From the Medical Research Council Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom
Received for publication, August 13, 2002, and in revised form, January 23, 2003
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ABSTRACT |
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Fission yeast Rhp23 and Pus1 represent two
families of multiubiquitin chain-binding proteins that associate with
the proteasome. We show that both proteins bind to different regions of
the proteasome subunit Mts4. The binding site for Pus1 was mapped to a
cluster of repetitive sequences also found in the proteasome subunit
SpRpn2 and the anaphase-promoting complex/cyclosome (APC/C) subunit
Cut4. The putative role of Pus1 as a factor involved in
allocation of ubiquitinylated substrates for the proteasome is discussed.
Ubiquitin-dependent protein degradation is a mechanism
employed in eukaryotic cells not only to recycle damaged and misfolded proteins but also to control cellular processes by specific breakdown of regulatory proteins (1). Ubiquitinylation is accomplished in
multiple steps (2). Initially ubiquitin is activated by a
ubiquitin-activating enzyme (E1) in an ATP-dependent
process whereby the E1 forms a thiol ester bond with the C terminus of ubiquitin. Subsequently the ubiquitin molecule is transferred to a
ubiquitin-conjugating enzyme (E2). E2s carrying ubiquitin associate
with substrate binding ubiquitin protein ligases (E3s) resulting in the
covalent attachment of ubiquitin to the substrate protein. Several
rounds of this conjugation process produce substrates carrying a chain
of ubiquitin moieties. There is a range of different E2s that associate
with various E3s, incorporating an element of substrate specificity to
this process.
Ubiquitin protein ligases can be divided in two major groups, HECT
domain E3s and RING finger domain E3s. The anaphase-promoting complex/cyclosome (APC/C)1
belongs to a family of multimeric ubiquitin protein ligases of the RING
finger type that also include the SCF and the VCB (2, 3). It has a
molecular mass of 700 kDa and consists of 11 different subunits.
Mitotic events are controlled by the APC/C via
multiubiquitinylation of cell cycle regulators like
Cut2/Pds1p/securin and B-type cyclins. Once a ubiquitin chain is
conjugated to these proteins they become targets for degradation by the
26 S proteasome enabling the continuation of downstream mitotic events.
The 2.5 MDa 26 S proteasome catalyzes the degradation of cellular
proteins in an ATP-dependent manner (4). Its proteolytic component is the 20 S core complex, a cylindrical structure comprising four stacked rings each containing seven proteins. The inner rings enclose a central chamber harboring the catalytic sites. Access to the
lumen is provided via the outer rings and regulated by the 19 S
regulatory complex that is attached to one or both ends of the 20 S
core (5).
The 19 S regulatory complex can be dissociated in two subcomplexes
called the base and the lid (6, 7). Six ATPases subunits are presumed
to form a structure that associates with the 20 S core. Together with
the two largest subunits of the proteasome SpRpn2/Rpn2/S1 and
Mts4/Rpn1/S2 these ATPases form the base complex that was suggested
to be participating in the unfolding and translocation of substrates.
The lid complex is believed to contact the base via the subunits
SpRpn2/Rpn2/S1, Mts4/Rpn1/S2, and Pus1/Rpn10/S5a. It is composed of a
number of non-ATPase subunits whose function remains rather enigmatic.
Proteasomes lacking the lid can cleave small peptides in an
ATP-dependent manner, but they are unable to degrade
ubiquitinylated proteins (6). Thus the lid is believed to be involved
in the recognition and processing of those substrates.
To date, one subunit of the proteasome has been found to possess the
ability to recognize multiubiquitinylated substrates, namely
Pus1/Rpn10/S5a (8). However, given the fact that in yeast the
corresponding RPN10/pus1+ genes are
not essential for cell viability, other mechanisms must clearly exist
for this crucial step in the degradation process (9). Recently, it has
been shown that the UBA/UBL domain proteins Rhp23/ Rad23p and
Dph1/Dsk2p can interact with both the proteasome and with
multiubiquitin suggesting that these factors play a role in the
recognition and delivery of substrates for proteolysis (10-13). The
Pus1/Rpn10p protein contains a stretch of 20 amino acids recently
defined as ubiquitin-interacting motif (UIM) that is involved in
multiubiquitin chain binding, whereas in Dph1/Dsk2p and Rhp23/Rad23p
the task is accomplished by a C-terminal ubiquitin pathway associated
(UBA) domain (9, 10, 14, 15, 16). Both Dph1/Dsk2p and Rhp23/Rad23p bind
to the proteasome with their N-terminal UBL domain (10, 17). As
Pus1/Rpn10p does not have a UBL domain it must use different structures
to associate with other proteasome subunits. Lambertson et
al. (18) were able to precipitate the proteasome from cells
deleted for RPN10 and RAD23 using tagged Rad23p
or Rpn10p, respectively, demonstrating that these proteins bind to the
proteasome independently.
So far little is known about how ubiquitinylated substrates arrive at
the proteasome. Some ubiquitinylating enzymes associate directly with
the proteasome as shown for the E2s Ubc1, Ubc2, Ubc4, and Ubc5 as well
as for the E3s Ubr1p, Ufd4p, and KIAA10 (19-23). It is also
conceivable that multiubiquitin-binding proteins provide a link between
components of the ubiquitinylation machinery and the proteasome as
implied by the finding that hPLIC proteins, the human homologues of
Dph1/Dsk2p, associate with E3 proteins (24). However, previously
neither Rhp23/Rad23p nor Pus1/Rpn10p have been reported to interact
with ubiquitinylating enzymes.
To improve our understanding of how the proteasome recruits ubiquitin
substrates we started to investigate the association of these proteins
with the 19 S regulatory complex. We characterize regions in proteasome
subunits that bind Pus1 and Rhp23 and describe the interaction of these
multiubiquitin-binding proteins with the APC/C.
Bioinformatics--
Sequence similarity searches were carried
out using Psi-BLAST (version 2.2.3) (25) using the BLOSUM62
substitution matrix for the first iteration. Sequences identified with
a BLAST E-value of <1e Schizosaccharomyces pombe Strains and Techniques--
Fission
yeast strains used in this study (wt, mts2-1,
mts4-1,
Standard genetic methods and media were used as described, and
S. pombe transformations were performed by the
lithium acetate procedure (26).
Plasmids--
The plasmid pREP1Rhp23 was produced as described
(10). To generate Mts4 constructs FL (full-length), C Yeast Two-hybrid Assay--
The full-length
rhp23+ gene and the UBL encoding domain of
rhp23+ were subcloned into the MCS of the pAS2
vector (Clontech). The yeast strain Y190
was transformed using this construct as bait. The obtained
transformants were then transformed again, this time with a series of
constructs encoding proteasomal subunits cloned into the pACT2 vector
(Clontech) as prey. The obtained double transformants were tested for growth on plates containing 25 mM 3-aminotriazole (Sigma) and for their ability to
activate the lacZ reporter by filter lifts assays. Only the Mts4 pACT2
prey construct scored positive in both of these tests.
In Vitro Binding Assays--
GST and GST fusion proteins were
expressed in Escherichia coli BL21 (DE3) pLysS,
bound to glutathione-Sepharose 4B beads (Amersham Biosciences) as
described by the manufacturer and protein/beads ratio was adjusted to
about 1 mg/ml. For assays using His-tagged constructs, proteins were
expressed in E. coli M15 cells (Qiagen) and lysed
in buffer A (50 mM Tris, pH 7.5, 100 mM NaCl,
10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, and CompleteTM protease inhibitors (Roche Applied
Science). 0.5 ml of the cleared extract were mixed with 15 µl of
beads bound to GST or GST fusion protein and incubated for 2 h at
4 °C. Protein concentration of the extracts varied between 10 and 50 mg/ml depending on the expression level of the His-tagged protein. The
beads were washed four times in buffer A and resuspended in 50 µl of
SDS sample buffer. 10 µl of the samples were separated on 10 or 12%
SDS gels and subjected to Western blot analysis. For binding assays
using total yeast extracts, cells were grown to an OD595
0.8, lysed in buffer B (1 mM dithiothreitol, 2 mM ATP and 10 mM MgCl2 in buffer A)
using glass beads (Braun) and cleared by centrifugation. The extracts were adjusted to a protein concentration of 10 mg/ml, and the binding
assay was performed as described for the bacterial extracts except that
Buffer B was used for washing the beads. Antisera used in Western blots
were polyclonal anti-Mts2, monoclonal anti-HA12CA5 (Roche Diagnostics),
and monoclonal anti-His6 (a gift from V. Van Heiningen).
Co-precipitation--
pREP1GST and pREP1GST-Pus1 were
transformed into cut9HA cells and total extracts of S. pombe transformants expressing gst+ and
cut9HA+ or gst-pus1+ and
cut9HA+ were prepared as described under
"In vitro Binding Assays." 1 ml of the extract
containing 5 mg of total protein were incubated with 15 µl of
glutathione-Sepharose (Amersham Biosciences) for 1 h at 4 °C.
The beads were washed four times in buffer B, resuspended in 50 µl of
SDS sample buffer, and 10 µl were loaded onto a 10% SDS gel. GST and
GST-Pus1 were visualized by Coomassie staining and Cut9HA by Western
blotting with an anti-HA antibody (12CA5, Roche Diagnostics).
The UBL Domain of Rhp23 Binds to the Proteasome Subunit
Mts4--
As a first step to characterize the interactions of Rhp23
with the proteasome we employed the yeast two-hybrid system. We tested
the ability of Rhp23 to bind to the proteasomal subunits Mts1 (Rpn9),
Mts2 (Rpt2p), Mts3 (Rpn12p), SpRpn2 (Rpn2), Mts4 (Rpn1), Pad1 (Rpn11),
Pus1 (Rpn10), SpRpt1 (Rpt1), SpRpt6 (Rpt6), and to the
proteasome-associated ubiquitin hydrolase Uch2. The two-hybrid assay
detected an interaction of Rhp23 with Mts4, a subunit of the
proteasomal base complex (data not shown). To test whether there is
also a genetic interaction between rhp23+ and
mts4+ we used the fission yeast
mts4-1 strain that carries a mutation in the gene encoding
Mts4 (27). By transforming mts4-1 cells with the plasmid
pREP1Rhp23, in which Rhp23 expression is driven by the thiamine
inducible nmt1 promoter, we were able to partially suppress
the temperature sensitive (ts) phenotype of the mutant (Fig.
1A). As a control we also
introduced the plasmid into mts2-1 cells, which have a
mutation in the gene for the proteasome subunit Mts2 and display a
temperature sensitive phenotype similar to mts4-1 (29).
However, transformation of mts2-1 with pREP1Rhp23 could not
rescue the mutant phenotype indicating that the interaction between
rhp23+ and mts4+ is
specific. In addition, the observed genetic interaction was dependent
on the Rhp23 UBL domain as when this domain was deleted from the
rhp23+ gene suppression of the mts4-1
mutant no longer
occurred.2
In order to determine whether this genetic interaction reflected a
direct physical interaction between Rhp23 and Mts4, we performed an
in vitro binding assay. A GST-Rhp23 fusion protein coupled
to glutathione-Sepharose was incubated with His-tagged full-length Mts4
as well as N- and C-terminal truncated versions of the protein
expressed in Escherichia coli. As shown, GST-Rhp23 precipitated Mts4 from the bacterial extracts (Fig. 1B)
demonstrating a direct interaction between these proteins. The use of
truncated versions of Mts4 revealed that Rhp23 binds within a region
stretching from amino acid position 181 to 407.
It has been shown that Rhp23 binds the proteasome via its UBL domain
(10). Therefore the binding of Rhp23 to Mts4 should also be dependent
upon the UBL domain. Truncated versions of Rhp23 fused to GST were used
in an in vitro binding assay similar to the one described
above. We found that only the truncation of Rhp23 containing the UBL
domain could precipitate Mts4 indicating that the UBL domain mediates
the binding between them (Fig. 1C).
To demonstrate that binding to the Mts4/Rpn1 subunit of the 26 S
proteasome is a general property of proteins that contain a UBL domain
we assayed the fission yeast Udp7 protein (SwissProt accession number
Q94580). The Udp7 protein, like the Rhp23 protein, contains a UBL
domain in its N terminus. When assayed in vitro GST-Udp7 was
able to precipitate Mts4 protein from the bacterial extracts
demonstrating an interaction (Fig. 1D). This result is
consistent with the hypothesis that the UBL domain is a general
Mts4/Rpn1 interaction domain.
Pus1 Binds Mts4 in a Region Containing Repetitive
Sequences--
It has been demonstrated in budding yeast that Rpn10p
(Pus1) interacts with Rpn1 (Mts4) in an in vitro binding
assay (20). Thus we investigated interactions between these two
subunits. Previously, a genetic interaction was detected between the
genes encoding these two proteins. The mts4-1 allele was
found to be synthetically lethal with a null allele of pus1
(9). The Pus1 binding site within Mts4 was determined in an in
vitro binding assay using the same Mts4 constructs employed for
mapping the Rhp23 binding site. An N-terminal GST fusion of Pus1
immobilized on glutathione-Sepharose was used to precipitate
full-length Mts4 as well as truncated versions of the protein. We could
narrow down the binding site for Pus1 on Mts4 to a stretch of amino
acids ranging from position 408 to 582 (Fig.
2, A and B). This
particular region contains a cluster of repeats that can also be found
in SpRpn2, the fission yeast homologue of the budding yeast Rpn2p subunit of the proteasome, and in Cut4 a subunit of the APC/C or
anaphase- promoting complex (30). Although the repeats are rather
weakly conserved they appear to be present only in orthologues of the
proteins mentioned. In the Prosite data base
(www.expasy.ch/ prosite/) this structure is referred to as
APC_SEN3_REPEAT (accession number PS50248), whereas the Pfam data base
(www.sanger.ac.uk/Pfam/) lists it as PC_rep for
proteasome/cyclosome repeat (accession number PF01851). We will
therefore call it PC repeat.
Pus1 Also Binds to PC Repeat Modules of SpRpn2 and
Cut4--
Having shown that Pus1 binds to one of the PC repeat modules
found in Mts4 we asked whether Pus1 might also interact with the PC
repeat modules present in SpRpn2 and Cut4. Moreover we wanted to narrow
down the region of Pus1 that is responsible for binding the PC repeat
structures. We expressed truncated versions of Pus1 with an N-terminal
GST fusion. These constructs were tested in a binding assay using
His-tagged PC repeat modules of Mts4, SpRpn2 and Cut4. Both the
full-length Pus1 and PusC Pus1 and Rhp23 Interact with APC/C--
We have
demonstrated that Pus1 can bind to the PC repeat module present in the
APC/C subunit Cut4 in vitro. If Pus1 binds to Cut4 in
vivo, it should be possible to use the GST-Pus1 fusion protein to
precipitate the APC/C from fission yeast extracts. Therefore we
incubated GST-Pus1 and GST-Rhp23 with extracts from cut9HA
cells that carry a genomically HA-tagged version of the APC/C subunit
Cut9 and tested whether these GST fusions could precipitate the APC/C
and/or the proteasome. Because Rhp23 and Pus1 bind to multiubiquitin
chains it had to be considered that they might interact with
multiubiquitinylated substrates attached to the APC/C. Taking that into
account, we also used Pus1N5 and Rhp23PP, which have mutations in their
ubiquitin binding domains and do not bind multiubiquitin (9, 10).
Furthermore, we tested truncated versions of Pus1, which lack the
C-terminal multiubiquitin binding motif (Pus1C
Rhp23, Rhp23PP, and Rhp23 but not Rhp23N
Given that Pus1 and Cut4 interact directly, we investigated whether any
genetic interactions exist using existing mutant alleles of these
genes. We crossed the
In order to elucidate whether the genetic data mirror an in
vivo interaction between Pus1 and the APC/C we performed
co-precipitation studies. Extracts from cut9HA cells
expressing either GST or GST-Pus1 were incubated with glutathione beads
and analyzed by SDS-PAGE and Western blotting. As presented in Fig.
3B we detected the APC/C subunit Cut9 in precipitates from
cut9HA/GST-Pus1 but not from
cut9HA/GST indicating an in vivo
interaction between Pus1 and the APC/C.
We have characterized the interactions of Pus1 and Rhp23,
representing two families of multiubiquitin-binding proteins, with the
proteasome and the APC/C. Our studies demonstrate that Rhp23 binds to
Mts4, a base component of the proteasomal 19 S regulatory complex. The
UBL domain of Rhp23, which is responsible for the interaction with the
proteasome, appears to bind the Mts4 protein between amino acid
positions 181 and 407. Using this sequence to search the data base
demonstrates that it is only found in other Mts4/Rpn1 orthologues from
other species. Interestingly, this sequence comprises part of a pfamB
domain, pfam B_4211. A multiple sequence alignment of the conserved
pfamB domain is shown in Fig. 4. A
homology search of the recently sequenced S. pombe genome
with this pfamB domain demonstrated that it was only present in the
Mts4 protein. Considering that there are a number of proteins carrying
UBL domains two conclusions could be drawn. Either the structures that
interact with the different UBL domains do not resemble each other, or
all proteins containing a UBL domain can bind to Mts4/Rpn1. In this
study we have shown that the UBL domain containing protein Udp7 is also
able to interact directly with Mts4/Rpn1 indicating that the latter
hypothesis seems more probable. Consistent with this hypothesis the
Dph1/Dsk2, Bag1, and Ubp6/Usp14 UBL containing proteins have already
been shown to bind the proteasome, presumably by interaction with the
Mts4/Rpn1 subunit, although this has only been shown for the budding
yeast Dsk2 protein (32-34).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
5 at convergence were filtered to
remove overly similar sequences (80% identity in alignment) and
aligned using ClustalW (1.74). The HMMer 2.0 package (version 2.2g;)
was used to generate and optimize Hidden Markov models (HMMs) from the
multiple sequence alignment. HMMsearch was used with the HMM to
identify further examples of the UBL binding region in the SPTR data base.
pus1, cut4-533,
cut9-665, and cut9HA) are derivatives of the wild-type heterothallic strains 972h
and
975h+.
1 (amino acids
1-755), C
2-(1-582), C
3-(1-180), N
1-(181-891),
N
2-(408-891), and PC-1-(408-582) cDNAs were amplified from the
pmts4+ plasmid (27) and subcloned into pQE30
(Qiagen) except Mts4PC1, which was subcloned into pET6H (Novagen).
Regions encoding the PC repeats of SpRpn2 (amino acids 385-744) and
Cut4-(873-1023) were amplified from genomic S. pombe DNA as
these regions of the gene do not contain introns and subcloned into
pQE30. GST-Pus1 full-length, N
1 (amino acids 84-243),
N
2-(144-243), and C
1-(1-193) were constructed by inserting the
amplified pus1+ cDNAs into pGEX-KG (Amersham
Biosciences). GST-Rhp23 proteins were produced as described (10).
pREP1GST and pREP1GST-Pus1 plasmids were generated by amplifying the
gst+ and gst-pus1+
cDNAs from pGEX-KGPus1 and inserting them into the pREP1 vector (28). GST-Udp7 was produced by amplifying the
udp7+ open reading frame from genomic S. pombe DNA and inserting in-frame into the pGEX-KG plasmid.
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Fig. 1.
The UBL domain of Rhp23 binds to proteasome
subunit Mts4. A, mts2-1 and mts4-1
cells were transformed with the empty pREP1 plasmid as a control or
with pREP1rhp23 as indicated. The cells were grown at 25 °C,
streaked to single colonies on minimal media plates, and grown at 25 or
34 °C respectively. B, bacterial extracts with His-tagged
Mts4 constructs FL (full-length), C 1, C
2, C
3, N
1, N
2,
and PC-1 were incubated with GST or GST-Rhp23 coupled to beads. After a
washing procedure samples were separated on a 10% SDS gel and
subjected to Western blot analysis using a monoclonal anti-His
antibody. 10% of the input extract was loaded onto the left
lane of each panel. For clarity the upper panel shows
the deletion constructs of the Mts4 protein, the amino acid coordinates
of each deletion construct, and whether it can bind with the GST-Rhp23
protein. C, His-tagged full length Mts4 protein expressed in
E. coli was incubated with N-terminal GST fusions of Rhp23,
Rhp23 N
, and Rhp23 C
1 and analyzed as described above. The
right hand panel shows a diagram of the Rhp23, the
Rhp23N
1and the Rhp23C
1 deletion constructs, the amino acid
coordinates of the deletions made, and the result of the GST binding
experiment. D, bacterial extracts with His-tagged Mts4
full-length protein were incubated with GST-Pus1, GST-Rhp23, GST-Udp7,
and GST and analyzed as described above.
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Fig. 2.
Pus1 interacts with the PC repeat regions of
Mts4, SpRpn2, and Cut4. A, GST and GST-Pus1 were used to
precipitate His-tagged Mts4 constructs from bacterial extracts as
described for Fig. 1A. (The smear detected in the extract
lane of the N 1 panel is presumably the result of nonspecific
proteolysis but does not interfere with binding by the GST-Pus1
protein.) B, PC repeat regions of Mts4, SpRpn2, and Cut4
were expressed as His-tagged proteins in E. coli and used
for in vitro binding assays (as described for Fig.
1B) involving N-terminal GST fusions of full-length Pus1,
N
1, N
2, and C
1. The bottom panel shows a diagram of
the different Pus1 constructs tested, the amino acid coordinates used
in the deletions, and the results of the GST binding experiments.
1 were capable of binding to all three PC
repeat modules tested, whereas no binding was observed using Pus1N
1
and Pus1N
2 (Fig. 2B). From these data we conclude that
the N-terminal region of Pus1 up to amino acid position 84 appears to
contain a region necessary for binding PC repeat modules.
1) as well as versions
of Rhp23 lacking either its UBL domain (Rhp23N
1) or both UBA domains
(Rhp23C
1). We were able to precipitate the APC/C and the proteasome
using Pus1, Pus1N5, or Pus1C
1 fusion proteins but not Pus1N
1 or
Pus1N
2 (Fig. 3A). These
findings indicate that the interaction of Pus1 with the APC/C and the
proteasome is independent of its ability to bind ubiquitin conjugates
but can be assigned to the Pus1 N terminus up to amino acid position
84, which is consistent with the results of the mapping studies
involving the Cut4 PC repeat region.
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Fig. 3.
Pus1 and Rhp23 can bind to the APC/C.
A, S. pombe extracts from cut9HA cells
expressing an HA-tagged Cut9 protein were incubated with N-terminal GST
fusions of Pus1 and Rhp23 constructs coupled to glutathione-Sepharose
as described in Figs. 1 and 2. In Pus1N5 the multiubiquitin binding
LALAL motif is substituted by NNNNN and in Rhp23PP the conserved
glycine residues 158 and 333 are changed to proline, causing a loss of
the multiubiquitin binding ability for both proteins. After washing,
beads were subjected to SDS-PAGE and Western blot analysis to detect
precipitated proteasome or APC/C by using anti-Mts2 or anti-HA antisera
respectively. B, extracts from cut9HA cells
expressing either gst+ or
gst-pus1+ were incubated with glutathione beads,
and precipitates were analyzed by SDS-PAGE and Western blotting. In the
left panel a Coomassie stain of the precipitates is shown,
visualizing GST and GST-Pus1 bands respectively. In the right
panel 10% of the input cut9HA/pREP1GST and
cut9HA/pREP1GST-Pus1 extracts was loaded, and on
the left panel 10 µl of the precipitates were loaded and
analyzed by anti-HA Western blotting.
1 were found to precipitate
the proteasome, confirming that the UBA domains of Rhp23 are not
involved in proteasome binding (10). However, the APC/C binding
activity of Rhp23 appears to be mediated by its multiubiquitin binding
UBA domains as we could detect APC/C when beads coated with Rhp23 or
Rhp23N
1 but not with Rhp23PP or Rhp23C
1 were used. Thus it is
likely that Rhp23 binds via multiubiquitinylated substrates associated
with the APC/C.
pus1 strain to the temperature sensitive mutant cut4-533 (31). Spores carrying both the
cut4-533 and the
pus1 allele were not viable.
As a control
pus1 was crossed to cut9-665
another temperature sensitive APC/C mutant (31). Synthetic lethality
was not observed between cut9-665 and
pus1
indicating a specific genetic interaction between
pus1+ and cut4+.
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Fig. 4.
Multiple sequence alignment of the conserved
pfamB region within the Rhp23 UBL binding region. Aligned
sequences are defined by a genus/species abbreviation followed by the
SPTR accession number. Sp, S. pombe; Sc, Saccharomyces
cerevisiae; Hs, Homo sapiens; Dm, Drosophila
melanogaster; Ce, Caenorhabditis elegans; Eh,
Entamoeba histolytica; Pf, Plasmodium falciparum;
Tc, Trypanosoma cruzi; At, Arabidopsis thaliana.
Black background indicates 100% identity, dark
gray 80%, and light gray 60% identity. Numbers to the
right of the alignment show the corresponding amino acid
coordinate for the protein sequence (as defined by the SPTR
accession).
Using protein cross-linking studies it has been reported that the budding yeast Dsk2 and Rad23 UBLs interact with both the Rpn1 and Rpn2 subunits (35). In contrast, in our in vitro binding experiments we found no interaction with the fission yeast Rpn2 orthologue and the fission yeast Rad23 orthologue, Rhp23. A recent study in budding yeast also indicated that the UBL domain of Rad23 specifically bound to the Rpn1 subunit and showed little affinity for the Rpn2 protein (32). However the UBL binding domain identified in Rpn1 by this group was different to that found by our binding studies of the Mts4 protein. However, in each case the UBL-binding region was defined by a series of nested deletions and the binding region left was internally consistent. Therefore, further experiments will have to be carried out to resolve this apparent paradox in the different UBL binding sites, which might reflect a difference in the budding and fission yeast proteins.
The structure of the UBL domain has recently been solved by NMR spectroscopy and shown to be related to ubiquitin (36). Therefore, we tested whether mono-ubiquitin or tetra-ubiquitin could bind to Mts4. In an in vitro binding assay we were not able to detect binding between those proteins (data not shown). In addition, in mammalian cells it has been shown that the hHR23 and S5a proteins, the human versions of the fission yeast Rhp23 and Pus1 proteins, can directly interact with each other (37). Using deletion analysis the S5a binding site was determined to be in the second UIM binding domain present in this protein. This domain is not present in the simpler versions of the fission yeast Pus1 and budding yeast Rpn10 proteins (9, 38). Consistent with this we observed no direct interaction between the fission yeast Pus1 and Rhp23 proteins.3
The proteasome subunits SpRpn2 and Mts4 appear to play a central role
in the structural organization of the 19 S regulatory complex. They
link the ATPases of the base and components of the lid and also harbor
binding sites for proteins that recognize multiubiquitinated substrates
(See Fig. 5). This provides a simple model for the delivery of ubiquitinated substrates between the lid
subcomplex and the ATPase ring. Presumably upon release from the
multiubiquitin-binding proteins the ubiquitnated subtrates are captured
by the multiubiquitin binding Rpt5/S6' ATPase, before the substrate
protein is unfolded by the action of the ATPase ring and the
polypeptide translocated to the 20 S catalytic complex for degradation
(39).
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Characterization of the Pus1 association with Mts4 revealed a specific interaction between Pus1 and a cluster of PC repeats in Mts4. These repetitive sequences contain an alternating pattern of large aliphatic residues and glycine or alanine (30). It has been suggested that these repeats form a structure possessing a concave surface of a hydrophobic nature which might represent a binding site. According to the Prosite data base, Mts4 contains two PC repeat clusters with five and three repeats, respectively, which we call here PC-1 and PC-2. The mapping experiments show that PC-1 is sufficient to bind Pus1. It is conceivable that PC-1 and PC-2 together form one binding site. This would imply that deletion of PC-2 does not interfere with the integrity of the binding site, but that PC-2 on its own is unable to form a functional binding structure itself. Mts4 binds not only Rhp23 and Pus1, its human homologue S2 was also shown to associate with the HECT E3 KIAA10 (22). SpRpn2 can bind Pus1 and for its budding yeast orthologue Rpn2p, an interaction with the RING finger E3 Ubr1p was demonstrated (20).
Data base searches revealed that PC repeats can be found only in the two largest subunits of the proteasome Mts4/Rpn1 and SpRpn2 and in the APC/C component Cut4/APC1. In binding studies we demonstrated that Pus1 could bind also to the PC repeat clusters of SpRpn2 and Cut4. This indicates that binding of Pus/Rpn10 represents a general and conserved function of these PC repeat structures.
The Pus1/Rpn10 protein represents a special case among regulatory subunits of the proteasome as it is the only subunit that also occurs in low molecular weight fractions of cell extracts (9, 38, 40). Furthermore it has been suggested that Rpn10p might have a role in linking the proteasome base and lid, based on the observation that the 19 S regulatory complex dissociates easily when purified from a null allele of RPN10 (6).
Our binding studies using truncated forms of Pus1 show that deletion of the N-terminal region up to amino acid position 84 is sufficient to abolish not only the ability of Pus1 to bind PC repeat modules but also its association with the proteasome and the APC/C. Glickman et al. (6) demonstrated that budding yeast Rpn10p deleted for its N-terminal region up to position 61 can still bind to the proteasomal base complex, but not to the lid. As the PC repeat-containing subunits of the proteasome are part of its base component the data suggests that in Pus1 the region between amino acids 61 and 84 is indispensable for the interaction with the PC repeat modules.
Interestingly the N terminus of Rpn10p up to position 50 has been shown
to be critical for the degradation of Ub-Pro--gal and resistance to
amino acid analogues (14). Therefore the ability of Pus1/Rpn10p to bind
the proteasome might be crucial for these phenotypic functions of the
protein. However, considering that Pus1 binds multiubiquitin chains as
well as associating with the APC/C and the proteasome, it is
conceivable that it has a role in the delivery of multiubiquitinylated
substrates to the proteasome. Pus1 and UBA/UBL proteins might represent
separate but, in terms of substrate specificity, overlapping pathways
that facilitate the transfer of substrates from ubiquitin protein
ligases to the proteasome.
Alternatively one could speculate that Pus1 acts as a factor involved in delivery of multiubiquitinylated substrates, be it from the APC/C to UBA/UBL proteins and to the proteasome or directly from E3s to the proteasome. Depending on which pathway is used to deliver the substrate, Pus1 might associate with the appropriate PC repeat structure in order to facilitate the process. Therefore one would predict that Pus1 binds to Mts4 when substrates are acquired via Rhp23 or KIAA10 or it uses the SpRpn2 PC repeat for the Ubr1p pathway.
Recently it has been demonstrated that valosin-containing protein, a
member of the AAA (ATPases associated with a variety of cellular
activities) family, can bind ubiquitin chains and is essential for the
ubiquitin-dependent degradation of certain proteasome
substrates (41). Future work will shed light on the relationship
between the various factors involved in the recruitment of
multiubiquitinylated proteins to the proteasome and elucidate further
the role of individual proteasomal subunits in this process.
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ACKNOWLEDGEMENTS |
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We thank M. Yanagida for the cut4-533, cut9-665, and cut9HA strains, V. Van Heiningen for the anti-His antibody, S. Bruce for technical support and Miranda Stone and Nick Hastie for useful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Medical Research Council funding (to C. G.) as well as by grants from the Danish Research Academy and the Lundbeck Foundation (to R. H. P.) and the Deutsche Forschungsgemeinschaft (to M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ Present address: Institut für Biochemie, Humboldt Universität, Monbijourstr. 2, D-10117 Berlin, Germany.
¶ Present address: Dept. of Biochemistry, August Krogh Inst., University of Copenhagen, Universitetsparken 13, 2100 Copenhagen, Denmark.
Present address: Paterson Inst. for Cancer Research, Christie
Hospital NHS Trust, Wilmslow Road, Manchester M20 9BX, UK.
** To whom correspondence should be addressed: MRC Human Genetics Unit, Western General Hospital, Crewe Rd., Edinburgh EH4 2XU, UK. Tel.: 44-131-332-2471; Fax: 44-131-343-2620; E-mail: Colin.Gordon@hgu.mrc.ac.uk.
Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M208281200
2 C. Gordon and R. Hartmann-Petersen, unpublished results.
3 M. Seeger and C. Gordon, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: APC/C, anaphase-promoting complex/cyclosome; UBL, ubiquitin-like; UBA, ubiquitin-associated; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin.
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