From the Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205
Received for publication, December 17, 2002, and in revised form, January 5, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most substrates of the 26 S proteasome are
recognized only following conjugation to a
Lys48-linked polyubiquitin chain. Rad23 is one member
of a family of proteins that possesses an N-terminal ubiquitin-like
domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA).
Recent studies have shown that UbLs interact with 26 S proteasomes,
whereas UBAs bind polyubiquitin chains. These biochemical properties
suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this
model, the effect of human Rad23A on the degradation of
polyubiquitinated substrates catalyzed by purified proteasomes is
exclusively inhibitory. Strong inhibition is dependent on the presence
of both UBAs, independent of the UbL, and can be explained by
competition between the UBA domains and the proteasome for binding to
substrate-linked polyubiquitin chains. The UBA domains bind
Lys48-linked polyubiquitin chains in strong preference to
Lys63 or Lys29-linked chains, leading to
selective inhibition of the assembly and disassembly of
Lys48-linked chains. These results place constraints
on the mechanism(s) by which UbL-UBA proteins promote
proteasome-catalyzed proteolysis and reveal new properties of UBA domains.
The conserved protein
Ub1 becomes covalently linked
through its C terminus (Gly76) to lysine residues of
substrates destined for degradation by the 26 S proteasome, a 2.5 MDa
assembly consisting of a central cylindrical 20 S core complex and two
distally positioned 19 S regulatory complexes (1). The 19 S complex
recognizes the substrate-linked Ub signal, unfolds the substrate
polypeptide, and translocates it into a sequestered active site chamber
within the 20 S catalytic complex (2). The Ub-proteasome pathway plays
a major role in intracellular regulation through its contribution to
the homeostasis of important regulatory proteins (1). Ub also functions
as a functionally distinct signal in other intracellular processes, including protein trafficking and DNA damage tolerance (reviewed in
Refs. 3 and 4).
In the most frequent mode of targeting to proteasomes, multiple Ubs are
chained together through Lys48-Gly76
isopeptide bonds, with the proximal Ub linked to a substrate lysine
residue (5, 6). The architecture of this Ub signal appears to be
specialized for targeting to proteasomes in vivo, whereas
other signal structures may be dedicated to distinct processes (reviewed in Refs. 3 and 4). Although a Lys48-linked chain
of at least four Ubs in length is an autonomous signal that affords
high affinity for purified proteasomes in vitro
(Kd of ~50 nM, Ref. 7), recent studies
have suggested that factors extrinsic to the 19 S complex might assist in targeting some polyubiquitinated substrates to proteasomes. Leading
candidates for such a role are members of the UbL-UBA protein family,
including Rad23/Rhp23 and Dsk2/Dph1.
Individual members of the UbL-UBA family were discovered by virtue of
their roles in distinct biological processes, including nucleotide
excision repair (Rad23, Ref. 8) and spindle pole body duplication
(Dsk2, Ref. 9), whose relationship to protein degradation remains
uncertain (see for example Refs. 10-13). All family members have an
N-terminal Ub-like (UbL) domain that binds to a specific site in the 19 S complex (14, 15), along with one or more Ub-associated (UBA) domains
(16) that bind polyUb chains (and mono-Ub) (10, 17-20). These
biochemical properties suggest that a substrate bound to a UBA domain
through its polyUb chain could be shuttled to the proteasome by means
of the UbL-19 S interaction (15, 17, 19, 21, 22).
Existing biological data both support and contradict this model. For
example, the turnover of certain model proteasome substrates is
retarded in rad23 The impact of UbL-UBA proteins on proteasome function has not
previously been examined in a defined biochemical system. Here we
report results from two different biochemical systems that are
inconsistent with a simple trans-targeting function for Rad23 and by
analogy, other UbL-UBA proteins. We also describe new properties of UBA
domains that have a high likelihood to be relevant for the biological
function(s) of these domains.
Proteins and Antibodies--
Bovine lactalbumin and bovine Ub
were from Sigma; Ubal was from BostonBiochem; MG-132 was from Peptides
International. Poly(His) antibody (H-15) was from Santa Cruz
Biotechnology. Polyclonal Rad23 antiserum was from P. Howley (Harvard
Medical School, Rad23A) or Affiniti (Rad23B). Rabbit polyclonal
antibodies against Ub were produced in rabbits by this group and
affinity-purified (29). P. Howley provided plasmids pGEX4T2-HHR23A and
pGEX-6p-1-HHR23B. Relative to the published sequence of human Rad23A
(30), the sequence of Rad23 used in this work carries T131A and E150K
mutations that do not lie in the UbL or UBA domains. Deleted open
reading frames (ORFs) were constructed by PCR and cloned between the
BamHI and SalI sites of pGEX4T2 (Amersham
Biosciences). All ORFs were verified by DNA sequence analysis. GST
fusion proteins were expressed in BL21pJY2 cells (31), absorbed onto
GSH beads (Sigma), and released by thrombin cleavage or eluted with GSH
and cleaved subsequently. In all experiments except Fig. 3,
below, Rad23 proteins were purified further on an FPLC Mono
Q column (Amersham Biosciences) using a salt gradient appropriate to
each protein. Bovine erythrocyte 26 S proteasomes and rabbit
reticulocyte fraction II were produced as described (7, 32). E2-25K,
His10-Mms2, UbcH5A, and the KIAA10 C-domain (CD) were
expressed in bacteria and purified as described (33-35). The yeast
Ubc13 ORF was cloned between the BamHI and SalI
sites of pGEX4T2 (Amersham Biosciences). Ubc13 was expressed in
BL21pJY2 cells, absorbed onto GSH beads, and released using thrombin.
Thrombin was inactivated with 1 mM phenylmethylsulfonyl fluoride. Ubc13 was dialyzed, concentrated, and used without further purification. The following mutant Ubs were expressed and purified by
established methods (35): K29C/K48R; K48R/D77R; K48C; D77; and
K63R. His6-tagged mouse E1 was expressed in Sf9
cells, using a virus provided by K. Iwai, and purified on nickel beads (Novagen).
Degradation Assays--
All results were replicated in two or
more independent experiments. Lactalbumin degradation assays contained
2.7 mg/ml fraction II protein, 23 µM Ub (where added),
~5 µg/ml 125I-lactalbumin (~106
cpm/µg), and buffer/cofactors as described (36). Ubiquitination and
degradation were assayed by SDS-PAGE/autoradiography and release of
acid-soluble radioactivity, respectively (36). Linear degradation rates
were corrected by subtracting the rate measured in a negative control
reaction lacking added Ub. Typically, Ub stimulated the degradation
rate by ~3-fold. A new version of H10-UbDHFR was used in
the present work. Relative to Ref. 7: 1) Val76 of Ub was
linked to DHFR with an Ala4 linker (junction sequence ... LRLGVAAAAMVRP ... in which L = Leu72 of
Ub and M = Met1 of mouse DHFR); 2) a protein kinase A
site was placed at the DHFR C terminus (final sequence ...
FEVYERRASVQ in which the serine residue is the
phosphorylation site). H10-UbDHFR was expressed, purified,
and conjugated to K48-Ub4 as described (7).
Degradation of Ub5DHFR by purified 26 S proteasomes was
assayed under previously described conditions (7) by either of two
methods: 1) Western blotting against the poly(His) tag of the
DHFR-fused Ub (37) (this Ub is degraded, Ref. 7); or 2) release of
32P incorporated at the kinase A site (~3 × 105 cpm/pmol). Ub5DHFR (10 µg) was labeled in
a 60-µl reaction containing: 20 mM Tris-HCl (pH 7.6), 0.1 M NaCl, 12 mM MgCl2, 20 units of protein kinase A (Sigma P-2645), 40 µCi of
[ Synthesis of K29-, K48-, and K63-Ub4--
K48- and
K63-Ub4 were produced as described (33, 38).
Cys48 at the distal terminus of K48-Ub4 was
alkylated with iodoacetamide, while K63-Ub4 carried a K63R
mutation at its distal terminus. K29-Ub4 was produced by
the general method used for K48-Ub4 except that the
KIAA10-CD/UbcH5A (E3/E2) conjugating system was used in place of
E2-25K, and all Ubs carried the K48R mutation to force the
dual-specificity E3 to use K29 in chain assembly (34). Incubations (pH
8) contained KIAA10-CD, UbcH5A, and E1 at 2, 1, and 0.1 µM, respectively. To reduce Ub consumption via
autoubiquitination of KIAA10-CD, we preincubated the conjugating
enzymes for 20 min with 29 µM lysine-less Ub (31) before
initiating chain assembly by adding K48R/D77-Ub and K29C/K48R-Ub (1.17 mM each). After 4 h of incubation (37 °C), enzymes
were removed by passage through Q-Sepharose (Amersham Biosciences).
K29-Ub2 was purified on an FPLC Mono S column (Amersham
Biosciences) as described (38) and divided into two portions. One
aliquot was deblocked at its proximal terminus using yeast Ub
hydrolase-1 (38) and then irreversibly blocked at its distal terminus
by alkylating Cys29 with iodoacetamide. The other aliquot
was deblocked at its distal terminus by alkylating Cys29
with ethyleneimine (Chemservice) (38). These two dimers were conjugated
to each other and purified as described for K29-Ub2.
Polyubiquitin Chain Binding by GST-Rad23 Pull-down--
All
Ub4 molecules used in chain binding had an extra amino acid
(Asp77) at the proximal chain terminus and carried a
blocking modification/mutation at the distal terminus (see preceding
paragraph). Each type of Ub4 was radioiodinated using
chloramine T (8-20,0000 cpm/pmol). GST-fused Rad23/mutants were bound
to GSH beads (10 pmol of protein/µl of beads). After washing with
binding buffer (20 mM Tris-HCl, pH 7.6, 50 mM
NaCl, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), loaded beads (5 µl)
were incubated with 1 µM 125I-Ub4
in a total volume of 115 µl, in binding buffer plus 1 mg/ml bovine
serum albumin, either for 2 h at room temperature or overnight at
5 °C. Unbound Ub4 was removed via three washes with
binding buffer, the beads were boiled in SDS-PAGE sample buffer, and
the released Ub4 was quantified by SDS-PAGE/phosphorimage analysis.
Chain Assembly Assays--
Incubations (37 °C) contained 117 µM Ub and 0.1 µM E1, under established
conditions (33, 38), for times as indicated in the legend to Fig. 6
("Results"). K48R-Ub was used to make Lys29- and
Lys63-linked chains, while K29R-Ub was used to make
Lys48-linked chains. Lys63-linked chains were
generated at pH 7.6 using His10-Mms2 and Ubc13 (3 µM each). Lys48 and Lys29 chains
were generated at pH 8 using KIAA10-CD (1 µM) and UbcH5A (0.5 µM). Chain products were detected by Ub Western blot.
Surface Plasmon Resonance--
Data were acquired on a Biacore
3000 instrument. R23D (5000 resonance units) was immobilized by amine
coupling (pH 4.5) to a CM5 sensor chip (Biacore) according to the
manufacturer's instructions. One flow cell of the chip was activated
and blocked in the absence of R23D to provide a control. Signals
generated in the control flow cell were subtracted from signals in the
R23D flow cell.
Rad23 Inhibits Ub-dependent Proteolysis in a
Reticulocyte Fraction II in a UBA Domain-dependent
Manner--
To assess the effects of Rad23 on Ub-dependent
proteolysis we first used Ub-depleted reticulocyte extract (fraction
II) in which the model substrate bovine lactalbumin is recognized by the N-end rule E3/E2 complex (E3
Mammals express two closely related isoforms of Rad23, known as Rad23A
and B (30). Rad23B complexes with the nucleotide excision repair factor
XPC/Rad4 (30), a function than can probably also be performed by Rad23A
(41). We employed human Rad23A in the studies described below, which
began with purification of the full-length protein and several
truncation mutants (Fig. 1, A
and B).
Adding a low concentration of full-length Rad23 (7 µM) to
fraction II significantly inhibited lactalbumin degradation (~50%, Fig. 2A) and higher
concentrations of Rad23 caused stronger inhibition (see Fig.
3, below). Rad23 failed to
stimulate degradation at any concentration tested. The lowest
concentration used, 0.7 µM (data not shown), represents
an excess of only ~3-fold over the estimated concentration of
proteasomes in this assay system (36). Thus, it is unlikely that we
failed to observe stimulation because of a gross excess of Rad23. An
earlier study in unfractionated rabbit reticulocyte lysate attributed
proteolytic inhibition by Rad23 to a competition between the UbL domain
and substrate-linked polyUb chains for access to proteasomes (24).
However, we found that deleting the UbL generated a stronger inhibitor,
indicating that the UbL is dispensable for inhibition (R23D, Fig.
2A; see also next paragraph). Degradation in the presence of
a saturating concentration of R23D was actually slower than in the
negative control without added Ub (Fig. 2A), probably
because some of the degradation in the negative control relies on a
trace of Ub in fraction II and is thus subject to inhibition by R23D.
Deletion analysis revealed that strong inhibition (
Titration analysis showed that R23D was a potent inhibitor, displaying
half-maximal inhibition at ~3 µM (Fig. 2B).
We tentatively attribute less potent inhibition by full-length Rad23,
versus UbL-deleted dual-UBA variants (Fig. 2A),
to an interaction that shields the UBA domains of the full-length
protein. (As shown below, inhibition of lactalbumin degradation is
probably mainly due to binding of lactalbumin-linked polyUb chains to
the Rad23 UBA domains.) Consistent with this model, we found that heat
treatment increased the inhibitory potency of full-length Rad23 but had little effect on inhibition by R23D (Fig. 3, data for 7 µM inhibitor). Rad23 was reported to homodimerize through
a UBA-UBA interaction (42), but recent NMR results exclude
self-interaction of minimal UBA domains (43). NMR results also exclude
self-association of minimal UbL domains (44). Stronger proteolytic
inhibition by R23D versus Rad23 (Figs. 2A and 3)
is therefore most simply explained by an intra- or intermoleular
interaction between the UbL and UBA domains that is disrupted upon
heating so as to increase accessibility of polyUb chains to the UBAs.
The proposed conformational transition could also increase
accessibility of the UbL, as suggested by the results of
Rad23-proteasome interaction studies (15). Further work will be
necessary to validate this conformational model.
Rad23 UBA Domains Inhibit the Degradation of a Polyubiquitinated
Substrate by Proteasomes--
In the fraction II system, the extract
provides both Ub-conjugating and proteasome activities. Rad23 was
previously found to inhibit E2-catalyzed histone polyubiquitination
(27), suggesting that UbL-UBA proteins could inhibit proteolysis by
inhibiting Ub-substrate conjugation. To rigorously determine if Rad23
inhibits 26 S proteasome activity, we studied the degradation of a
polyubiquitinated model substrate by purified proteasomes. The
high affinity of Ub5DHFR for proteasomes
(Km, ~35 nM) is due solely to the
binding of its polyUb chain to the 19 S complex (7), where the chain
contacts the S6'/Rpt5 ATPase (26). The structure of Ub5DHFR
resembles that of conjugated forms of UbPro
Low levels of Rad23 are found associated with affinity-purified
proteasomes from budding yeast (46), consistent with the demonstrated
interaction of the Rad23 UbL with the 19 S complex (14, 15).
Stimulation of degradation by Rad23 could have been missed in the above
described experiment (Fig. 4A) if our purified proteasomes
were already saturated with Rad23. However, quantitative Western
blotting showed that Rad23A, although detectable in the purified
proteasomes, was substantially substoichiometric relative to an
integral 19 S subunit, S6'/Rpt5 (Fig.
5B, estimated content ~0.3
mol of Rad23A/mol19S), while Rad23B was not detected (data not shown).
Therefore failure to observe stimulation by Rad23 is not due to prior
saturation of proteasomes with Rad23.
Inhibition by Rad23 could be explained if its UbL binds to the chain
receptor of the 19 S complex and prevents access of the DHFR-linked
polyUb chain (24). Alternatively, the UBA(s) could prevent substrate
access by sequestering the DHFR-linked chain. Failure of the isolated
UbL domain to inhibit lactalbumin degradation in fraction II and potent
inhibition by the UbL-deleted variant R23D in that system (Fig.
2A) argue in favor of the second model. Also in accord with
this model, the isolated UbL did not inhibit Ub5DHFR
degradation by purified proteasomes (Fig. 4A, lanes
9 and 10 versus 4). Moreover,
excess UbL did not inhibit the cross-linking of a reactive polyUb chain
to S6'/Rpt5,2 whereas
Ub5DHFR does block cross-linking (26). These results show
that the UbL domain binds to a distinct site from that which binds
polyUb chains as a prelude to proteolysis. They are consistent with
findings that different subunits of the 19 S complex contact Lys48-linked chains (S6'/Rpt5) and the Rad23 UbL (S2/Rpn1)
(15, 26).
We confirmed that the UBA domains are responsible for inhibition by
showing that R23D (lacking the UbL) is a more potent proteasome inhibitor than full-length Rad23 (Fig. 4A, lanes
7 and 8 versus 5 and
6). The same relationship holds for lactalbumin degradation in fraction II (Fig. 2A). Inhibition by R23D was more potent
(smaller K0.5) at a lower Ub5DHFR
concentration, indicative of a competitive effect (Fig. 4B,
squares versus diamonds). The qualitative agreement of
structure/function results in the two experimental systems (Figs.
2A and 4A) suggests that proteasome inhibition is
the main underlying cause of the proteolytic inhibition in fraction II. However, it remains possible that other effects of Rad23 (see below)
make a contribution to inhibition in fraction II. The more potent
inhibition seen with purified proteasomes (Figs. 2B
versus 4B) is likely explained by the high
concentration of proteasomes and competing poly(Ub) chains in fraction
II (36). Overall, our results show that R23D inhibits proteasomes
through UBA-mediated sequestration of substrate-linked polyUb chains,
an explanation consistent with the demonstrated ability of UBA domains
to bind Lys48-linked chains (17, 19, 20).
Rad23 UBA Domains Modulate Chain Assembly and
Disassembly--
Besides inhibiting proteolysis, overexpression of
Rad23 (with or without its UbL) raises the intracellular level of
ubiquitinated proteins (20, 21, 27, 28, 47). Similarly, 7 µM R23D, a concentration that completely inhibited
lactalbumin degradation (Fig. 2B), strongly increased the
level of ubiquitinated lactalbumin in fraction II (Fig.
6A, lane 8 versus
2). The major known in vivo consequences of Rad23
overexpression are therefore recapitulated in fraction II (Figs.
2A and 6A). The presence of R23D also altered the
size distribution of conjugates: levels of adducts apparently carrying
three to five ubiquitins increased strongly (Fig. 6A, lane 8 versus 2). The level of substrate
conjugates reflects the balance between substrate polyubiquitination
catalyzed by the relevant E3/E2 complex and conjugate degradation and
chain disassembly catalyzed by proteasomes and DUBs, respectively.
Although inhibiting proteasomes should raise conjugate levels, the
effect of R23D greatly exceeded the effect of a saturating
concentration of a proteasome catalytic site inhibitor, MG-132 (Fig.
6A, lane 8 versus 6). The striking effect seen in
Fig. 6A (lane 8) suggested that R23D might affect
additional steps in the Ub-proteasome pathway.
To test if R23D also inhibited DUB activity, we monitored the
disassembly of an unanchored Lys48-linked Ub4
chain (K48-Ub4) in fraction II. This reaction is primarily catalyzed by isopeptidase T/Ubp14 (48). At the concentration employed
(4 µM), R23D inhibited Ub4 disassembly
significantly (Fig. 6B, lane 8 versus 6,
Ub4 band), but less strongly than the specific DUB inhibitor Ubal (2.3 µM; lane 7).
Because R23D and Ubal show the opposite relative potency in stabilizing
ubiquitinated lactalbumin (lanes 4 and 8 in Fig.
6A versus lanes 7 and 8 in Fig. 6B),
we suspect that the DUBs that act on ubiquitinated lactalbumin are more
sensitive to R23D than is isopeptidase T. Indeed, Ubal did not further
increase the level of ubiquitinated lactalbumin in the presence of R23D
(Fig. 6A, lane 10 versus 8), suggesting that the
relevant DUBs are already fully inhibited by R23D alone. Note that
isopeptidase T acts exclusively on unanchored polyUb chains (48, 49)
and so should be irrelevant in the stabilization of ubiquitinated
lactalbumin. Our results thus suggest that R23D may inhibit
deubiquitination generally. This could be explained if R23D binds to
polyUb chains and hinders their accessibililty to DUBs. A similar model
was proposed above to explain proteasome inhibition by Rad23 UBA
domains (above).
Even when combined, MG-132 and Ubal did not cause the accumulation of
specific lactalbumin conjugates (data not shown). We therefore
suspected that R23D might also affect polyUb chain extension. To test
this possibility we used an E3/E2 complex (34) that assembles
unanchored Lys48-linked chains. Addition of R23D to chain
assembly assays strongly inhibited the production of chains more than
three Ubs in length (Fig. 6C, lanes 1-4 versus
5-8). R23D also abolished the formation of extremely high
molecular weight products that result from autopolyubiquitination of
the E3 (region marked E3-Ubn in Fig.
6C, lanes 5-8).
We attribute the inhibition of free chain assembly to selective binding
of K48-Ub3 and longer chains because when the same E3 was
constrained to assemble Lys29-linked chains (through the
use of K48R-Ub), the assembly of unanchored chains was unaffected or
even stimulated, as seen most clearly for Ub3-6 in Fig.
6C (lanes 9-12 versus 13-16; note that Lys29-linked chains are synthesized more slowly than
Lys48-linked chains). However, auto-polyubiquitination was
still abolished when this reaction involved Lys29-linked
chains (lanes 13-16). Three conclusions follow from these chain assembly results. First, it is unlikely that R23D inhibits intrinsic E3 activity, because unanchored Lys48- and
Lys29-linked chains are assembled at the same active site
(34). Second, R23D apparently does not bind to unanchored
Lys29-linked chains. This conclusion was verified by two
independent methods as discussed below. Third, unlike inhibition of
free chain synthesis, inhibition of autopolyubiquitination is
independent of chain linkage and is therefore unlikely to be caused by
R23D binding to chains.
Selective binding of R23D to K48-Ub3 and longer
chains, along with inhibition of deubiquitination, probably contributes
to the stabilization of specific lactalbumin conjugates seen in
fraction II (Fig. 6A). Although our results are reminiscent
of a finding by Madura and co-workers (27) that Rad23 inhibited
E2-catalyzed histone polyubiquitination (27), the chains observed in
that study were not Lys48-linked. Thus, our results provide
the first direct evidence that UBA domains can modulate both positive
and negative reactions of proteolytic signal homeostasis.
Linkage-specific Binding of polyUb Chains to Rad23 UBA
Domains--
Rad23 mutants with two UBA domains inhibited proteolysis
more strongly than single UBA proteins (Fig. 2A), suggesting
that the presence of a second UBA domain might confer stronger chain binding. Indeed, such an effect was observed in GST pull-down assays of
125I-K48-Ub4 binding (Fig.
7A). A similar differential
was seen in other studies, in which GST-Rad23 and GST-R23D bound
1.5-2-fold more Ub4 than GST-UbL-UBA1 and GST-UBA2 (data
not shown). We did not detect binding of Ub1 (data not
shown, but see Fig. 7C, below), consistent with
the much higher estimated relative affinity of UBA domains for poly(Ub)
chains (Kd~20 nM for
K48-Ub4, Ref. 17 versus ~10 µM
for Ub1, Ref. 10). These results confirm the established
ability of a single UBA domain to bind a Lys48-linked chain
(17) and suggest for the first time that two UBA domains may afford
more robust binding. It remains to be determined if this effect can
fully explain the stronger proteolytic inhibition caused by versions of
Rad23 with two UBA domains (Fig. 2A).
Although Lys48-linked chains are the principal proteasomal
targeting signal (5, 6), Lys29-linked chains have been
implicated in the proteasomal degradation of UFD substrates (45).
Lys63-linked-chains, in contrast, appear to be
non-proteolytic signals (3, 50-53). However, the molecular
determinants of linkage specificity in polyUb chain signaling remain
obscure. To determine if UBA domains have potential to regulate
signaling by noncanonical chains, we assembled K29- and
K63-Ub4 and tested these chains for binding to immobilized
GST-UBA1-2 protein. Remarkably, the canonical chain was strongly
favored in its binding (Fig. 7B). Similar results were
obtained in assays with GST-fused versions of R23D, UBA2, and UbL-UBA1
(data not shown), indicating that this linkage specificity is intrinsic
to each UBA domain of Rad23.
Additional lines of evidence confirm this linkage specificity of
binding. First, R23D-dependent inhibition of
Ub4 disassembly was limited to the Lys48-linked
tetramer (Fig. 6B, lanes 4 and 12 versus 8). Since all three chains can be
disassembled by isopeptidase T at the same active
site,3 linkage-specific
inhibition is most simply explained by selective binding of R23D to the
K48-Ub4 substrate. Second, R23D strongly inhibited the
assembly of Lys48-linked chains of n > 3, but
had very little effect on the assembly of chains linked through
Lys29 or Lys63 (Fig. 6C, lanes
13-16 and 21-24 versus respective
controls). Finally, direct assays of binding by surface plasmon
resonance confirmed that K48-Ub4 was strongly preferred by
R23D over K63/K29-Ub4, although significant
K63-Ub4 binding was detectable by this more sensitive
method (Fig. 7C).
Effects of Rad23 on Proteasome-catalyzed Proteolysis--
UbL-UBA
proteins can play a positive role in proteasome-catalyzed degradation
(19, 22, 23), but their mechanism of action remains poorly defined. An
attractive model postulates that UbL-UBA proteins shuttle
polyubiquitinated substrates to proteasomes by using their UbL and UBA
domains to interact with the 19 S complex and poly(Ub) chains,
respectively. The current work represents the first biochemical test of
this hypothesis.
A principal finding of this work is that the influence of purified
Rad23 on proteasome activity in vitro is exclusively
inhibitory (Figs. 2 and 4), reflecting a competition between the Rad23
UBA domains and the proteasome's polyUb receptor for substrate-linked chains. Our results show that the binding of poly(Ub) chains to UBA
domains can also inhibit other chain recognition events, leading to
defects in the assembly and disassembly of substrate-linked chains
(Figs. 4 and 6). Since substrate ubiquitination and deubiquitination (54, 55) are both required for degradation, these effects may also
contribute to the proteolytic inhibition seen when Rad23 is
overexpressed in yeast cells. Indeed, a recent study attributed the
Rad23-mediated stabilization of an unstable variant of Rad4/XPC to
blockade of ubiquitination (56). However, overexpression effects may
also depend on yet to be determined UbL-UBA properties, as suggested by
the results of a recent suppression analysis. Funakoshi et
al. (19) found that some mutations which suppressed the growth
inhibitory effects of Dsk2 overexpression mapped to the Pre2 catalytic
subunit of the 20 S complex and the Rpn1 subunit of the 19 S complex.
If the toxicity of Dsk2 overexpression was due solely to proteasome
inhibition, then suppressing mutations might be expected to increase
proteasome activity. Instead, each of the suppressing mutations
crippled Leu
Overall, our findings suggest that the ability of Rad23 to promote
degradation in vivo depends on interactions, factors, or modifications beyond those which can be recapitulated by the in vitro systems used here, despite their apparently faithful
behavior. Our results place constraints on the mechanism(s) by which
Rad23 (and by analogy, other UbL-UBA family members) promote
proteolysis
How do UbL-UBA proteins promote substrate proteolysis? Further work
will be necessary to answer this question. The phenotypes of
single-gene knockouts are rather substrate-specific: among model
substrates, UbPro Linkage-specific PolyUb Chain Binding by Rad23 UBA
Domains--
Our results provide the first rigorous evidence that UBA
domains can bind polyUb chains in a linkage-dependent
manner, despite a previous report that the UBA domain of Dsk2 binds
Lys48-linked chains preferentially (19). Funakoshi et
al. (19) found that overexpression of K29R- or K63R-Ub in S. cervisiae did not inhibit the binding of endogenous ubiquitinated
proteins to a GST-UBA fusion protein, whereas overexpression of K48R-Ub did inhibit. However, because Lys63-linked chains
constitute only a few percent of the chains in budding yeast and
endogenous Lys29-linked chains have never been detected
(52, 53), diminished binding of alternatively-linked chains in this
experiment would have been impossible to detect against the high
background of bound Lys48-linked chains. Thus, the previous
results (19) show only that Lys48-linked chains are
competent in binding.
Our conclusion that Rad23 does not bind Lys29-linked polyUb
chains contradicts another recent report (22). Rao and Sastry (22)
detected a yeast two-hybrid interaction between Ub and each UBA domain
of Rad23. The interactions were abolished by G76V or K29R/K48R
mutations in the Ub bait, effects that were interpreted to indicate
that interaction relied on assembly of the bait Ub into a chain through
either Lys29 or Lys48. It is possible that
interactions in this system may have relied on an unidentified cellular
factor(s); or the mutational effects could have reflected
structure/function properties of conjugating/deconjugating enzymes as
opposed to the chemical structure(s) of chain products. The rigorously
established linkages of the Ub4 molecules used in the
present work provide high confidence in our conclusion that
Lys29-linked chains have negligible affinity for the Rad23
UBA domains.
The UBA domain is the first protein element found to interact with one
type of polyUb chain in preference to another. In view of increasing
evidence that different polyUb chains can act as functionally distinct
signals (3, 45, 50-53), the molecular basis of this preference is of
significant interest. Mueller and Feigon (43) suggested that a
hydrophobic surface patch of the UBA interacts with a hydrophobic
surface patch of Ub. If this model is correct, then the patches must be
differentially accessible in different chains. Alternatively, the
selective interaction of Rad23 UBAs with Lys48-linked
chains could rely on contacts at the Ub-Ub junctions or with
conformationally-induced determinants (not necessarily the hydrophobic
patch) that are unique to these chains. A recent NMR analysis provided
evidence that Lys48-linked chains can adopt specific
conformations in solution (61). Whether or not this property proves
relevant to the interaction specificity seen here, selective binding to
UBA domains appears likely to contribute to linkage specificity in
polyUb chain signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or dsk2
yeast (19, 22,
23). Complementation of this phenotype generally requires that both the
UbL and UBA domains be present (17, 21-23). UbL-UBA proteins interact
physically with S5a/Rpn10 (24), a subunit of the 19 S complex that is
also found outside the 19 S (25). UbL-UBA proteins also interact functionally with S5a/Rpn10, in that deletion of individual UbL-UBA genes together with the S5a/Rpn10 gene results in
a synergistic stabilization of certain proteasome substrates (17, 20,
21, 23). However, these interactions, although suggestive, do not lead
to a defined functional model, in part because the specific role of
S5a/Rpn10 in proteasome-catalyzed proteolysis remains uncertain (see
Refs. 25 and 26). Still other findings appear to be inconsistent with a
critical role for UbL-UBA proteins in targeting substrates to
proteasomes. For example, deletion of individual UbL-UBA genes, even
together with the gene encoding Rpn10/S5a, does not inhibit the
turnover of naturally short-lived proteins (23). Moreover,
overexpression of UbL-UBA proteins inhibits, rather than stimulates,
the turnover of natural and model proteasome substrates in yeast
cells (19, 21, 27, 28).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP, and 10 mM dithiothreitol.
Carrier protein (bovine serum albumin or ovalbumin) was added to
improve the recovery of Ub5DHFR. After 10 min at 37 °C,
the incubation was spin-filtered through Sephadex G-25 to remove
unincorporated radioactivity and stored at 5 °C for up to 1 week.
The concentration of Ub5DHFR was estimated by SDS-PAGE and
Coomassie Blue staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/E2-14K, equivalent to yeast Ubr1/Ubc2), conjugated to Lys48-linked polyUb chains and
degraded by proteasomes (5, 32, 36, 39). Previous studies have
implicated UbL-UBA proteins in the turnover of N-end rule substrates.
For example, Leu
Gal, which is partially stabilized in
dsk2
yeast (19), is polyubiquitinated through the N-end
rule pathway (40).
View larger version (38K):
[in a new window]
Fig. 1.
Rad23 and mutant proteins. A,
domain structures and true molecular masses. B, Coomassie
Blue-stained gel. Adjacent lanes show ~2 and 1 µg,
respectively, of the indicated purified proteins (Rad23 and some
mutants migrate anomalously in SDS-PAGE).
50% at 7 µM) was associated with the presence of two UBAs (Rad23,
R23D, UBA1-2 in Fig. 2A). The UBAs, rather than the region
between them, mediate inhibition, as shown by comparing the effects of
UBA1-2 and ID in Fig. 2A.
View larger version (15K):
[in a new window]
Fig. 2.
Potent inhibition of
125I-lactalbumin degradation in reticulocyte fraction II
requires both UBA domains of Rad23. A, degradation
assays ("Experimental Procedures") were supplemented with the
indicated Rad23 protein (7 µM). Rates of
Ub-dependent degradation are expressed relative to the
control lacking Rad23. B, concentration dependence of
inhibition by R23D.
View larger version (10K):
[in a new window]
Fig. 3.
Effect of heat treatment on proteolytic
inhibition by full-length Rad23. Full-length Rad23 or R23D (1 mg/ml) was treated at 100 °C for 1 min in buffer containing 20 mM Tris-HCl (pH 7.6). The solutions were cooled, and
aliquots were diluted into lactabumin degradation assays. Rates are
expressed relative to the control lacking any Rad23. The weaker
inhibition by 7 µM R23D here (versus Fig.
2A) can be explained by the use of a less pure R23D
preparation ("Experimental Procedures").
Gal, a Ub fusion degradation (UFD) substrate whose
intracellular degradation depends on the extension of a polyUb chain
from either Lys48 or Lys29 of a non-cleavable
Ub moiety (45). UbPro
Gal is strongly stabilized in
rad23
or dsk2
yeast (22, 23). We therefore
anticipated that a stimulatory effect of Rad23 would be manifested with
Ub5DHFR. Instead, full-length Rad23 inhibited the
degradation of this substrate in a concentration-dependent
fashion (Fig. 4A, lanes
5 and 6 versus 4).
View larger version (19K):
[in a new window]
Fig. 4.
Rad23 inhibits the degradation of
Ub5DHFR by purified 26 S proteasomes. A, Western
blot. Assays contained 0.1 µM unlabeled
Ub5DHFR and 12 nM 26 S proteasomes.
B, concentration dependence of inhibition by R23D. Assays
contained 1.6 nM 26 S proteasomes and 15 nM
(diamonds) or 100 nM (squares)
32P-Ub5DHFR. Degradation was monitored as the
production of acid-soluble radioactivity ("Experimental
Procedures").
View larger version (26K):
[in a new window]
Fig. 5.
Rad23A is a substoichiometric constituent of
purified 26 S proteasomes. A, purified proteins
(Coomassie Blue stain). Lane 1, 7 µg; lane 2,
0.2 µg. B, Western blot (anti-Rad23A). Lanes
contained the indicated amounts of protein. The amount of 19 S in the
26 S proteasome was calculated assuming 2 mol of 19 S/mol of 26 S and a
molecular mass of 700 kDa for the 19 S complex.
View larger version (87K):
[in a new window]
Fig. 6.
Effects of Rad23 on polyUb chain homeotasis.
A, R23D promotes the accumulation of Ub-lactalbumin
conjugates (autoradiograph). The indicated effectors were added at 3 µM (Ubal), 150 µM (MG-132), or 7 µM (R23D). Control, no added inhibitor;
cont., labeled contaminant; 2, 4, and
6, forms of lactalbumin migrating as expected for
conjugation to the indicated number of Ubs. B, effect of
R23D on Ub4 disassembly. 125I-Ub4
of the indicated linkage (0.8 µM) was incubated with 0.3 mg/ml fraction II protein for 10 min at 37 °C with no further
additions, or in the presence of Ubal (2.3 µM) or R23D (4 µM). C, effect of R23D on poly(Ub) chain
assembly (Ub Western blot). Mono-Ub (117 µM)
was incubated with the indicated chain-assembling enzymes for the
indicated times, under conditions described under "Experimental
Procedures." Incubations were done in the presence or absence of R23D
(20 µM) as indicated. K29R-Ub was used to make
Lys48-linked chains; K48R-Ub was used for
Lys29/Lys63-linked chains. Numbers show
n values of the indicated chains.
E3-Ubn denotes very large autopolyubiquitination
products whose formation is inhibited by R23D (see the text).
View larger version (15K):
[in a new window]
Fig. 7.
PolyUb chain binding to Rad 23. A,
two UBA domains afford stronger binding of K48-Ub4.
Immobilized, GST-fused Rad23 mutants were used to pull down
125I-Ub4 as described under "Experimental
Procedures." B, linkage specificity. GST-UBA1-2 protein
(Fig. 1A) was used in pull-down assays (as in panel
A) with each radiolabeled Ub4. C, surface
plasmon resononace analysis of Ub1 and Ub4
binding to R23D (in HEPES-buffered saline, pH 7.4). Analyte
Ub4 molecules were 0.12 µM, while
Ub1 was 0.48 µM (duplicates in each case).
Association denotes injection of analyte;
dissociation denotes shift to buffer alone (see
"Experimental Procedures").
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Gal turnover much more severely than did overexpression
of Dsk2 (19). Thus, UbL-UBA overexpression probably inhibits growth by
a mechanism(s) in addition to generic proteasome inhibition.
the current findings argue strongly against a simple
trans-targeting event in which the sole participants are the UbL-UBA
protein, the substrate's Lys48-linked chain and the
proteasome. Certain in vivo results are difficult to
reconcile with this simple model as well. In particular, it does not
easily accommodate functional specificity of different UbL-UBA family
members, but Rad23 and Dsk2 are both needed for Ub
Gal turnover in
budding yeast (22, 23). Nor does a simple trans-targeting model explain
the strong functional interaction between S5a/Rpn10 and UbL-UBA
proteins (17, 20, 21, 23), particularly given the lack of evidence that
S5a/Rpn10 plays a major role in polyUb chain recognition by proteasomes
(25, 26).
Gal is strongly affected, Leu
Gal is less affected, and naturally short-lived proteins, when assayed in bulk, are
unaffected (19, 22, 23). Yet all of these turnover events depend, in
whole or in part, on Lys48-linked chains. Functional
distinctions among different substrates could be explained if yet
unknown determinants (besides the substrate-linked polyUb chain)
influence substrate interactions with UbL-UBA proteins. The possibility
of additional interactions is suggested by the diverse set of
Rad23-interacting proteins already identified, including XPC/Rad4 (30),
the HIV-Vpr protein (57), a cytosolic protein deglycosylase (58), and a
DNA glycosylase (59). Alternatively, events that cannot yet be
recapitulated in vitro, such as a post-translational modification, may be important for the proteolysis-promoting effects of
Ubl-UBA proteins. Nor can it be excluded that these proteins may
stimulate proteolysis by modulating ubiquitination or deubiquitination (above). Finally, UbL-UBA proteins could promote the proteolysis of
certain substrates by inhibiting the proteolysis of other substrates. One such model is suggested by the involvement of Lys29
linkages in UFD substrate turnover (3, 45, 60) and the inability of the
Rad23 UBA domains to bind Lys29-linked chains. Our findings
suggest that Rad23 will neither inhibit the assembly of
Lys29-linked chains nor hinder the possible interaction of
these chains with proteasomes. Thus, any substrates targeted to
proteasomes via Lys29-linked chains should be immune to the
inhibitory effects discovered in the present work. Moreover, by
sequestering substrates conjugated to Lys48-linked chains,
proteasome-bound Rad23 could indirectly promote the proteolysis of
substrates conjugated to Lys29-linked chains. However,
although certain UFD substrates are targeted by polyUb chains
initiating at Lys29 (45), it is not yet known if
Lys29 linkages are important for targeting to proteasomes,
versus other interactions (60).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank P. Howley for a generous gift of Rad23 plasmids and antibodies; I. Orlov and M. Cloney for assistance with Biacore studies; M. Hochstrasser and R. Cohen for helpful discussions; and Y. Lam and C. Tsui for permission to cite unpublished results.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant DK46984 from the National Institutes of Health.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.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Johns Hopkins University, 615 North Wolfe St.,
Baltimore, MD 21205. Tel.: 410-614-4554; Fax: 410-955-2926; E-mail:
cpickart@jhmi.edu.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M212841200
2 Y. Lam and C. Pickart, unpublished data.
3 C. Tsui and C. Pickart, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Ub, ubiquitin;
Gal,
-galactosidase;
DHFR, dihydrofolate reductase;
DUB, deubiquitinating enzyme;
E1, ubiquitin-activating enzyme;
E2, ubiquitin-conjugating enzyme, E3, ubiquitin-protein ligase;
polyUb, polyubiquitin (refers to a branched, isopeptide-linked ubiquitin chain);
UBA, ubiquitin-associated domain;
Ubal, ubiquitin aldehyde;
UbL, ubiquitin-like domain;
GST, glutathione S-transferase;
FPLC, fast protein liquid chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425-479[CrossRef][Medline] [Order article via Infotrieve] |
2. | Baumeister, W., Walz, J., Zuhl, F., and Seemuller, E. (1998) Cell 92, 367-380[Medline] [Order article via Infotrieve] |
3. | Pickart, C. M. (2000) Trends Biochem. Sci. 25, 544-548[CrossRef][Medline] [Order article via Infotrieve] |
4. | Hicke, L. (2001) Nat. Rev. Mol. Cell. Biol. 2, 195-201[CrossRef][Medline] [Order article via Infotrieve] |
5. | Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583[Medline] [Order article via Infotrieve] |
6. | Finley, D., Sadis, S., Monia, B. P., Boucher, P., Ecker, D. J., Crooke, S. T., and Chau, V. (1994) Mol. Cell. Biol. 14, 5501-5509[Abstract] |
7. |
Thrower, J. S.,
Hoffman, L.,
Rechsteiner, M.,
and Pickart, C. M.
(2000)
EMBO J.
19,
94-102 |
8. | Watkins, J. F., Sung, P., Prakash, L., and Prakash, S. (1993) Mol. Cell. Biol. 13, 7757-7765[Abstract] |
9. | Biggins, S., Ivanovska, I., and Rose, M. D. (1996) J. Cell Biol. 133, 1331-1346[Abstract] |
10. | Bertolaet, B. L., Clarke, D. J., Wolff, M., Watson, M. H., Henze, M., Divita, G., and Reed, S. I. (2001) Nat. Struct. Biol. 8, 417-422[CrossRef][Medline] [Order article via Infotrieve] |
11. | Russell, S. J., Reed, S. H., Huang, W., Friedberg, E. C., and Johnston, S. A. (1999) Mol. Cell 3, 687-695[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Gillette, T. G.,
Huang, W.,
Russell, S. J.,
Reed, S. H.,
Johnston, S. A.,
and Friedberg, E. C.
(2001)
Genes Dev.
15,
1528-1539 |
13. |
Lommel, L.,
Chen, L.,
Madura, K.,
and Sweder, K.
(2000)
Nucleic Acids Res.
28,
4839-4845 |
14. | Schauber, C., Chen, L., Tongaonkar, P., Vega, I., Lambertson, D., Potts, W., and Madura, K. (1997) Nature 391, 715-718[CrossRef] |
15. | Elsasser, S., Gali, R. R., Schwickart, M., Larsen, C. N., Leggett, D. S., Muller, B., Feng, M. T., Tubing, F., Dittmar, G. A. G., and Finley, D. (2002) Nat. Cell Biol. 4, 725-730[CrossRef][Medline] [Order article via Infotrieve] |
16. | Hofmann, K., and Bucher, P. (1996) Trends Biochem. Sci. 21, 172-173[CrossRef][Medline] [Order article via Infotrieve] |
17. | Wilkinson, C. R. M., Seeger, M., Hartmann-Petersen, R., Stone, M., Wallace, M., Semple, C., and Gordon, C. (2001) Nat. Cell Biol. 3, 939-943[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Chen, L.,
Shinde, U.,
Ortolan, T. G.,
and Madura, K.
(2001)
EMBO Rep.
2,
933-938 |
19. |
Funakoshi, M.,
Sasaki, T.,
Nishimoto, T.,
and Kobayashi, H.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
745-750 |
20. | Saeki, Y., Saitoh, A., Toh-e, A., and Yokosawa, H. (2002) Biochem. Biophys. Res. Commun. 293, 986-992[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Chen, L.,
and Madura, K.
(2002)
Mol. Cell. Biol.
22,
4902-4913 |
22. |
Rao, H.,
and Sastry, A.
(2002)
J. Biol. Chem.
277,
11691-11695 |
23. |
Lambertson, D.,
Chen, L.,
and Madura, K.
(1999)
Genetics
153,
69-79 |
24. |
Hiyama, H.,
Yokoi, M.,
Masutani, C.,
Sugasawa, K.,
Maekawa, T.,
Tanaka, K.,
Hoeijmakers, J. H. J.,
and Hanaoka, F.
(1999)
J. Biol. Chem.
274,
28019-28025 |
25. | van Nocker, S., Sadis, S., Rubin, D. M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R. D. (1996) Mol. Cell. Biol. 16, 6020-6028[Abstract] |
26. | Lam, Y. A., Lawson, T. G., Velayutham, M., Zweier, J. L., and Pickart, C. M. (2002) Nature 416, 763-767[CrossRef][Medline] [Order article via Infotrieve] |
27. | Ortolan, T. G., Tongaonkar, P., Lambertson, D., Chen, L., Schauber, C., and Madura, K. (2000) Nat. Cell Biol. 2, 601-608[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Clarke, D. J.,
Mondesert, G.,
Segal, M.,
Bertolaet, B. L.,
Jensen, S.,
Wolff, M.,
Henze, M.,
and Reed, S. I.
(2001)
Mol. Cell. Biol.
21,
1997-2007 |
29. |
Haas, A. L.,
and Bright, P. M.
(1985)
J. Biol. Chem.
260,
12464-12473 |
30. | Masutani, C., Sugasawa, K., Yanagisawa, J., Sonoyama, T., Ui, M., Enomoto, T., Takio, K., Tanaka, K., van der Spek, P. J., Bootsma, D., Hoeijmakers, J. H. J., and Hanaoka, F. (1994) EMBO J. 13, 1831-1843[Abstract] |
31. | You, J., Cohen, R. E., and Pickart, C. M. (1999) BioTechniques 27, 950-954[Medline] [Order article via Infotrieve] |
32. |
Hershko, A.,
Heller, H.,
Elias, S.,
and Ciechanover, A.
(1983)
J. Biol. Chem.
258,
8206-8214 |
33. |
Hofmann, R. M.,
and Pickart, C. M.
(2001)
J. Biol. Chem.
276,
27936-27943 |
34. |
You, J.,
and Pickart, C. M.
(2001)
J. Biol. Chem.
276,
19871-19878 |
35. | Haldeman, M. T., Xia, G., Kasperek, E. M., and Pickart, C. M. (1997) Biochemistry 36, 10526-10537[CrossRef][Medline] [Order article via Infotrieve] |
36. | Beal, R. E., Toscano-Cantaffa, D., Young, P., Rechsteiner, M., and Pickart, C. M. (1998) Biochemistry 37, 2925-2934[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Sloper-Mould, K. E.,
Jemc, J.,
Pickart, C. M.,
and Hicke, L.
(2001)
J. Biol. Chem.
276,
30483-30489 |
38. |
Piotrowski, J.,
Beal, R.,
Hoffman, L.,
Wilkinson, K. D.,
Cohen, R. E.,
and Pickart, C. M.
(1997)
J. Biol. Chem.
272,
23712-23721 |
39. |
Beal, R.,
Deveraux, Q.,
Xia, G.,
Rechsteiner, M.,
and Pickart, C. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
861-866 |
40. | Varshavsky, A., Turner, G., Du, F., and Xie, Y. (2000) Biol. Chem. 381, 779-789[Medline] [Order article via Infotrieve] |
41. |
Ng, J. M. Y.,
Vrieling, H.,
Sugasawa, K.,
Ooms, M. P.,
Grootegoed, J. A.,
Vreeburg, J. T. M.,
Visser, P.,
Beems, R. B.,
Gorgels, T. G. M. F.,
Hanaoka, F.,
Hoeijmakers, J. H. J.,
and van der Horst, G. T. J.
(2002)
Mol. Cell. Biol.
22,
1233-1245 |
42. | Bertolaet, B. L., Clarke, D. J., Wolff, M., Watson, M. H., Henze, M., Divita, G., and Reed, S. I. (2001) J. Mol. Biol. 313, 955-963[CrossRef][Medline] [Order article via Infotrieve] |
43. | Mueller, T. D., and Feigon, J. (2002) J. Mol. Biol. 319, 1243-1255[CrossRef][Medline] [Order article via Infotrieve] |
44. | Walters, K. J., Kleijnen, M. F., Goh, A. M., Wagner, G., and Howley, P. M. (2002) Biochemistry 41, 1767-1777[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Johnson, E. S.,
Ma, P. C.,
Ota, I. M.,
and Varshavsky, A.
(1995)
J. Biol. Chem.
270,
17442-17456 |
46. |
Verma, R.,
Chen, S.,
Feldman, R.,
Schieltz, D.,
Yates, J.,
Dohmen, J.,
and Deshaies, R.
(2000)
Mol. Biol. Cell
11,
3425-3439 |
47. |
Elder, R. T.,
Song, X.-Q.,
Chen, M.,
Hopkins, K. M.,
Lieberman, H. B.,
and Zhao, Y.
(2002)
Nucleic Acids Res.
30,
581-591 |
48. |
Amerik, A. Y.,
Swaminathan, S.,
Krantz, B. A.,
Wilkinson, K. D.,
and Hochstrasser, M.
(1997)
EMBO J.
16,
4826-4838 |
49. | Wilkinson, K. D., Tashayev, V. L., O'Connor, L. B., Larsen, C. N., Kasperek, E., and Pickart, C. M. (1995) Biochemistry 34, 14535-14546[Medline] [Order article via Infotrieve] |
50. | Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C., and Chen, Z. J. (2000) Cell 103, 351-361[Medline] [Order article via Infotrieve] |
51. | Hoege, C., Pfander, B., Moldovan, G.-L., Pyrowolakis, G., and Jentsch, S. (2002) Nature 419, 135-141[CrossRef][Medline] [Order article via Infotrieve] |
52. | Spence, J., Sadis, S., Haas, A. L., and Finley, D. (1995) Mol. Cell. Biol. 15, 1265-1273[Abstract] |
53. | Spence, J., Gali, R. R., Dittmar, G., Sherman, F., Karin, M., and Finley, D. (2000) Cell 102, 67-76[Medline] [Order article via Infotrieve] |
54. |
Verma, R.,
Aravind, L.,
Oania, R.,
McDonald, W. H.,
Yates, J. R.,
Koonin, E. V.,
and Deshaies, R. J.
(2002)
Science
298,
611-615 |
55. | Yao, T., and Cohen, R. E. (2002) Nature 419, 403-407[CrossRef][Medline] [Order article via Infotrieve] |
56. | Lommel, L., Ortolan, T., Chen, L., Madura, K., and Sweder, K. S. (2002) Curr. Genet. 42, 9-20[CrossRef][Medline] [Order article via Infotrieve] |
57. | Dieckmann, T., Withers-Ward, E. S., Jarosinski, M. a., Liu, C.-F., Chen, I. S. Y., and Feigon, J. (1998) Nat. Struct. Biol. 5, 1042-1047[CrossRef][Medline] [Order article via Infotrieve] |
58. |
Suzuki, T.,
Park, H.,
Kwofie, M. A.,
and Lennarz, W. J.
(2001)
J. Biol. Chem.
276,
21601-21607 |
59. |
Miao, F.,
Bouziane, M.,
Dammann, R.,
Masutani, C.,
Hanaoka, F.,
Pfeifer, G.,
and O'Connor, T. R.
(2000)
J. Biol. Chem.
275,
28433-28438 |
60. | Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H. D., Mayer, T. U., and Jentsch, S. (1999) Cell 96, 635-644[Medline] [Order article via Infotrieve] |
61. | Varadan, R., Walker, O., Pickart, C. M., and Fushman, D. (2002) J. Mol. Biol. 324, 637-647[CrossRef][Medline] [Order article via Infotrieve] |