotrieve] |
19. | Henikoff, S., and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10915-10919[Abstract] |