(Received for publication, September 11, 1995; and in revised form, November 15, 1995)
From the
The ubiquitin/proteasome system is the main eukaryotic
nonlysosomal protein degradation system. Substrate selectivity of this
pathway is thought to be mediated in part by members of a large family
of ubiquitin-conjugating (E2) enzymes, which catalyze the covalent
attachment of ubiquitin to proteolytic substrates. E2 enzymes have a
conserved 150-residue so-called UBC domain, which harbors the
cysteine residue required for enzyme-ubiquitin thioester formation.
Some E2 enzymes possess additional carboxyl-terminal extensions that
are involved in substrate specificity and intracellular localization of
the enzyme. Here we describe a novel family of E2 enzymes from higher
eukaryotes (Drosophila, mouse, and man) that have
amino-terminal extensions but lack carboxyl-terminal extensions. We
have identified four different variants of these enzymes that have
virtually identical UBC domains (94% identity) but differ in their
amino-terminal extensions. In yeast, these enzymes can partially
complement mutants deficient in the UBC4 E2 enzyme. This indicates that
members of this novel E2 family may operate in UBC4-related proteolytic
pathways.
In eukaryotes, selective protein degradation is largely mediated
by the ubiquitin/proteasome system (for reviews, see (1, 2, 3, 4, 5) ).
Degradation by this system was recently found to be instrumental in a
variety of cellular functions such as DNA repair, cell cycle
progression, signal transduction, transcription, and antigen
presentation. Known substrates of this pathway include transcription
factors (MAT2, GCN4, c-Jun, p53, NF-
B), protein kinases
(Mos), cyclins, inhibitors of cyclin-dependent kinases (SIC1, p27), and
subunits of trimeric G proteins (for review, see (1, 2, 3, 4, 5) ).
Moreover, the ubiquitin/proteasome system also eliminates abnormal
proteins, e.g. misfolded, mislocalized, or misassembled
proteins.
Substrate recognition by this pathway involves a specialized recognition and targeting apparatus, the ubiquitin-conjugating system, which operates spatially detached from the proteasome. Proteins recognized by this system are earmarked by the covalent attachment of ubiquitin, a small and highly stable protein. In most cases, ubiquitination involves the formation of multiubiquitin chains attached to the substrate that are subsequently recognized by a specific receptor of the (26 S) proteasome. Proteins bound to the receptor are then probably unfolded and translocated into the central cavity of the proteasome where they are degraded to small polypeptides. Ubiquitin chains are released from substrates and recycled to single ubiquitin moieties (for review, see (5) ).
Ubiquitin
conjugation involves a reaction
cascade(1, 3, 6, 7) . Initially,
ubiquitin-activating (E1) ()enzyme hydrolyses ATP and forms
a thioester bond between itself and ubiquitin. Ubiquitin is then passed
on to ubiquitin-conjugating (E2) enzymes and often subsequently to
ubiquitin ligases (E3). Each step involves the formation of a
thioester-linked ubiquitin-enzyme (E1, E2, or E3)
intermediate(7) . E2 and/or E3 enzymes finally catalyze
isopeptide formation between the carboxyl terminus of ubiquitin and
-amino groups of internal lysine residues of target proteins. Both
E2 and E3 enzymes exist as protein families, and diverse combinations
of E2
E3 enzyme complexes are thought to define the substrate
specificity of the conjugation system.
In the yeast Saccharomyces cerevisiae, 12 different genes for
ubiquitin-conjugating enzymes (UBC genes) have been detected to
date(3, 6) . Genetic studies revealed that the encoded
enzymes mediate strikingly diverse functions such as DNA
repair(8) , sporulation(3, 8) , cell cycle
progression(9, 10) , peroxisome
biogenesis(11) , membrane-protein degradation(12) ,
heat shock resistance(13) , and cadmium tolerance(15) .
One of the most prominent E2 enzymes from yeast is UBC4(13) . A
principal function of UBC4 and the highly related UBC5 enzyme appears
to be the degradation of abnormal proteins as indicated by the
sensitivity of ubc4 ubc5 double mutants to heat shock,
canavanine (an arginine analog), and cadmium(13, 15) .
In addition, UBC4/5-mediated proteolysis is important for some
regulatory processes. One example is the UBC4/UBC5-mediated degradation
of the yeast transcription factor MAT2 involved in mating type
control (16) . UBC4/UBC5 homologs have been described from
several organisms including Drosophila (UbcD1; (17) ), Caenorhabditis elegans (ubc-2; (18) ) and man (UbcH5; (19) ). The function of
these enzymes in these organisms is not known, but, in the cases
tested, the respective genes can fully complement yeast ubc4 ubc5 mutants. Interestingly, vertebrate UBC4 homologs can mediate p53 (19) and cyclin (20) ubiquitination in vitro,
suggesting that UBC4/5-mediated degradation may be of central
regulatory importance.
Here we report the identification of a novel family of ubiquitin-conjugating enzymes from higher eukaryotes that are related in function and sequence to yeast UBC4/5. In contrast to UBC4 (and its homologs from higher eukaryotes) these enzymes have amino-terminal extensions. The UBC domain of these enzymes from Drosophila, mouse, and man is exceptionally highly conserved, exhibiting 94% sequence identity over a stretch of 149 amino acid residues. In contrast, the amino-terminal extensions of four different members of this family exhibit little sequence similarity and may define substrate specificity or are involved in the regulation of these enzymes. In each species, probably all four (or possibly more) different members of this UBC subfamily are expressed, suggesting that each one of these enzymes fulfils special tasks.
Figure 1: Nucleotide and predicted amino acid sequences of UbcD2, UbcM2, and UbcM3cDNAs. Nucleotide numbers starting at the first nucleotide of the coding region are given on the left. In-frame stop codons in the 5`-untranslated region are underlined. Active site cysteine residues required for thioester formation are shown in boldface. The nucleotide sequences have been submitted to the Genbank/EMBL data base with the accession numbers X92663 UbcD2, X92664 UbcM2 and X92665 UbcM3.
Figure 4:
Complementation of the yeast ubc4 ubc5 mutant by expression of UbcD2, UbcM2, UbcM3 and an amino-terminal truncated UbcM3. A, growth
of yeast ubc4 ubc5 double mutant on YPGal plates at normal
growth temperature (30 °C) and heat shock temperature (37 °C)
expressing the following UBCs (in clockwise orientation). Vector as
negative control, amino-terminal truncated UbcM3
(pUbcM31-47), UbcM3 (pUbcM3), UbcM2
(pUbcM2), UbcD2 (pUbcD2), and yeast UBC4
(pUBC4) as positive control. B, Western blot analysis
of total yeast proteins from ubc4 ubc5 cells expressing UbcM3 and UbcM3
1-47, respectively, with
antibodies generated against the conserved UBC domain of
UbcD2/UbcM2/UbcM3 family. Additional protein bands cross-reacting with
the antiserum serve as loading control. Size references are given on
the right.
Figure 2: Northern blot analysis. UbcD2, UbcM2, and UbcM3 transcripts are shown with their respective sizes. The positions of RNA size markers are indicated on the right.
A comparison of the deduced amino acid sequences of UbcD2, UbcM2, and UbcM3 shows that they are highly related (Fig. 3). Unlike previously identified E2 enzymes(3, 6) , these new enzymes possess amino-terminal extensions in addition to the UBC domain. The UBC domains of the three enzymes are almost identical in sequence (94% identity over 149 amino acid residues; between 72 and 79% at the DNA level). UbcD2 differs from the UbcD2/UbcM2/UbcM3 consensus by 6, UbcM2 by 2, and UbcM3 by 3 residues (Fig. 3). In contrast to the extreme conservation of the UBC domains, the amino-terminal extensions of the three enzymes (designated extensions A, B, and C; Fig. 3) show little sequence similarity among each other and differ in size (Fig. 3). The weak sequence similarity of the extensions is largely restricted to clusters of serine/threonine and basic residues. No significant sequence similarities between the extensions and known sequences in the data bases were found except for short consensus sequences for phosphorylation sites. Further data base searches detected (in addition to a human UbcM2 homolog designated UbcH9) a partial open reading frame from a human cDNA fragment (designated UbcH8), and this represents a probable fourth member of this enzyme family. This partial sequence exhibits a 100% match to the corresponding sequences of the UBC domains of UbcD2, UbcM2, and UbcM3 and an amino-terminal extension (designated extension D) nonidentical, but related to, extension B of UbcM2 and UbcH9 (Fig. 3). This suggests that the novel E2 enzyme family described here has at least four distinct members.
Figure 3:
Sequence similarity between members of the
novel UBC-family. Primary sequences of UbcD2, UbcM2, UbcM3, and three
additional members of this family designated UbcH8, UbcH9 (translated
from partial cDNA sequences, accession numbers Z44894 and H12272) and
UbcH6 are compared with yeast UBC4(14) . Upper
panel, schematic diagram of the primary sequences. The highly
conserved UBC domain of the new UBC family is shown as a white box and corresponds to the entire sequence of UBC4 (light
gray). The four different amino-terminal extensions are shown as
boxes in distinct gray shades and are designated A, B, C, and D extensions. Numbers above the
boxes correspond to amino acid residues. Lower left panel, sequence comparison of the amino-terminal extensions of UbcD1,
UbcM2, UbcM3, and UbcH8. Residues that are identical in at least two
proteins are boxed. Gaps (indicated by dashes) were
permitted to optimize alignments. Amino-terminal extensions of the
homologs UbcM2 and UbcH9 (extension B) differ by only one residue
(K31E) and of the homologs UbcM3 and UbcH6 (extension C) by two
residues (G26T/S27N). Lower right panel, sequence comparison
of the UBC domains of different family members with UBC4. Unavailable
sequence data of the partial UbcH8 clone is indicated by a dotted line. Residues that are identical in at least three
proteins are boxed. The sequences were aligned using the
BoxAlign program (GCG package). Residue numbers are given on the left.
To study the activity of UbcD2, UbcM2 and UbcM3 in yeast, we cloned the respective reading frames into yeast high copy number, 2 micron based expression vectors. When yeast cells were transformed with these plasmids, all three genes could complement the growth deficiency and heat sensitivity of ubc4 ubc5 double mutants (Fig. 4A). Although complementation was only partial as indicated by slight growth defects at 30- and, in particular, at 37 °C, UbcD2, UbcM2, and UbcM3 are likely to function in similar proteolysis pathways as UBC4 (or UbcD1). The incomplete complementation of ubc4 ubc5 by these genes may indicate that UbcD2, UbcM2, and UbcM3 only interact with a subset of UBC4's substrates or that they may fail to collaborate with certain components of a UBC4-dependent degradation pathway (e.g. ubiquitin ligases, E3), or both.
In addition
to a comparably weak sequence similarity to UBC4/UBC5 (identity of 64 versus 80% for UbcD1, Fig. 5) the three enzymes differ
from UBC4 and its homologs by the presence of amino-terminal
extensions. These extensions could possibly function as regulatory
(either activating or repressing) or interacting domains with specific
components of the ubiquitin-conjugation system. To test these
possibilities, we constructed a derivative of UbcM3 lacking the
amino-terminal extension (UbcM31-47). When yeast cells were
transformed with UbcM3 and truncated UbcM3
1-47, both gene products were expressed with
the expected sizes to similar levels (Fig. 4B).
Remarkably, full-length and truncated UbcM3 enzymes complemented the
yeast ubc4 ubc5 mutant to a similar extend (Fig. 4A). Thus the amino-terminal extension of UbcM3
(and probably also of UbcD2 and UbcM2) does not modulate the activity
of the enzyme to function in UBC4-dependent pathways in yeast.
Figure 5: Phylogenetic tree of the UBC4-related subfamily. Relatedness was calculated by the algorithm provided by the DNA Star package and compared with distantly related yeast UBC2/RAD6. Only the UBC domains were compared. Complementation of yeast ubc4 ubc5 mutants (growth at 30 and 37 °C) is indicated; ++, full complementation; +, partial complementation by overexpression; -, no complementation (see text for experimental details).
Previously identified ubiquitin-conjugating enzymes (3, 6) are small proteins (14-32 kDa),
which either consist of the UBC domain only (class I E2 enzymes) or
they possess additional carboxyl-terminal extensions (class II
enzymes). Here we describe a novel family of ubiquitin-conjugating
enzymes from higher eukaryotes that have amino-terminal but lack
carboxyl-terminal extensions (designated class III enzymes). We have
cloned three members of this class and identified a fourth in the data
base. The UBC domain of these novel enzymes is virtually identical, but
the amino-terminal extensions show limited sequence similarity. The
recent identification of human homologs to UbcM2 (UbcH9; Fig. 3)
and UbcM3 (UbcH6; Fig. 3), (
)which are homologous to
their respective murine counterparts over their entire lengths
(including the extensions), and the extreme conservation of the UBC
domains of different members of this family strongly suggest that a
homolog of each of these novel four UBCs may be present in each of
these species, i.e. Drosophila, mouse, and man. We have
unsuccessfully tried to identify yeast homologs to these enzymes using
different PCR strategies. Thus we assume this family probably evolved
relatively late in evolution and may be unique to multicellular
organisms. Intriguingly, these enzymes are among to the most highly
conserved proteins of these organisms. The UBC domains of UbcD2 from Drosophila and UbcM2, and UbcM3 from mice share 94% identical
amino acid residues (homologs of other UBCs are typically 70-80%
identical in sequence; (17, 18, 19) ,
29-32; see Fig. 5). The extreme conservation of UbcD2,
UbcM2, and UbcM3 is even more remarkable given the likely possibility
that the true homologs, i.e. the enzymes with similar
extensions are yet to be identified. Proteins of similar high
conservation, e.g. histones or ubiquitin, either have multiple
interacting partners or most of their amino acid residues participate
in intramolecular contacts. Both types of interactions are thought to
prevent evolutionary amino acid sequence drift. We thus assume that the
novel UBC enzymes interact with several proteins. Candidates for
binding partners are components of the ubiquitin/proteasome system or
substrates. Since overexpressed UbcD2, UbcM2, and UbcM3 can partially suppress UBC4/UBC5 deficiency in
yeast, these enzymes and UBC4/UBC5 probably have many substrates in
common. However, the presence of multiple, highly conserved extensions
of these enzymes suggests that they are likely to carry out specialized
functions distinct from those of UBC4. What these functions are is not
known at present, but the gene expression pattern of the Drosophila UBCs may provide some clues. Interestingly, UbcD1, the UBC4 homolog, is continuously expressed throughout development
consistent with a ``housekeeping'' function of the encoded
enzyme. (
)Transcripts of UbcD3(bendless), another UBC4-related gene (which is actually unable to rescue ubc4
ubc5 mutants; Fig. 5)
can also be detected at
all developmental stages of Drosophila development. In
contrast, UbcD2 appears to be exclusively expressed at
postlarval (L3) stages, but in eggs the transcript is supplied
maternally.
Thus the functions of these class III enzymes
may be predominantly restricted to distinct tissues in pupae or adult
flies.
The significance of the amino-terminal extensions is currently unclear, but their conservation between species (e.g. the extensions B and C; Fig. 3) indicate that they are probably relevant to their cellular functions. The carboxyl-terminal extensions of class II E2 enzymes are known either to contribute to their substrate specificity (UBC2, UBC3; (9) and (33) -35) or they mediate intracellular localization (UBC6; (12) ). The prevalence of putative phosphorylation sites within the extensions of the UbcD2, UbcM2, and UbcM3 enzymes may indicate that the enzymatic activity or a possible interaction with other proteins is possibly controlled by enzyme phosphorylation. Alternatively, these sequences rich in serine, threonine, and basic residues may represent binding sites for specific components of the ubiquitin-conjugating system or proteolytic substrates. Class I ubiquitin-conjugating enzymes have highly conserved three dimensional structures with exposed amino termini(36) . This suggests that the highly charged amino-terminal extensions of the class III enzymes described here may fold into separate domains, which are probably readily accessible to interacting partners.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X92663[GenBank], X92664[GenBank], and X92665[GenBank].