THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 271, No. 40, Issue of October 4, pp. 24811–24816, 1996
Printed in U.S.A.
Molecular Cloning of the cDNA and Chromosome Localization
of the Gene for Human Ubiquitin-conjugating Enzyme 9*
(Received for publication, June 24, 1996, and in revised form, July 22, 1996)
Zhao-Yi Wang‡§, Qing-Qing Qiu‡, Wolfgang Seufert¶i, Takahiro Taguchi**, Joseph R. Testa**, S.
A. Whitmore‡‡, David F. Callen‡‡, Douglas Welsh§, Thomas Shenk§ §§, and Thomas F. Deuel‡§¶¶
From the ‡Department of Medicine, Beth Israel Hospital and the Harvard Medical School, Boston, Massachusetts 02215,
the §Department of Molecular Biology, Princeton University, Princeton, New Jersey 08560, the ¶Institut für Genetik und
Mikrobiologie, Universität München, 80638 München, Germany, the **Department of Medical Oncology, Fox Chase
Cancer Center, Philadelphia, Pennsylvania 19111, and the ‡‡Department of Cytogenetics and Molecular Genetics, Women
& Children’s Hospital, North Adelaide S.A. 5006, Australia
The ubiquitin-dependent protein degradation system has
been recognized as a complete enzymatic pathway that is responsible for the selective degradation of abnormal and shortlived proteins (1, 2). In this process, ubiquitin is covalently
linked to target proteins prior to their degradation through the
combined action of three classes of proteins, the ubiquitinactivating enzyme (E1),1 ubiquitin-conjugating enzymes (E2),
and in some cases, the ubiquitin-protein ligases (E3) that are
believed to be important in substrate recognition. E1 catalyzes
the ATP-dependent formation of a thioester bond between the
C-terminal glycine of ubiquitin and the active site cysteine of
* This work was supported in part by the National Institutes of
Health (to T. F. D.). The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBankTM/EBI Data Bank with accession number(s) U66818
and U66867.
i Heisenberg Fellow at the Deutsche Forschungsgemeinschaft.
§§ Investigator of the Howard Hughes Medical Institute and an
American Cancer Society Professor.
¶¶ To whom correspondence should be addressed. Tel.: 617-667-1227;
Fax: 617-667-1276.
1
The abbreviations used are: E1, ubiquitin-activating enzyme; E2,
ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; PAGE,
polyacrylamide gel electrophoresis; HA, hemagglutinin; kb, kilobase(s);
Mb, megabase(s); FISH, fluorescence in situ hybridization.
E1. “Activated” ubiquitin is then transferred to the active site
cysteine of an E2 enzyme which itself, or in conjunction with an
E3 protein, catalyzes the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and a lysine e-amino
group in the protein targeted for degradation by the 20 S
proteosome complex. The diversity and multiple protein-protein interactions of the ubiquitin-dependent proteolytic system
suggests that a high degree of regulation is required to achieve
the specificity needed to control the fate of the different proteins that are degraded through ubiquitination. It is likely that
ubiquitin-conjugating (E2) enzymes may contribute to the determination of which groups of protein are targeted for selective degradation.
The ubiquitin-conjugating enzymes (E2s) are a family of
proteins characterized by a highly conserved catalytic site containing an invariant active site cysteine (3). In yeast Saccharomyces cerevisiae, at least 10 different E2s have been identified that are involved in essential cellular processes such as
DNA repair, cell cycle control, and stress responses (4), suggesting that E2 enzymes may be key players in establishing the
diversity of the ubiquitin-proteolytic system. Although genetic
analysis in yeast has revealed that ubiquitin conjugation is
essential to cell viability (5, 6), only a few cellular targets of the
ubiquitin protein ligase system have been identified. These
include cyclin B and the yeast transcription factor MATa2 (7,
8). Recently, the ubiquitin-dependent proteolytic system has
also been shown to actively participate in the degradation of
p53 (9), in the degradation and processing of cystic fibrosis
transmembrane conductance regulator (10, 11), and in the
processing of NF-kB (12), suggesting more widespread roles of
this system.
The Wilms’ tumor suppressor gene encodes a zinc finger
DNA-binding protein (13, 14) that functions in transient transfection assays as a transcriptional repressor of several growthrelated genes such as insulin-like growth factor II (15), insulinlike growth factor IR (16), platelet-derived growth factor
A-chain (17), colony stimulating factor 1 (18), and transforming
growth factor b1 (19). Previously, we identified two separate
domains (residues 85–179 and 250 –266) of WT1 that function
independently as transcriptional repressors (20, 21). When expressed independently of its zinc finger domain, the proximal
repressor domain (amino acids 85–179) relieved the repressor
activity of wild type WT1 in a dose-dependent manner (22),
suggesting the presence of an interactive protein with WT1 in
vivo. In order to identify and clone the gene encoding this
interactive protein, we have used the yeast two-hybrid system
with the proximal repressor domain of WT1 as “bait.” We
report here the isolation and characterization of cDNA clones
that encode the human homolog of the yeast ubiquitin-conju-
24811
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We report a novel human gene whose product specifically associates with the negative regulatory domain of
the Wilms’ tumor gene product (WT1) in a yeast twohybrid screen and with WT1 in immunoprecipitation
and glutathione S-transferase (GST) capture assays. The
gene encodes a 17-kDa protein that has 56% amino acid
sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes. The human
gene fully complements yeast that have a temperaturesensitive yUBC9 gene mutation to fully restore normal
growth, indicating that we have cloned a functionally
conserved human (h) homolog of yUBC9. Transcripts of
hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were
found in all human tissues tested. A single copy of the
hUBC9 gene was found and localized to human chromosome 16p13.3. We conclude that hUBC9 retains striking
structural and functional conservation with yUBC9 and
suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor
system.
24812
Molecular Cloning and Chromosome Localization of the cDNA for hUBC9
gating enzyme (yUBC9) that specifically interacts with the
proximal repressor domain of WT1 in the yeast two-hybrid
system and with WT1 in co-immunoprecipitation and glutathione S-transferase assays. The results suggest that UBC9 may
be involved in the transcription regulation mediated by WT1.
MATERIALS AND METHODS
b-Galactosidase activity
Fusion partner
Vector alone
1WT (85-179)
1WT (250-266)
1Lamin C
a
Vector
a
ND
0.3 6 0.2
0.2 6 0.2
0.3 6 0.1
hUBC9
0.2 6 0.1
57.9 6 16.2
0.3 6 0.2
1.5 6 0.5
ND, not determined.
Fluorescence in Situ Hybridization (FISH)—Metaphase chromosome
spreads from normal human lymphocytes were prepared according to
the method of Fan et al. (25). Chromosomal hybridization and detection
of immunofluorescence were carried out as described previously (26).
The chromosome preparations were stained with both diamidino-2phenylindole and propidium iodide and observed by fluorescence
microscopy.
Chromosome Mapping by Somatic Cell Hybrid—A panel of mouse/
human somatic cell hybrids containing breakpoints on the distal tip of
the short arm of chromosome 16 (28) were digested with HindIII,
transferred to GeneScreen Plus (DuPont NEN), and probed with 32Plabeled 1.1-kb hUBC9 cDNA. Controls used were human, mouse, and
the hybrid CY18 which has chromosome 16 as the only human
chromosome.
RESULTS
To identify the WT1 interactive protein previously described
(22), a human placental cDNA/Gal4 transcriptional activation
domain fusion library was screened in the yeast two-hybrid
system with the proximal repressor domain of WT1 as bait.
Eleven of 2 3 106 clones screened were strongly positive in the
yeast two-hybrid screen, and seven of these clones encoded
portions of the same protein. Each of the encoded proteins of
the 7 positive clones interacted strongly with the bait but failed
to interact with the LexA DNA-binding domain alone, with
lamin C, or with the second repressor domain of WT1 (residues
250 –260) (Table I). These data suggest that the interaction of
the protein products of the 7 related positive clones with the
proximal repressor domain of WT1 is specific.
To obtain full-length cDNAs, a placental cDNA library was
probed with one of the positive clones. Eight independent
clones with inserts of 0.6, 1.1, and 1.8 kb were isolated. The
1.8-kb and 1.1-kb cDNAs were fully sequenced. The two cDNA
clones share the initial 15 base pairs upstream of the translation start site and the entire coding as well as 39-untranslated
regions. However, the two cDNA clones differ in the more
59-untranslated regions, presumably the result of alternative
splicing. The longest open reading frame of each clone predicts
a protein product of 157 amino acids (Fig. 1A). To establish that
the cDNAs encoded protein, each of the cDNA clones was tested
by in vitro transcription/translation. Each cDNA encodes a
protein that migrated identically in SDS-polyacrylamide gels
with a mobility consistent with the predicted 157-amino acid
protein products (data not shown).
The predicted amino acid sequence of the two cDNA clones
was compared with protein sequences deposited in GenBankTM. A 56% identity was established between the predicted
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Yeast Two-hybrid Screening—The proximal repressor domain of human WT1 (residues 85–179) was coupled with the LexA DNA binding
domain (pLexADB/WT-N) to serve as bait. To construct pLexADB/
WT-N, the cDNA fragment encoding residues 85–179 of WT1 was
obtained by digesting pSGWT-N (21) with XbaI, blunt-ending it with
the Klenow fragment and then digestion with EcoRI, and cloning the
fragment into pStop116 digested with EcoRI and SmaI. pStop116 was
modified from plasmid BTM116 (constructed by Drs. Paul Baily and
Stanley Fields) by introducing stop codons into each of the three reading frames within the polylinker region. Yeast strain L40 was transformed with pLexADB/WT-N and subsequently with the Gal4 activation domain/human placenta cDNA library (Clontech). Positive clones
were sequenced using dideoxynucleotide diphosphates and Sequenase
2.0. A lgtII human placenta cDNA library was provided by Dr. Evan
Sadler (Washington University, St. Louis) and screened as described
(23). Two million yeast transformants were screened. Sixty-five positive
clones were identified in initial screening, and eleven of these clones
were confirmed as positive. The 11 positive clones were further tested
for specificity with plasmids encoding hybrids of the LexADB with
Lamin C and with the second negative regulatory domain of WT1
(residues 250 –266). The pLexADB/WT-N alone failed to activate transcription of reporter genes containing LexA binding sites in yeast.
b-Galactosidase assay (24) was used to test the interactive specificity of
the DNA-binding domain fusion partners with the vector alone and
with the vector containing the Gal4 activation domain fused with cDNA
from positive clones. The plasmids that scored positive in these assays
were reintroduced to yeast and to quantify b-galactosidase activity
shown in Table I as a further test of specificity.
Glutathione S-Transferase Assay—To construct GST-hUBC9, pBShUBC9 was digested with EcoRI and subcloned into the EcoRI site of
the pGEX-KG vector (29). GST-hUBC9 was expressed and purified from
Escherichia coli strain DH5a. Extracts from 2 3 106 (human embryonic
kidney) cells transfected with cytomegalovirus promoter-driven expression vectors encoding full-length and the WT1D1– 84, WT1D1–294, and
WT1D297– 429 deletion mutants of WT1 (22) were incubated with
Sepharose beads containing 2–3 mg of GST and GST-hUBC9 in lysis
buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1%
Nonidet P-40, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml
leupeptin, 1 mg/ml antipain) for 2–3 h at room temperature. After
extensive washing with lysis buffer and lysis buffer with 0.5 M NaCl,
sample were boiled in SDS-PAGE loading buffer for analysis in 15%
SDS-polyacrylamide gels. The proteins were transferred to nitrocellulose membranes, immunoblotted with polyclonal anti C-terminal
and N-terminal WT1 antibodies WT(C-19) and WT 180 (Santa Cruz),
and visualized with an alkaline phosphatase-conjugated secondary
antibody.
Co-immunoprecipitation/Western Blot Analysis—An expression vector encoding the influenza virus hemagglutinin (HA)-tagged hUBC9
was constructed by cloning the 1.1-kb EcoRI fragment of hUBC9 into
the EcoRI site of expression vector pGCN in frame with HA tag. The cell
lysates were prepared from 293 cells co-transfected with WT1 and
HA-tagged hUBC9 plasmids and immunoprecipitated with anti-WT1
(WTC-19) and an anti-Gal4 DNA-binding domain antibody as control.
The Western blot analysis was performed with anti-HA monoclonal
antibody.
Northern and Southern Blot Analysis—The human tissue Northern
blot was obtained from Clontech and probed with the full-length 1.1-kb
hUBC9 cDNA according to the manufacturer’s recommendation. Human genomic DNA was purchased from Promega, digested with different restriction enzymes as indicated, and separated on a 1% agarose
gel. Southern blots were performed as described (23) with the 1.1-kb
cDNA as probe.
Yeast Complementary Assay—Strain W9432 (MATa, ubc9-D1::TRP1,
pSE362 (ARS1, CEN4, HIS3)-ubc9-1) carries a replacement of the
genomic yUBC9 coding sequence by the TRP1 marker and a plasmidborne copy of the yUBC9-1 allele (1.5-kb XbaI-SspI fragment) (10) and
is otherwise to Trp-303. The human UBC9 cDNA (1.1-kb EcoRI fragment) and the yeast UBC9 gene (0.6-kb EcoRI-XbaI fragment) were
fused to the GAL1 promoter in vectors p416GAL1 (ARSH4, CEN6,
URA3) and pSE936 (ARS1, CEN4, URA3), respectively.
TABLE I
Binding specificity of hUBC9 to the negative regulatory domain of
WT1 in the yeast two-hybrid system
Residues 85-179 of human WT1 were coupled with the LexA DNA
binding domain (pLexADB/WT-N) to serve as a bait for screening in
yeast. Two million yeast transformants were screened, and 65 positive
clones were identified in initial screening. After secondary screening,
the 11 positive clones were tested for specificity with plasmids encoding
hybrids of the LexA DB with lamin C and with a second negatively
regulatory domain of WT1 (250-266). pLexADB/WT-N alone failed to
activate transcription of reporter genes containing LexA binding sites
in yeast. b-Galactosidase activity units are shown for the DNA-binding
domain fusion partner co-expressed with the vector alone and with the
vector containing Gal4 activation domain fused with hUBC9. The plasmids recovered were reintroduced to yeast to recheck specificity and for
quantitation of b-gal actosidase activity shown.
Molecular Cloning and Chromosome Localization of the cDNA for hUBC9
24813
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FIG. 1. A, full-length nucleotide and predicted amino acid sequences of two cDNA clones encoding a protein interactive with the WT1 repressor
domain. The vertical line from cytosine 792 of the longer and cytosine 73 of the shorter form indicates the putative splice site. The longest reading
frame of the cDNAs predicts a protein of 157 amino acids. B, comparison of predicted amino acid sequences of hUBC9 and of the yUBC9 (GCG
program, Madison, WI). The conserved active site cysteines are boxed.
amino acid sequence of our clones and yeast ubiquitin-conjugating enzyme 9 (yUBC9), a nuclear protein that is required for
cell growth and that is involved in the degradation of S- and
M-phase cyclins (6). The predicted protein product contains the
active site cysteine residue (residue 93 boxed, Fig. 1B) necessary for thioester formation and the ubiquitin conjugating activity of all E2 enzymes. To establish that our clone encodes a
functional UBC9 homolog, the 1.1-kb human cDNA was expressed in a yeast carrying a temperature-sensitive (ts) yUBC9
mutation (6). The human UBC9 homolog fully restored normal
growth to the yeast strain carrying the yUBC9 ts mutant at the
otherwise nonpermissive temperature (Fig. 2). This result indicates that the cDNA we isolated encodes a biologically active
human homolog of yUBC9 that is structurally and functionally
highly conserved with yUBC9. We have designated our gene as
human (h) UBC9.
We compared the predicted amino acid sequence of hUBC9
with other E2 enzymes. Identification in regions of amino acid
sequence were established within a number of other ubiquitinconjugating enzymes, some of which are shown in Fig. 3. The
UBCs from human, pea, yeast S. cerevisiae, and Arabidopsis
thaliana retain a striking degree of amino acid identity in
several domains between the UBC9 and UBC4 families (Fig. 3),
suggesting perhaps some degree of functional overlap among
UBC9 and UBC4.
To further establish the specificity of the interaction of
hUBC9 with the N-terminal repressor domain of WT1 that was
revealed in the yeast two-hybrid screening, glutathione
S-transferase (GST) capture assays were used. Human UBC9
was expressed as a GST-fusion protein, coupled to glutathione-
24814
Molecular Cloning and Chromosome Localization of the cDNA for hUBC9
FIG. 2. Human UBC9 complements the loss of yeast UBC9 gene
function. Yeast strain W9432 carrying the temperature-sensitive
ubc9-1 allele was transformed with control vectors (row 1, p416GAL1;
row 4, pSE936) or constructs expressing either a human UBC9 cDNA
(row 2) or the yeast UBC9 gene (row 3). To compare growth of these
strains, cells were spotted in a dilution series on galactose-containing
plates and incubated for 3.5 days at the permissive temperature (23 °C)
or 2 days at the restrictive temperature (34 °C).
Sepharose beads, and incubated with extracts from 293 cells
that express the full-length (or fragments) of WT1. Eluates
were analyzed by Western blots with a rabbit anti-WT1 polyclonal antisera. Repeated experiments demonstrated that
GST-hUBC9 “captures” WT1 from lysates of 293 cells (Fig. 4A).
In control experiments, the GST-hUBC9 fusion protein failed
to capture the C-terminal DNA-binding domain of WT1 that
lacks the N-terminal amino acid repressor domain used as bait
in the yeast two-hybrid screening. GST alone failed to capture
WT1 (Fig. 4A), and GST-hUBC9 failed to capture proteins from
control 293 cells that did not express WT1 (Fig. 4A). These data
support the specificity of the interaction between the N-terminal repressor domain of WT1 and hUBC9.
Co-immunoprecipitation also was used to establish the specificity of the interactions of hUBC9 and WT1. hUBC9 was fused
at its amino terminus with an influenza (flu) hemagglutinin
(HA) “tag,” the flu-“tagged” hUBC9 was co-expressed with WT1
in 293 cells, and extracts of co-expressing cells were immunoprecipitated with anti-WT1 polyclonal antisera and washed
extensively. The immunoprecipitates were separated on SDSpolyacrylamide gels, blotted, and analyzed in Western blots
with an anti flu-tag monoclonal antibody. The flu-tagged
hUBC9 was identified in lysates from cells that co-expressed
WT1 and flu-tagged hUBC9 (Fig. 4B). The flu-tagged hUBC9
was not observed in the same lysates when a polyclonal antiGal4 antiserum was substituted for the anti-WT1 antisera in
the immunoprecipitation step, and immunoprecipitate of lysates of cells that expressed hUBC9 alone (without WT1) did
not contain proteins recognized by anti-HA antibody (Fig. 4B).
Taken together, the results of the yeast two-hybrid screening,
GST fusion protein capture assay, and co-immunoprecipitation
strongly support the conclusion that the N-terminal repressor
domain of WT1 and hUBC9 interact specifically both in vivo
and in vitro.
Northern blots from different human tissues were used to
identify the tissues that expresses hUBC9. Transcripts of 4.4,
2.4, and 1.3 kb were identified in each of the tissues that were
examined (Fig. 5A). Heart and smooth muscle have higher
levels of hUBC9 transcript relative to the other tissues analyzed, whereas the 2.4-kb mRNA isoform is relatively less
expressed in kidney, suggesting that the hUBC9 is ubiquitously expressed but at different levels in different tissues.
To determine the chromosomal locus of hUBC9, we first
probed blots of human genomic DNA with the 1.1-kb cDNA
fragment and observed single hybridization bands in BglII and
BamHI digests (Fig. 5B), suggesting that hUBC9 is a singlecopy gene. The human UBC9 was then mapped in human
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FIG. 3. A multiple sequence alignment of the Arabidopsis
UBC9 sequence, the yeast UBC9 and UBC4 sequences, and the
human UBC9 and UBC4 sequences. The sequences were aligned
using the PILEUP algorithm. PILEUP is part of the Wisconsin Sequence Analysis Package. An amino acid is placed in the consensus
sequence if it is located at that position in four of the five aligned
sequences.
FIG. 4. The interaction of WT1 and hUBC9. A, in vitro interactions of WT1 and hUBC9. The full-length hUBC9 cDNA was fused
in-frame with the GST and expressed as the GST-hUBC9 fusion protein. GST and GST-hUBC9 fusion proteins were bound to glutathioneSepharose beads. Extracts from 293 cells that expressed WT1 and
deletion mutants of WT1, WT1D1– 84, WT1D297– 429, and WT1D1–294,
were incubated with GST and GST-fusion protein bound to Sepharose
beads for 2–3 h. Following washing with lysis buffer, beads were eluted
with SDS gel loading buffer and separated by 15% SDS-PAGE and
transferred to nitrocellulose filters for immunoblotting with antibodies
against N- and C-terminal portions of WT1 and visualized by fluorography. B, co-immunoprecipitation of WT1 and hUBC9 from 293 cells
expressing WT1 and HA-hUBC9. Extracts of 293 cells were first immunoprecipitated with either anti-WT1 (WTC-19) or a nonspecific antibody (anti-Gal4 DNA binding domain), separated on 15% SDS-PAGE,
blotted, and probed with anti-HA tag monoclonal antibody.
Molecular Cloning and Chromosome Localization of the cDNA for hUBC9
24815
FIG. 6. Chromosomal localization of hUBC9 probe by FISH on
human metaphase chromosomes. Note specific hybridization of fluorescein-labeled probe to subband 16p13.3. The photograph represents
a computer-enhanced, merged image (arrow), and diamidino-2-phenylindole-stained chromosomes.
DISCUSSION
FIG. 5. A, Northern blot of hUBC9 in different human tissues. A
human tissue Northern blot (Clontech) was probed with the 1.1-kb
hUBC9 cDNA. Each lane contained 2 mg of poly(A)1 RNA from heart
(H), brain (B), placenta (Pl), lung (L), smooth muscle (SM), kidney (K),
and pancreas (Pa). The b-actin cDNA was used to probe the same blot
as control. The size markers are indicated on the left side of the blot. B,
Southern blot analysis of the hUBC9 gene. 10 mg of human genomic
DNA were digested with HindIII (H), EcoRI (E), BglII (Bg), or BamHI
(B), and DNA fragments were separated by electrophoresis on 1%
agarose gel. The blot was probed with the 1.1-kb hUBC9 cDNA probe.
metaphase chromosomes by FISH. The hUBC9 probe hybridized on the short arm of human chromosome 16 in 22 of 26
metaphase spreads examined. Among 94 fluorescent signals on
all chromosomes, 55 (58.5%) were located on band 16p13. Most
signals localized distally at subband 16p13.3 (Fig. 6). A mouse/
human somatic cell panel with average distance between
breakpoints of 1.2 Mb and a potential resolution of 1 Mb was
used to further fine map the gene. Major bands of approximately 20 kb and 8.5 kb and mouse bands of 9.5 kb, 6 kb, and
3.2 kb were observed in the Southern blots. The human band of
The data strongly suggest that the interactive protein previously identified (22) that modulates WT1 repressor activity is
hUBC9. This conclusion is based upon the specificity of the
interaction of the N-terminal but not the C-terminal repressor
domain of WT1 with hUBC9, the failure of lamin C to interact
with WT1 in the yeast two-hybrid system, and upon the specificity of the interaction of hUBC9 and WT1 demonstrated in
co-immunoprecipitation and GST capture assays. The data also
indicate significant identity of primary structure and retention
of the invariant active site cysteine residue between the predicted amino acid sequence of our cDNA clones and yUBC9.
Our cDNA clone functionally complements yeast carrying a
temperature-sensitive UBC9 gene mutant, indicating that
hUBC9 is fully functional as an E2 enzyme in yeast and that
we have cloned the human homolog of the yUBC9. Human
UBC9 retains a high degree of amino acid identity with other
UBCs that have been cloned and is most closely related to the
UBC4 family. The potential importance of the highly conserved
domains that we have identified among UBCs is unclear, but
these domains may be important in selective interactions with
specific target proteins.
The ability of our cDNA to fully rescue yeast that contain a
temperature-sensitive mutant is a striking example of functional conservation from yeast to human and suggests a highly
important role of the UBC9s throughout evolution. In yeast,
UBC9 is involved in cell cycle progression. Yeast UBC9 is
required for degradation of S- and M- phase cyclins and for
viability (6). In this context, it is important to note that WT1
blocks cell cycle progression and that this block is relieved by
expression of exogenous cyclin E and CDK2 as well as cyclin
D1/CDK4 (27). Because of the striking conservation of primary
structure and because hUBC9 fully complements yeast with
yUBC9 temperature-sensitive mutations to allow normal progression of yeast through the cell cycle, we suggest that hUBC9
may have a similar role in eukaryotic cells. The results also
suggest that the interaction of WT1 with hUBC9 may be important in the cell cycle block imposed by WT1.
Human hUBC9 is an extraordinarily conserved protein in
evolution from yeast to man. Whereas its precise role in human
cells is unknown, the requirement of UBC9 in yeast for normal
cell cycle progression and the specific interactions with WT1
suggest important roles of hUBC9 in mammalian cells and
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20 kb was present on chromosome 16 and was present in the
hybrids CY189 and CY193 but absent from the hybrids CY14,
CY192, and additional hybrids with more proximal breakpoints
of CY200, CY193 to CY189, indicating that hUBC9 maps between (data not shown).
24816
Molecular Cloning and Chromosome Localization of the cDNA for hUBC9
raise the possibility of important roles of ubiquitination in
regulation of transcription.
Acknowledgments—We thank Drs. Kendall Blumer, Andre Shaw,
and Daniel Link for help in establishing the yeast two-hybrid system.
We also thank Dr. Nobuo Horikoshi for helpful advice.
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Human Ubiquitin-conjugating Enzyme 9
Zhao-Yi Wang, Qing-Qing Qiu, Wolfgang Seufert, Takahiro Taguchi, Joseph R. Testa, S.
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J. Biol. Chem. 1996, 271:24811-24816.
doi: 10.1074/jbc.271.40.24811
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