From the Deutsches Krebsforschungszentrum, Angewandte Tumorvirologie, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany
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ABSTRACT |
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Members of the hect domain protein family are
characterized by sequence similarity of their C-terminal regions to the
C terminus of E6-AP, an E3 ubiquitin-protein ligase. An essential
intermediate step in E6-AP-dependent ubiquitination is the
formation of a thioester complex between E6-AP and ubiquitin in the
presence of distinct E2 ubiquitin-conjugating enzymes including human
UbcH5, a member of the UBC4/UBC5 subfamily of E2s. Similarly, several
hect domain proteins, including Saccharomyces cerevisiae
RSP5, form ubiquitin thioester complexes, indicating that hect domain
proteins in general have E3 activity. We show here, by the use of
chimeric E2s generated between UbcH5 and other E2s, that a region of
UbcH5 encompassing the catalytic site cysteine residue is critical for
its ability to interact with E6-AP and RSP5. Of particular importance
is a phenylalanine residue at position 62 of UbcH5 that is
conserved among the members of the UBC4/UBC5 subfamily but is not
present in any of the other known E2s, whereas the N-terminal 60 amino acids do not contribute significantly to the specificity of these interactions. The conservation of this phenylalanine residue throughout evolution underlines the importance of the ability to interact with
hect domain proteins for the cellular function of UBC4/UBC5 subfamily members.
Modification of proteins by the covalent attachment of ubiquitin
in many cases targets these proteins for proteasome-mediated degradation (reviewed in Refs. 1-5) or, as shown for some membrane proteins, for internalization and degradation via the lysosomal pathway
(6, 7). Ubiquitin conjugation (ubiquitination) requires the combined
action of ubiquitin-activating enzymes
E11 (or UBA),
ubiquitin-conjugating enzymes E2 (or UBC), and, probably in most cases,
ubiquitin-protein ligases E3 (or UBR). In the first step, ubiquitin is
activated by E1 at the expense of ATP via the formation of a high
energy thioester intermediate between E1 and ubiquitin. Ubiquitin is
then transferred to one of a number of E2s, preserving the thioester
bond. Finally, ubiquitin is covalently linked to a substrate
protein either by the E2 alone or in conjunction with an E3.
This sequential mode of ubiquitin transfer indicates that E2s and in
particular E3s play a major role in substrate recognition.
Proteins that have been identified with E3 activity include yeast UBR1
(8), mammalian E6-AP (9), members of an E6-AP related family of
putative E3s termed hect (homologous to E6-AP C terminus) domain proteins (10, 11), a protein
complex termed APC (anaphase promoting
complex) or cyclosome (reviewed in Refs. 12 and 13), and
the recently identified SCF complexes (14-17). Based on their mode of
action, it appears that E3s can be classified into at least two
categories. Some E3s may function as docking proteins by binding
specifically to E2s and substrate proteins, thereby facilitating
E2-catalyzed ubiquitination of the substrates. Such E3s appear to be
represented by the SCF complexes (14, 15, 18). Other E3s are loaded
with ubiquitin by E2s via thioester formation, indicating that these
E3s directly catalyze the attachment of ubiquitin to substrate
proteins. E3s with the capacity to form ubiquitin thioester complexes
include E6-AP and hect domain proteins (10, 11, 19, 20). Despite their
different mode of action, a common feature of all known E3s is that
each E3 appears to specifically interact with a distinct E2 or distinct
subsets of E2s. For instance, SCF complexes bind specifically to
UBC3CDC34 (14-16, 18), whereas E6-AP and other hect domain
proteins, including Saccharomyces cerevisiae RSP5, have been
shown in vitro to interact with UbcH5 and/or UbcH7 and
related E2s (9-11, 21-23). UbcH5 belongs to an evolutionally
conserved subfamily of E2s that are highly similar to S. cerevisiae UBC4/UBC5 (9, 21, 24), whereas based on amino acid
sequence comparisons, the genome of S. cerevisiae does not
encode an obvious ortholog to UbcH7 (22).
Members of the hect domain family are large proteins ranging in size
from 90 kDa to more than 500 kDa and are found in all eukaryotic
organisms examined (10, 11, 19). They are characterized by a C-terminal
region of approximately 350 amino acids in length, which shows
significant similarity to the C terminus of E6-AP, and therefore has
been termed the hect domain (10). The hect domain appears to represent
the catalytic domain of these otherwise unrelated proteins because the
hect domain is necessary and sufficient to form ubiquitin thioester
complexes in the presence of its cognate E2s (11). Similar to E2s, it
appears that a cell contains a number of different hect domain
proteins. Based on data base searches, the genome of S. cerevisiae encodes five different hect domain proteins (4),
whereas the human genome codes for at least 20 hect domain proteins
(11).
All known E2s are characterized by a conserved core domain of
approximately 150 amino acids in size, termed the UBC domain, and can
be grouped into four classes based on the presence or absence of
additional N-terminal and/or C-terminal sequences (25). Because UbcH5
belongs to the so-called class I E2 enzymes, which consist of the UBC
domain only, we were interested to determine the regions of UbcH5
that specify its ability to interact with members of the hect domain
family. Here we report that a region surrounding the catalytic site
cysteine residue, as well as a phenylalanine residue at position 62 that is characteristic for members of the UBC4/UBC5 subfamily, are
critical for the ability of UbcH5 to interact with E6-AP and RSP5
in vitro. This indicates that the structural conformation of
the thioester-forming region of E2s determines whether or not a given
E2 can functionally interact with hect domain proteins.
Plasmids--
The pET-3a expression plasmid for UbcH5 has been
described previously (21). Expression plasmids for UbcM3 and UbcH1 were constructed by ligating PCR products into pET-3a (UbcH1 and UbcM3) and
pGEX-2T (UbcM3). The valine residue at position 129 of UbcM3 (or
position 83 with respect to the amino acid sequence of UbcH5; see Fig.
1A), the serine residue at position 83 of the chimeric E2 35 (see Fig. 1B), and the asparagine residue at position 65 of
UbcH1 (see Fig. 1A) were altered to serine (3-V83S), valine (35-S83V), and phenylalanine (1-N65F), respectively, by
PCR-directed mutagenesis. The respective cDNAs were cloned into
pGEX-2T. The cDNAs used as template for UbcM3 (kindly
provided by S. Jentsch) and UbcH5 in PCR have been described
(21, 26). A UbcH1 cDNA was obtained by RNA PCR using
cytoplasmic RNA prepared from HeLa cells. Expression plasmids encoding
the various chimeric E2s were constructed by ligating PCR products into
pET-3a (355, 533, 335, 553) or pGEX-2T (355, 533, 335, 553, 35, 353, 335-V83S, 35-S83V, 155, 155-N65F). The cDNAs encoding the various
chimeric E2s consist of the following nucleotides (nt) of the cDNA
of the respective E2s (with nt 1 defined as A in the initial ATG): 553, nt 1-320 of UbcH5 fused to nt 459-582 of UbcM3;
533, nt 1-186 of UbcH5 fused to nt 325-582 of
UbcM3; 355, nt 1-324 of UbcM3 fused to nt
187-444 of UbcH5; 35 and 35-S83V, nt 1-369 of
UbcM3 fused to nt 232-444 of UbcH5; 353, nt
1-369 of UbcM3 fused to nt 232-320 of UbcH5
fused to nt 459-582 of UbcM3; 335 and 335-V83S, nt 1-458 of UbcM3 fused to nt 321-444 of UbcH5; 155 and
155-N65F, nt 1-195 of UbcH1 fused to nt 187-444 of
UbcH5. All PCR-generated cDNAs were sequenced to rule
out the possibility that second site mutations were introduced during
PCR amplification.
Protein Expression--
UbcH1, UbcH5, UbcM3, and the chimeric
E2s 355, 335, 533, 553 were expressed in Escherichia coli
BL21 (DE3) using the pET expression system as described previously
(21, 22). For ubiquitination and ubiquitin thioester assays, crude
bacterial extracts containing the respective E2s were used. GST fusion
proteins (E2s, ubiquitin, and E6-E7) were expressed in E. coli DH5
E6-AP, RSP5, RSP5 Thioester and Ubiquitination Assays--
Formation of thioester
adducts of the different E2s, E6-AP, RSP5, and RSP5
Ubiquitination assays using bacterially expressed GST-E6-E7 as a
substrate were performed as described (9).
Generation of Chimeric E2s Consisting of Different Parts of UbcH5
and UbcM3--
Similar to UbcH5, murine UbcM3 and its putative human
homolog UbcH6 are members of the UBC4/UBC5 subfamily of E2s (22, 26). Unlike UbcH5, however, UbcM3 and UbcH6 contain an N-terminal extension of 46 amino acids in addition to the UBC domain (Fig.
1A). Despite its similarity to
UbcH5 at the amino acid sequence level, it was shown that UbcH6 is only
weakly active in E6-AP-dependent ubiquitination (22).
Furthermore, this difference between UbcH5 and UbcH6 was not due to the
N-terminal extension of UbcH6 because a respective deletion mutant of
UbcH6 was as inefficient in E6-AP-dependent ubiquitination
as full-length UbcH6. UbcH6 and UbcM3 are identical at the amino acid
sequence level, except for two amino acid exchanges in the N-terminal
extension (22, 26) and thus can be considered functionally equivalent.
For cloning reasons, a cDNA encoding UbcM3 was used for the
following rather than a cDNA encoding UbcH6.
To determine the region(s) of UbcH5 that is responsible for the
observed difference to UbcM3, initially two cDNAs encoding two
different chimeric E2s between UbcH5 and UbcM3 were generated and
cloned into the bacterial pET expression vector system. The respective
E2s were termed 533 and 355 (the nomenclature reflects the relative
contribution of the UBC domain of UbcH5 and UbcM3 to the UBC domain of
the respective chimeric E2; Fig. 1B) and consist of the
N-terminal 62 amino acids of UbcH5 fused to the C-terminal 85 amino
acids of UbcM3 and of the N-terminal 108 amino acids of UbcM3 fused to
the C-terminal 85 amino acids of UbcH5, respectively. The chimeric E2s
were expressed in bacteria and tested for their ability to interact
with E6-AP.
An essential intermediate step in E6-AP-dependent
ubiquitination is the formation of a thioester complex between E6-AP
and ubiquitin in the presence of E1 and UbcH5 or UbcH7 (19, 22). As
shown in Fig. 2, 355 was able to transfer
ubiquitin to E6-AP with an efficiency similar to UbcH5, whereas 533 was
almost inactive under the conditions used. It should be noted that
similar amounts of active E2s were used as determined by their ability
to form thioester complexes with ubiquitin in the presence of E1 (data not shown). Taken together, this clearly shows that the difference between UbcM3 and UbcH5 with respect to their ability to interact with
E6-AP maps to the C-terminal 85 amino acids of UbcH5.
A Region Encompassing the Catalytic Site Cysteine Residue Is
Critical for Interaction of UbcH5 with E6-AP and RSP5--
To further
delineate the region of UbcH5 that is critical for functional
interaction with E6-AP, additional chimeric E2s were constructed (Fig.
1B). Because some of the chimeric E2s were expressed at
rather low levels, if at all, in the pET expression system (not shown),
making it difficult to assess the amount of active E2, the E2s were
expressed as GST fusion proteins. The fusion proteins were concentrated
by affinity chromatography and then the GST part was removed by
thrombin cleavage to ensure that the ability of the respective E2s to
interact with E6-AP was not influenced by their fusion to GST. This
showed that a chimeric E2 (termed "35"; Fig.
3A) containing the C-terminal
70 amino acids of UbcH5 was as active as 355 in E6-AP/ubiquitin
thioester assays, whereas a chimeric E2 containing the C-terminal 40 amino acids of UbcH5 (335) was significantly less active. This
indicates that a region of UbcH5 comprising amino acids 78-107 is
critical for interaction with E6-AP. Indeed, the chimeric E2 353 in
which the corresponding region of UbcM3 was replaced by amino acids
78-107 of UbcH5 was nearly as active as 355 in transferring ubiquitin
to E6-AP.
Amino acid sequence comparison of UbcH5 with UbcM3 (Fig. 1A)
reveals that amino acid region 78-107 of UbcH5 contains six amino acid
exchanges to the corresponding region of UbcM3. The most significant
difference would appear to be the presence of a serine residue at
position 83 of UbcH5, whereas UbcM3 contains a valine residue at the
corresponding position. Therefore, point mutants of 35, 335, and UbcM3
were generated by substituting the corresponding amino acid with either
valine (35-S83V) or serine (335-V83S or 3-V83S). Indeed, 35-S83V was
unable to load E6-AP with ubiquitin (Fig. 3A). In contrast,
thioester complex formation of E6-AP with ubiquitin was observed in the
presence of 335-V83S and 3-V83S, although with a reduced efficiency
compared with 355 (Fig. 3A) or UbcH5 (not shown). This
indicates that the serine residue at position 83 of UbcH5 is necessary
but not sufficient for an efficient interaction of UbcH5 with
E6-AP.
Taken together, the data presented above indicate that the structural
conformation of the region surrounding the catalytic site cysteine
residue is important for UbcH5 to functionally interact with E6-AP.
Furthermore, the amino acid sequence differences in this region are
mainly responsible for the observed difference between UbcH5 and UbcM3
with respect to their ability to interact with E6-AP. This notion is
further supported by the finding that only those chimeric E2s that were
able to load E6-AP with ubiquitin were able to support
E6-AP-dependent ubiquitination of an artificial substrate
protein (see Fig. 5 and Fig. 1B).
The yeast hect domain protein RSP5, which except for the hect domain is
not related to E6-AP at the amino acid sequence level, was previously
shown to form thioester complexes with ubiquitin in the presence of
both UbcH5 and UbcM3 but with significantly less efficiency in the
presence of UbcM3 (16, 22). Therefore, ubiquitin thioester assays with
RSP5 were performed using the various chimeric E2s. As shown in Fig.
3B, the same chimeric E2s that could interact with E6-AP
were able to load RSP5 with ubiquitin, whereas those that did not
interact with E6-AP also did not interact with RSP5. This indicates
that similar amino acid sequences or structures of UbcH5 are required
for its interaction with both E6-AP and RSP5.
A Phenylalanine Residue Conserved among All Members of the
UBC4/UBC5 Subfamily of E2s Is Essential for Interaction with E6-AP and
RSP5--
Replacement of the N-terminal 62 amino acids of UbcH5 with
the corresponding region from UbcM3 resulted in a chimeric E2 that was
capable of transferring ubiquitin to both E6-AP and RSP5 (355; Figs. 2
and 3). This shows that this region is not responsible for the observed
difference between UbcH5 and UbcM3. Because UbcH5 and UbcM3 belong to
the same subfamily of E2s, however, the possibility that the N-terminal
62 amino acids of UbcH5 contain a determinant(s) that is critical for
UBC4/UBC5 subfamily members to interact with hect domain proteins
cannot be excluded. This possibility was addressed by testing whether
or not the N-terminal region of UbcH5 can be functionally replaced by
the corresponding region of an unrelated E2. A chimeric protein was
constructed consisting of the N-terminal 65-amino acid residues of
UbcH1, the human homolog of S. cerevisiae
UBC2RAD6 (28), and the C-terminal 85 amino acids of UbcH5
(155; Fig. 1). UbcH1 was used because it had previously been shown that
UbcH1 or its Arabidopsis thaliana homolog UBC1 can neither
transfer ubiquitin to E6-AP or RSP5 nor support
E6-AP-dependent ubiquitination (9, 11). Ubiquitin thioester
assays showed that, although 155 itself can form thioester complexes
with ubiquitin with an efficiency similar to that of UbcH5 (data not
shown), it was not able to transfer ubiquitin to either E6-AP or RSP5
(Fig. 4). Similarly, 155 was inactive in
E6-AP-dependent ubiquitination (Fig.
5). Thus, the N-terminal 62 amino acids
of UbcH5, as well as the corresponding region of UbcM3, contain a
determinant that is required for interaction with hect domain family
members.
Alignment of various UBCs (not shown) reveals that a phenylalanine
residue at position 62 of UbcH5 (Fig. 1A) is conserved among
the members of the UBC4/UBC5 subfamily but is not present in any of the
other known E2s. For instance, UbcH1 contains an asparagine residue at
the corresponding position (Fig. 1A). Therefore, this
asparagine residue was substituted by phenylalanine in both UbcH1
(1-N65F) and 155 (155-N65F). Functional analysis of these mutants
revealed that 155-N65F interacted with E6-AP with an efficiency similar
to UbcH5 in both ubiquitin thioester assays (Fig. 4A) and
ubiquitination assays (Fig. 5), whereas 1-N65F was as inactive as
UbcH1. The results obtained for RSP5 were slightly different in that
155-N65F was able to transfer ubiquitin to RSP5 but with an
approximately 2-fold reduced efficiency compared with UbcH5 (Fig.
4A). Taken together, these results indicate that a
phenylalanine residue at position 62 is crucial for the ability of
UbcH5 to interact with E6-AP and RSP5.
S. cerevisiae UBC4 and UBC5 are involved in stress-related
functions (24, 29). Yeast strains carrying mutations in either one of
these genes are viable and lead to only moderate phenotypes, whereas
ubc4 ubc5 double mutants grow slowly and are inviable at
elevated temperatures (24). It has previously been reported that
members of the UBC4/UBC5 subfamily of E2s, including
Drosophila UbcD1, can rescue the deficiencies of yeast
ubc4 ubc5 mutants (30, 31). Furthermore, the genome of
S. cerevisiae does not encode a functional homolog of E6-AP
but, in addition to RSP5, encodes four additional hect domain proteins
(4). Therefore, the various chimeric E2s were tested for their ability
to complement the ubc4 ubc5 phenotype. In yeast, however,
the chimeric E2s 35, 335, and 155 and their respective point mutated
forms were expressed at vastly different
levels.2 Thus, it was not
possible to assess whether the ability of the various chimeric E2s to
interact with E6-AP and RSP5 in vitro correlates with their
ability to complement the phenotype of ubc4 ubc5 mutants
in vivo.
It is assumed that the specificity of
ubiquitin-dependent degradation is mediated at least in
part by the E2 and E3 enzymes of the ubiquitin conjugation system (1,
2, 4, 5). Furthermore, it appears that distinct E3s interact only with
certain E2s, suggesting that a specific interplay between E2s and E3s contributes to the specificity and selectivity of protein
ubiquitination. Members of the hect domain family of putative E3s have
been shown to specifically interact in vitro with members of
the UBC4/UBC5 subfamily of E2s including human UbcH5 (9-11, 22). The
mutational analysis of UbcH5 presented in this study shows that the
amino acid composition of a region encompassing the thioester-forming cysteine residue (amino acid residues 78-107) and a phenylalanine residue at position 62 are essential determinants for its ability to
functionally interact with hect domain proteins. The presence of a
phenylalanine residue at the respective position is evolutionally conserved among the members of the UBC4/UBC5 subfamily but is not
present in any of the other known E2s. This supports the notion that
the ability of UbcH5 and related proteins to interact with hect domain
proteins is important for their physiological function.
The three-dimensional structure of several class I E2s, including
S. cerevisiae UBC4 and A. thaliana UBC1, the
putative homolog of UbcH1 and S. cerevisiae
UBC2RAD6, has been determined (32, 33). This revealed that
the overall structure of the UBC domain is conserved and contains four
Amino acid residues 107/108 are located within the second Based on the results obtained with the chimera 155 and its mutant form
155-N65F, it appears that the N-terminal 61 amino acids of UbcH5 are
not critical for its ability to interact with hect domain proteins, as
indicated by the fact that this region can be replaced by the
corresponding region of a functionally unrelated E2. Although this does
not exclude the possibility that the N-terminal region of E2s is
directly involved in contacting hect domain proteins, it suggests that
this region is required for properties common to all E2s,
e.g. interaction with E1 and/or proper folding. The distinct
property of UbcH5 to functionally interact with hect domain proteins,
however, is determined by its C-terminal 87 amino acid residues.
Furthermore, the presence of a phenylalanine residue at position 62 of
UbcH5 seems to be of particular importance. Interestingly, all known
members of the UBC4/UBC5 subfamily, including UbcM3, contain a
phenylalanine residue at the corresponding position, and this seems to
be unique to members of this subfamily. The results presented strongly
indicate that the presence of a phenylalanine residue at this position
is necessary for the ability of an E2 to interact with E6-AP and RSP5.
However, it is not sufficient as demonstrated by the results obtained
with 1-N65F, a mutant form of UbcH1 in which an asparagine
residue at the respective position was replaced by phenylalanine.
An essential intermediate step in E6-AP-dependent
ubiquitination is the transfer of ubiquitin from UbcH5 to E6-AP
resulting in thioester complex formation of E6-AP with ubiquitin (19). Thus, it is not surprising that the region of UbcH5 surrounding the
putative ubiquitin-donating cysteine residue at position 85 is critical
for the ability of UbcH5 to functionally interact with E6-AP and RSP5
and presumably with hect domain proteins in general. SCF complexes also
have E3 activity (14, 15) but, unlike hect domain proteins, SCF
complexes do not appear to form thioester complexes with ubiquitin but
instead function as docking proteins by stably binding both substrate
proteins and their cognate E2, UBC3CDC34.
UBC3CDC34 is a class II enzyme containing a C-terminal
extension of approximately 70 amino acids in addition to the UBC domain
(34). Consistent with the notion that in SCF-dependent
ubiquitination the final transfer of ubiquitin to substrates is
probably directly catalyzed by UBC3CDC34, the interaction
of SCF complexes with UBC3CDC34 is not mediated by the UBC
domain. Rather, it was shown that the C-terminal extension of
UBC3CDC34 is necessary and sufficient to mediate its
interaction with SCF complexes (18). Thus, it appears that, according
to their mode of action, different E3 proteins recognize different
regions on their cognate E2s. Interestingly, it was recently reported
that UBC4 is involved in UBR1-dependent ubiquitination of
certain proteins (35). UBR1 was originally isolated as an E3 that, in
conjunction with UBC2RAD6, mediates the recognition of
substrates according to the N-end rule (8, 36, 37). Although UBR1 does
not appear to be related to hect domain proteins at the amino acid
sequence level, it has been reported that UBR1 can form thioester
complexes with ubiquitin (5). It will be interesting to see whether
this property of UBR1 requires the activity of UBC4/UBC5 and whether
the regions of UBC4/UBC5 that are required for interaction with hect
domain proteins are also involved in the interaction with UBR1.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
and affinity-purified on glutathione-Sepharose
(Amersham Pharmacia Biotech). UbcM3, UbcM3-V83S, and the various
chimeric E2s were cleaved with thrombin (Novagen) while bound to
glutathione-Sepharose to yield the respective free E2s. Upon cleavage,
the supernatants containing the free E2s were dialyzed against 50 mM Tris-HCl, pH 7.6, 30 mM NaCl, 1 mM dithiothreitol, and the dialyzed proteins stored at
4 °C. For generation of radiolabeled E6-E7 or radiolabeled
ubiquitin, the respective GST fusion protein was radioactively labeled
while bound to glutathione-Sepharose using protein kinase A (Sigma) in
the presence of [
-32P]ATP (9). The radiolabeled fusion
proteins were then eluted with 10 mM glutathione and used
directly in ubiquitination assays (E6-E7) or cleaved with thrombin to
yield free ubiquitin. Upon cleavage, thrombin was inactivated by
incubation at 75 °C for 15 min, precipitated proteins were spun out
by centrifugation, and the ubiquitin-containing supernatant was used in
thioester assays.
C6, and wheat germ E1 were prepared from Sf9
cells infected with the respective recombinant baculoviruses (RSP5 and
wheat germ E1 encoding recombinant baculoviruses were kindly provided
by J. M. Huibregtse and W. Krek, respectively). Partial purification
of E6-AP, RSP5, and RSP5
C6 was performed as described previously (9,
10, 27), and partial purification of E1 was performed as described for
bacterially expressed E1 (9).
C6 with ubiquitin
was determined essentially as described previously (19). Reaction
mixtures contained approximately 20-50 ng of E1, 300 ng of
radiolabeled ubiquitin, the respective E2s, and 300 ng of E6-AP or
100-300 ng of RSP5 as indicated. With respect to their enzymatic
activity, similar amounts of the different E2s were used as assessed by
their ability to form thioester complexes with ubiquitin in the
presence of E1.
RESULTS
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Fig. 1.
Generation of chimeric E2s between UbcH5 and
UbcM3 or UbcH1. A, the amino acid sequences of UbcH5
(21), S. cerevisiae (sc) UBC4 (24), UbcM3 (26),
and UbcH1 (28) were aligned with respect to UbcH5. Amino acids are
shown in the single-letter codes. Differences in amino acid sequence
are indicated, and identities are denoted by dashed lines.
The active site cysteine residue (position 85) is marked with an
asterisk. The phenylalanine residue at position 62 and the
serine residue at position 83 of UbcH5, which are critical for
interaction with E6-AP and RSP5, as well as the respective residues of
scUBC4, UbcM3, and UbcH1 are boxed. Arrows
indicate the sites that were used to generate chimeric E2s between
UbcH5 and UbcM3 or UbcH1, respectively. The sequence of the N-terminal
extension of UbcM3 (46 amino acids in size) is only partially shown,
with 40 amino acid residues abbreviated as X40.
B, schematic representation of the chimeric E2s generated.
For ease of presentation, the threonine residue at position 47 of
UbcM3, which corresponds to the initial methionine of UbcH5, was
defined as position 1 in those chimeric E2s that contain parts of UbcM3
at the N terminus. It should be noted, however, that these chimeric E2s
also contain the extra N-terminal 46 amino acids of UbcM3 as indicated
in A. The nomenclature reflects the relative contribution of
the UBC domain of UbcH5, UbcM3, or UbcH1 to the UBC domain of the
respective chimeric E2. The various chimeric E2s were generated in
bacteria and tested for their interaction with E6-AP or RSP5, as
assessed by their ability to load E6-AP or RSP5 with ubiquitin in the
presence of E1 (see "Experimental Procedures"). +, transfers
ubiquitin to E6-AP and RSP5 with an efficiency similar to UbcH5; +/ ,
significantly less efficient (more than 3-fold reduced) in transferring
ubiquitin to E6-AP and RSP5 than UbcH5;
, thioester complex formation
of E6-AP and RSP5 with ubiquitin was not observed under the conditions
used.
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Fig. 2.
Ubiquitin thioester complex formation of
E6-AP in the presence of the chimeric E2s 355 and 533. Thioester
reactions contained 32P-labeled ubiquitin, partially
purified recombinant E1, crude extracts from bacteria expressing
various E2s as indicated, and partially purified E6-AP (for details,
see "Experimental Procedures"). Similar amounts of the respective
E2s were used as determined by their ability to form thioester
complexes with ubiquitin in the presence of E1 (not shown). After 5 min
at 25 °C, reactions were stopped in the absence or presence (not
shown) of a reducing agent and subjected to SDS-polyacrylamide gel
electrophoresis followed by autoradiography as described previously
(19). The reactions stopped in the presence of a reducing agent are not
shown, but it should be noted that under these conditions, the
ubiquitin adducts of E6-AP could not be detected, indicating that the
observed ubiquitin adducts indeed represent thioester complexes.
Positions of free 32P-labeled ubiquitin (ub) and
of the respective thioester complexes with E1, the different E2s, and
E6-AP are indicated. Due to the percentage of the SDS-polyacrylamide
gel used, the ubiquitin adducts of UbcH5 and 533 were not separated
from free ubiquitin.
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Fig. 3.
Ability of chimeric E2s between UbcM3 and
UbcH5 to transfer ubiquitin to E6-AP and RSP5. The chimeric E2s
indicated were expressed as GST fusion proteins in E. coli.
After purification by affinity chromatography, the GST part of the
fusion proteins was removed by cleavage with thrombin (see
"Experimental Procedures"). As a source of E6-AP and RSP5, E6-AP
and the C-terminal truncation mutant of RSP5, RSP5 C6, were expressed
in the baculovirus system and partially purified by anion exchange
chromatography as described (9, 10). Finally, the various E2s were
tested for their ability to transfer 32P-labeled ubiquitin
to E6-AP (A) or RSP5
C6 (B) as described in the
legend to Fig. 2. Reactions stopped in the absence of a reducing agent
only are shown. Positions of free ubiquitin (ub) the
thioester adducts of 32P-labeled ubiquitin with E1, the
various E2s, E6-AP, and RSP5
C6 are indicated.
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Fig. 4.
Ability of chimeric E2s between UbcH1 and
UbcH5 to transfer ubiquitin to E6-AP and RSP5. The various E2s
were tested as indicated for their ability to load E6-AP (A)
and RSP5 (B) with 32P-labeled ubiquitin in a
standard ubiquitin thioester assay (see "Experimental Procedures").
The reactions stopped in the absence of a reducing agent only are
shown. Similar amounts of the respective E2s were used as determined by
their ability to form thioester complexes with ubiquitin in the
presence of E1 (not shown). Positions of the ubiquitin thioester
adducts of E1, E6-AP, RSP5, and the different E2s are indicated. As a
source for E6-AP and RSP5, E6-AP and the full-length form of RSP5 were
expressed in the baculovirus system and partially purified by anion
exchange chromatography. Under the conditions used, RSP5 copurifies
with E1 present in the insect cell extracts, explaining why levels of
E1 used in the RSP5 thioester assay were apparently higher than those
used in the E6-AP thioester assay. Furthermore, to clearly separate
RSP5 thioester adducts from E1 thioester adducts, the reactions were
run on an SDS-polyacrylamide gel with a lower percentage than the
respective reactions with E6-AP. Therefore, the E2 thioester adducts of
ubiquitin with the different E2s were not separated from free ubiquitin
in B. Finally, it should be noted that the C-terminally
truncated form of RSP5, RSP5 C6, used in the experiment shown in Fig.
3, interacts significantly less efficiently with 155-N65F (2-3-fold)
than the full-length form of RSP5. The reason for this difference is
presently unclear.
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Fig. 5.
Effectiveness of chimeric E2s in
E6-AP-dependent ubiquitination. Ubiquitination
reactions contained recombinant E1, ubiquitin, baculovirus-expressed
E6-AP, and a 32P-labeled GST-E6-E7 fusion protein as
substrate (9). Reactions were started by the addition of the various
E2s as indicated. Relative amounts of the respective E2s are given
(1×, 2×, and 4×) as determined by their ability to form thioester
complexes with 32P-labeled ubiquitin in the presence of E1
(not shown). After 2 h at 25 °C, reactions were stopped, and
the complete reaction mixtures were subjected to SDS-polyacrylamide gel
electrophoresis followed by autoradiography. The position of GST-E6-E7
is indicated. The multi-ubiquitinated forms of GST-E6-E7 are marked
with asterisks.
DISCUSSION
-helices and a four-stranded antiparallel
-sheet. The putative
catalytic site cysteine residue is located in a long extended stretch
between the fourth
-strand and the second
-helix. As breakpoints
to generate chimeric E2s between UbcH5 and UbcM3 or UbcH1, the amino acid residues at position 62/63, at position 77/78, and at position 107/108 (numbering according to the UbcH5 sequence; Fig. 1) were used.
Amino acid residues 62/63 are located in a loop connecting the third
and the fourth strand of the
-sheet, whereas amino acid residues
77/78 are located in the stretch between the fourth
-strand and the
second
-helix. Such chimeric E2s were expressed in bacteria to
appreciable levels and were active in thioester complex formation with
ubiquitin. This demonstrates that the fusion between different E2s at
these positions does not grossly interfere with proper folding of the
resulting chimeric E2s.
-helix and
thus fusion of the respective parts of different E2s at this site may
disturb the overall structure of some of the resulting chimeric E2s.
This was clearly not the case for the chimeric E2 353, because it was
nearly as active as UbcH5 in transferring ubiquitin to both E6-AP and
RSP5, respectively. This shows that the difference between UbcH5 and
UbcM3, with respect to their ability to interact with hect domain
proteins, is mainly determined by a region surrounding the catalytic
site cysteine residue at position 85 of UbcH5. In particular, the
presence of a serine residue at position 83 seems to be critical
because a mutant form of the chimeric E2 35 bearing a valine at this
position, the corresponding amino acid residue of UbcM3, interacts with
E6-AP and RSP5 as inefficiently as UbcM3. Conversely, substitution of
this valine residue in UbcM3 with serine significantly enhances its
ability to interact with E6-AP and RSP5. It is notable that the
presence of a bulky hydrophobic residue at position 83 appears to be a peculiarity of UbcM3 and E2s that are highly similar to UbcM3 (26),
apparently not being found in other members of the UBC4/UBC5 subfamily
as well as in unrelated E2s. Because the cellular functions of UbcM3
and its related E2s are unknown, the significance of this difference is
presently unclear. The data obtained with the chimeric E2 353, however,
do not exclude the possibility that the C-terminal 40 amino acids of
UbcH5/UbcM3 are critically involved in the interaction with E6-AP and
RSP5. To address this possibility a chimeric E2 analogous to 353 was
generated by replacing the C-terminal UbcM3 part by the corresponding
region of UbcH1. However, this chimeric E2 termed 351 was not expressed
in bacteria to significant levels. A reasonable explanation for this is
that fusion of the C terminus of UbcH1 to the respective part of UbcH5
results in a structural distortion of the resulting chimeric protein.
In any case, because of the lack of detectable expression, it cannot be
excluded that the C-terminal 40 amino acids of UbcH5/UbcM3 are
critically involved in the interaction with E6-AP and RSP5.
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ACKNOWLEDGEMENTS |
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We thank D. Heiss for technical assistance and N. J. Whitaker and S. E. Schwarz for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft.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.
Present address: Burnham Inst., Program on Apoptosis and Cell
Death Research, 10901 N. Torrey Pines Rd., La Jolla, CA 90237.
§ To whom correspondence should be addressed: Deutsches Krebsforschungszentrum, Angewandte Tumorvirologie F0701, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany. Tel.: 49-6221-424-834; Fax: 49-6221-424-822; E-mail: m.scheffner{at}dkfz-heidelberg.de.
2 U. Nuber, K. Matuschewski, S. Jentsch, and M. Scheffner, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; SCF, SKP1, CDC53, F-box protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; nt, nucleotide(s).
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REFERENCES |
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