Cloning and Characterization of ELL-associated Proteins EAP45 and EAP20

A ROLE FOR YEAST EAP-LIKE PROTEINS IN REGULATION OF GENE EXPRESSION BY GLUCOSE*

Takumi KamuraDagger §, Dennis BurianDagger §, Hamed KhaliliDagger , Susan L. Schmidt||, Shigeo SatoDagger , Wen-Jun LiuDagger , Michael N. ConradDagger , Ronald C. ConawayDagger , Joan Weliky ConawayDagger **DaggerDagger, and Ali Shilatifard||§§

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 yjr102cDelta , snf8Delta , or vps36Delta 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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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.


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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.

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).


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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.

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.


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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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