26
Review
TRENDS in Biochemical Sciences
Vol.28 No.1 January 2003
Transferring substrates to the 26S
proteasome
Rasmus Hartmann-Petersen1, Michael Seeger2 and Colin Gordon3
1
August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen O, Denmark
Medical Faculty Charite, Humboldt University, Monbijoustrasse 2, D-10117 Berlin, Germany
3
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
2
Ubiquitin-dependent protein degradation is not only
involved in the recycling of amino acids from damaged
or misfolded proteins but also represents an essential
and deftly controlled mechanism for modulating the
levels of key regulatory proteins. Chains of ubiquitin
conjugated to a substrate protein specifically target it
for degradation by the 26S proteasome, a huge multisubunit protein complex found in all eukaryotic cells.
Recent reports have clarified some of the molecular
mechanisms involved in the transfer of ubiquitinated
substrates from the ubiquitination machinery to the
proteasome. This novel substrate transportation step in
the ubiquitin –proteasome pathway seems to occur
either directly or indirectly via certain substrate-recruiting proteins and appears to involve chaperones.
Covalent attachment of proteins to the small and
evolutionarily conserved protein, ubiquitin, plays an
essential role in a variety of cellular processes. These
include the degradation of bulk proteins, cell cycle control,
DNA repair, antigen presentation, vesicle transport and
the regulation of signal-transduction pathways and
transcription [1]. Ubiquitination is mediated by a cascade
of three consecutively acting enzymes called E1, E2 and E3
[2]. Several rounds of ubiquitin conjugation produce multiubiquitinated substrates carrying branched chains of
ubiquitin moieties, which are connected via isopeptide
bonds. There is a range of different E2 enzymes that can
associate with a variety of E3 enzymes, incorporating
substrate specificity to the process. To become multiubiquitinated, some proteasome substrates may also
require the action of a chain elongation factor (E4) [3].
Multi-ubiquitination is a reversible process and several
de-ubiquitinating enzymes appear to play important
regulatory roles in trimming or editing the length of the
ubiquitin chains on target proteins [4]. Within the cytosol
and nucleus of all eukaryotic cells the 2.5-MDa 26S
proteasome catalyses the degradation of multi-ubiquitinconjugated proteins in an ATP-dependent manner [5]. This
multi-subunit complex contains a proteolytic active 20S
core complex consisting of a cylindrical stack of four
heptameric rings. The inner rings harbour the catalytic
sites, which face an enclosed chamber that is accessible
only through narrow pores at either end. These pores are
Corresponding author: Colin Gordon ([email protected]).
gated by a 19S regulatory complex which is attached to one
or both ends of the 20S cylinder [6].
Tight regulation of access to the active sites of the 26S
proteasome obviously limits the hazard of uncontrolled
proteolysis within the cell. However, this regulation has a
cost in the form of the energy expended, because substrates must become unfolded before they can reach the
active sites. This substrate unfolding or anti-chaperonelike activity of the 26S proteasome is carried out by the 19S
particles bound to the ends of the 20S complex [7].
On its own, the 19S particle can be dissociated into two
subcomplexes called the base and the lid [8]. The base
subcomplex contains six ATPase subunits and is believed
to participate in the substrate-unfolding step of the
degradation pathway [9,10]. The lid subcomplex covers
the base and is thought to be involved in the recognition
and ubiquitin chain processing of substrates before their
translocation and degradation.
By performing in vitro reconstitution experiments, it
has been shown that the minimal requirements for protein
degradation is a suitable substrate, the ubiquitinating
enzymes (E1, E2 and E3) and the 26S proteasome [11].
However, recent data have demonstrated that additional
accessory proteins are needed for optimal degradation of
many substrates in vivo.
Multi-ubiquitin-binding proteins
Considering the variety of processes in which ubiquitin is
involved, it is puzzling that until recently, only a limited
number of proteins had been reported to interact specifically with multi-ubiquitin chains. However, during the
past year, several novel ubiquitin-binding proteins have
been described, and the protein domains responsible for
ubiquitin recognition have been characterized on both a
functional and a structural level [12 – 15] (discussed in
detail below).
On their own, purified proteasomes display a strong and
specific affinity for multi-ubiquitin chains, but when
assayed individually only one subunit of the 26S proteasome, called S5a in mammals [16] and Rpn10 and Pus1 in
budding [17] and fission yeast [18], respectively, binds
ubiquitin chains. The S5a/Pus1/Rpn10 subunit is most
likely localized at the base – lid interface of the 19S particle
[8,19]. The ubiquitin-binding properties of S5a/Pus1/
Rpn10 have been well described [16,20]. The interaction
site has been mapped to a small domain called the
http://tibs.trends.com 0968-0004/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(02)00002-6
TRENDS in Biochemical Sciences
(b)
Multi-ubiquitin
S5a
Ubiquitin/UBX-domain
VCP/Cdc48
Pus1/Rpn10
(c)
(d)
26S proteasome
Multi-ubiquitin
Rhp23/Rad23
Hsp70
(a)
27
Vol.28 No.1 January 2003
26S proteasome
Review
Rhp23/Rad23
Bag1L
Mud1
Bag1M
Ddi
Bag1S
SpBg1a
UBL domain
Aspartate protease homology domain
UBA domain
Ubiquitin binding domain
UIM domain
AAA ATPase domain
Bag domain
von Willebrand factor domain
Ti BS
Fig. 1. Domain organization of ubiquitin- and/or proteasome-binding proteins that might be involved in transferring multi-ubiquitinated substrates to the 26S proteasome.
described both in the fission yeast Schizosaccharomyces
pombe [13] and in budding yeast [14,15,23,24] (Fig. 2).
Both proteins bind strongly to multi-ubiquitin chains.
However, neither Rhp23 nor Dph1 is an actual subunit of
the 26S proteasome, but instead transiently associates
with the complex. This combination of abilities to bind both
ubiquitinated substrates and proteasomes indicates that
these proteins could provide an alternative pathway to
S5a/Pus1/Rpn10 in mediating substrate transfer to the
proteasome. Accordingly, neither rhp23 þ nor dph1 þ is an
essential gene in S. pombe, but the combined disruption of
the genes encoding Rhp23, Dph1 and Pus1 is a lethal event
in fission yeast and the pus1 2rhp23 2 double-null mutant
ubiquitin-interacting motif, or UIM domain (Fig. 1) [12].
This subunit, like the whole 26S proteasome, has a
preference for chains of four or more ubiquitin moieties
[21]. Obviously, these important discoveries favoured S5a
as the substrate receptor of the 26S proteasome. However,
when the corresponding genes were deleted in either
budding or fission yeast or in plants, the cells were viable
and showed only slight defects in protein degradation
[17,18,22]. Collectively this indicated that other factors
besides S5a/Pus1/Rpn10 must be involved in recognizing
the multi-ubiquitin signal and presenting substrates to
the 26S proteasome. Indeed, two such additional factors,
called Rhp23/Rad23 and Dph1/Dsk2, were recently
(a)
Ub
Ub
Ub
Ub
E3
Pus1/Rpn10
(b)
Rhp23/Rad23
S6′/Rpt5
(c)
Dph1/Dsk2
26S
proteosome
(d)
Hsp70
(e)
(f)
S6′/Rpt5
Bag1
VCP/Cdc48
Ub
Ub
Ub
Ub
Bypass
Ti BS
Fig. 2. Model of pathways regulating the transfer of substrates to the 26S proteasome. When multi-ubiquitinated (Ub chain) substrates (black thread) are transferred from
E3 enzymes to the 26S proteasome for destruction, they can take one of several possible routes. (a) S5a/Pus1/Rpn10 can directly recognize the substrate. Alternatively, substrate recognition could be mediated via the multi-ubiquitin/proteasome-binding proteins (b) Rhp23/Rad23 or (c) Dph1/Dsk2. (d) Misfolded or aggregation-prone substrates
could become ubiquitinated by E3 enzymes, such as CHIP (not shown), and delivered by Hsp70 chaperones to the proteasome via the linker protein Bag1. (e) The ringshaped ATPase complex VCP/Cdc48 might disassemble ubiquitinated proteins and deliver them to the 26S proteasome. (f) Some E3 ubiquitin–protein ligases bind the 26S
proteasome and might deliver their products directly for degradation. Once the ubiquitinated substrates are delivered to the 26S proteasome, they are then recognized by
the S60 /Rpt5 ATPase 19S subunit. Finally, a few proteins such as ornithine decarboxylase undergo rapid proteasome-dependent but ubiquitin-independent hydrolysis [64],
indicating that some proteins are recognized by the proteasome via alternative pathways (not shown).
http://tibs.trends.com
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Review
TRENDS in Biochemical Sciences
accumulates multi-ubiquitinated substrates, indicating
that together these three proteins constitute an essential
substrate recognition site of the 26S proteasome (Fig. 2)
[13].
The genes for Rhp23 and Dph1 are well conserved from
fission yeast to humans; their budding yeast orthologues
are called Rad23 and Dsk2, respectively. Unlike
S5a/Pus1/Rpn10, neither Rhp23/Rad23 nor Dph1/Dsk2
contains any UIM domains, but instead utilizes a
ubiquitin-pathway-associated (UBA) domain to interact
with ubiquitin chains (Fig. 1). However, in budding yeast,
another ubiquitin-binding protein, Ddi1, might also be
involved as it contains a proteasome-binding UBL domain
[25] (discussed in detail below). Curiously, the fission yeast
version of Ddi1 (Mud1) does not contain such a domain
(Fig. 1).
The three-dimensional structure of the UBA domain of
the human Rhp23/Rad23 orthologue has been solved and
reveals a bundle of three tightly packed helices, exposing a
hydrophobic surface patch which may constitute the
ubiquitin interaction site [26,27].
For several of the proteins containing UBA domains,
binding experiments have revealed a strong affinity for
multi-ubiquitin chains, with dissociation constants in the
nanomolar range [13] (and our unpublished results). For
example, preliminary results from our laboratory have
shown that both Ucp6 (SWISS-PROT Q10187) and Ucp7/
Swa2 (SWISS-PROT O13773) bind ubiquitin chains. UBA
domains have also been reported to possess a much weaker
affinity for free mono-ubiquitin moieties [25]. Although the
physiological significance of these weak interactions is
unclear, they might prove to play a regulatory role in
proteasomal substrate recognition.
In a recent report, the binding of a ubiquitinated
substrate to purified 26S proteasome was investigated by
chemical cross-linking experiments. These experiments
provided evidence that S60 /Rpt5, one of the ATPase
subunits of the 19S base subcomplex, interacted with the
multi-ubiquitin chains in an ATP-dependent manner [28].
However, using electrophoretic mobility shift assays, it has
recently been shown that S5a/Pus1/Rpn10 is essential for
the interaction between 26S proteasomes and ubiquitin
conjugates [29]. Thus it appears that both the S5a/Pus1/
Rpn10 and the S60 /Rpt5 subunit can mediate proteasomal
substrate recognition.
In yeast, it has been found that S5a/Pus1/Rpn10 exists
in two forms, either as a proteasome subunit or in a free
form [18,30], indicating that in addition to the role S5a/
Pus1/Rpn10 plays in proteasomal substrate recognition,
the free protein may be involved in ubiquitin-binding
events either upstream of the proteasome or in events
unrelated to protein degradation.
The role of ubiquitin chain linkages
Ubiquitin has seven lysine residues (K6, K11, K27, K29,
K33, K48 and K63), each which could potentially form an
isopeptide bond with the C-terminal glycine of the next
ubiquitin moiety in the polyubiquitin chain [2,31]. Only
linkages to K11, K29, K48 and K63 have been found in
nature and each polyubiquitin chain seems to have
linkages to the same lysine residue in each ubiquitin
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Vol.28 No.1 January 2003
moiety [2]. The most common form of ubiquitin chain is the
K48-type chain and both it and K29-linked chains signal
degradation in proteasomes [32]. By contrast, K11- and
K63-linked chains are apparently only relevant for DNA
repair and endocytosis [2].
So far, K48-linked ubiquitin chains have been used
exclusively for multi-ubiquitin interaction studies. Future
studies are therefore necessary to determine the relative
affinities of the ubiquitin-binding proteins for the various
forms of ubiquitin chains.
Proteasome-binding proteins
Both Rhp23/Rad23 and Dph1/Dsk2 associate with 26S
proteasomes through what appears to be a general
proteasome-binding module known as a ubiquitin-like
(UBL) domain [13,15,33– 35]. The structural similarities
of the UBL domain and ubiquitin fit with the observation
that like ubiquitin, the UBL domain of human Rad23
interacts with the second UIM domain of the human S5a
protein [25,34,36]. However, three observations make it
unlikely that S5a/Pus1/Rpn10 functions as the proteasome’s general UBL receptor. First, studies in budding
yeast [23] have revealed that tagged Rad23 can precipitate
proteasomes from cells from which RPN10 has been
deleted. Second, the region of the S5a protein that
interacts with human Rad23 is in the second UIM domain,
a sequence not found in the shorter forms of the S5a
orthologues of both budding and fission yeast. Finally, as
stated previously, null mutations in the budding yeast
RPN10 and fission yeast orthologue pus1 þ are viable,
unlike mutations in most other 26S proteasome genes
which encode essential genes. Consistently, both Rhp23/
Rad23 and Dph1/Dsk2 were recently shown to interact
with the essential 19S base subunit called S2/Mts4/Rpn1
[29].
Chaperones are involved
Proteasomes and chaperones represent the two main
pathways open to misfolded proteins. Although degradation and refolding have been studied in great detail
separately, little is known about the integration of the two
processes. However, recently some regulatory proteins of
the Hsp70 chaperones have been linked to the ubiquitin –
proteasome pathway [37]. One example is the Bag1
protein, which in its C-terminus contains a Bcl2-associated athanogene (BAG) domain that mediates binding to
the ATPase domain of Hsp70 [38,39] and subsequent
release of substrates from Hsp70 [40]. In higher eukaryotes, Bag1 exists in three isoforms: large, medium and
small (Fig. 1). Evolutionarily, Bag1 appears to be conserved from fission yeast to mammals, although, surprisingly, budding yeast does not appear to have a Bag1
orthologue. Interestingly, Bag1 contains a UBL domain at
its N-terminus, which mediates association with 26S
proteasomes from HeLa cells [41]. Hence, Hsp70 chaperones could associate with the proteasome via Bag1 and
deliver aggregation-prone substrates for degradation
(Fig. 2), or perhaps represent a rescue pathway for
refolding partially denatured proteasome substrates.
Another component of the Bag1/Hsp70 complex is the
CHIP protein. The CHIP protein can interact directly with
Review
TRENDS in Biochemical Sciences
both the Bag1 and Hsp70 proteins. The CHIP protein is
thought to act as an E3 ubiquitin ligase that ubiquitinates
unfolded proteins, targeting them to the 26S proteasome
for degradation [42– 44].
Recent studies from several laboratories have greatly
advanced our understanding of a hexameric ATPase
complex, called the valosin-containing protein (VCP) in
humans and Cdc48 in yeast.
In budding yeast, Cdc48 is an essential protein, which
appears to be involved in a series of seemingly unrelated
cellular processes such as homotypic fusion of endoplasmic
reticulum (ER) and Golgi membranes and ubiquitindependent cleavage and activation of the membranebound transcription factors Spt23 and Mga2 [45,46].
Association with various adaptor proteins could achieve
this functional diversity of VCP/Cdc48 so that functionally
distinct complexes are formed. Accordingly, VCP/Cdc48 in
complex with its co-factor p47 or the budding yeast
orthologue, Shp1, appears to mediate membrane fusion
[45], and association with two other co-factors, Ufd1 and
Npl4, yields a Cdc48Ufd1/Npl4 complex involved in ubiquitindependent activation of membrane-bound transcription
factors [46].
Interestingly, several studies have revealed that the
cytosolic ubiquitin –proteasome pathway mediates the
degradation of misfolded proteins from the ER secretory
pathway [47]. To accomplish this task, lumenal ER
substrates must somehow be transported back across the
ER membrane for degradation in the cytosol by the
proteasome. This pathway is called the ER-associated
protein degradation, or ERAD, pathway and appears to
require the Cdc48Ufd1/Npl4 complex [48 –50].
The Shp1 protein, which directs Cdc48 to membrane
fusion events, contains a C-terminal domain called UBX.
This domain has little sequence similarity to ubiquitin but
has nonetheless been shown to have a three-dimensional
structure very similar to that of ubiquitin [51]. The UBX
domain mediates the interaction between Shp1 and the
N-domain of Cdc48. Hence, one would expect Cdc48 to
interact with ubiquitin through a molecular mimicry
mechanism, and a direct interaction between ubiquitin
and Cdc48 has indeed been found [52]. Cdc48 co-purifies
with 26S proteasomes [53,54]. Hence, like Rhp23/Rad23
and Dhp1/Dsk2, the VCP/Cdc48 complex possesses this
twofold ability to interact with multi-ubiquitin chains and
the 26S proteasome simultaneously (Fig. 2).
Finally, an enzyme called peptide:N-glycanase (Png1),
which cleaves oligosaccharide chains from glycoproteins,
was recently found to associate with the mouse homologue
of Rhp23/Rad23, as well as with several subunits of the
26S proteasome and a likely VCP/Cdc48 co-factor protein
containing both a UBA domain and a UBX domain [55].
Hence, this enzyme might be required for deglycosylation
of misfolded glycoproteins in the ERAD pathway before
degradation.
Linking the ubiquitination machinery with the
proteasome
Clearly, if these ubiquitin- and proteasome-binding proteins are involved in transferring substrates from the
ubiquitination machinery to the 26S proteasome, one
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Vol.28 No.1 January 2003
29
might imagine them to interact with various E3 enzymes
(or E4 enzymes) to collect their cargo. However, such
interactions are likely to be transient and reports describing them are scarce. Nevertheless, evidence has been
presented that the yeast E4 ubiquitinating enzyme, Ufd2,
binds Cdc48 [3] and, as mentioned, the Hsp70 chaperone
co-factor CHIP acts as an E3 ubiquitin ligase in the
ubiquitination of various chaperone substrates [41,43,44].
Also, the human orthologues of Dph1/Dsk2 have been
reported to interact with an E3 enzyme called E6AP [35],
and it is likely that both Rhp23/Rad23 and Dph1/Dsk2
interact with a range of E3 enzymes via attached
ubiquitinated substrates.
Finally, a protein called Cic1 in budding yeast appears
to play an important role in regulating the degradation of
certain substrate proteins. It binds both 26S proteasomes
and subunits of a multi-subunit E3 complex, called the
SCF complex [56].
A growing list of ubiquitinating proteins has been found
to associate with 26S proteasomes. For example, several
E2 enzymes have been shown to associate with the
proteasome [57]. In humans, the KIAA10 E3 binds to the
proteasome subunit S2 [58], and in budding yeast the Ubr1
E3 binds subunit Rpn2 [59]. Another example is the UBLdomain-containing protein, Parkin, an E3 that is defective
in several patients with Parkinson’s disease. Finally, both
the multi-subunit E3 enzyme called the anaphase-promoting complex and the SCF complex have been found to copurify with 26S proteasomes from budding yeast [54].
However, direct association between E3 enzymes and the
26S proteasome does not necessarily make substratetransferring proteins obsolete. When bound to the substrates attached to the E3 enzymes, these proteins could
regulate the degree of substrate ubiquitination and, in
cooperation with chaperones, ensure a vectorial channelling
of substrates to the proteasome and, as such, coordinate
the link between ubiquitination and degradation.
Confusingly, Rad23 has been shown to inhibit the multiubiquitination of histone H2B in vitro. Dependent on the
presence of a UBA domain but not of the UBL domain,
Rad23 suppressed the formation of di-, tri- and multiubiquitinated, but not mono-ubiquitinated, forms of H2B
[60]. Additional in vivo experiments in which Rad23 was
overexpressed revealed reduced multi-ubiquitination and
degradation of certain proteasome substrates [60]. Collectively, this indicates that the ubiquitin-binding activity of
Rad23 might inhibit the action of E3 enzymes, whereas the
effect on protein degradation could be a result of swamping
the UBL binding site of the 26S proteasome with the
overexpressed Rad23. Contrary to this, overexpression of
Dph1/Dsk2 is toxic in both budding yeast and fission yeast
and is accompanied by a dramatic increase in the total
amount of multi-ubiquitin [14,61], indicating that rather
than increasing the flux through the proteasome, excess
Dsk2 might inhibit de-ubiquitinating enzymes. Perhaps
Dsk2 shares this function with other ubiquitin-binding
proteins.
Intriguingly, S5a has also been shown to inhibit the
degradation of ubiquitinated lysozyme and cyclin B in
vitro [62]. These data suggest that in excess, these proteins
might specifically interfere with multi-ubiquitination and
30
Review
TRENDS in Biochemical Sciences
conjugate recognition by the 26S proteasome. Perhaps
Rhp23/Rad23 (and less so Dph1/Dsk2) have a feedback
effect on the E3 enzymes and need to transfer the multiubiquitinated substrate protein to the 26S proteasome to
relieve the E3 enzymes of product inhibition.
There is no evidence as yet to indicate that the interaction
of Rhp23/Rad23, Dph1/Dsk2 and S5a/Pus1/Rpn10 with
multi-ubiquitin chains is regulated. However, gel-filtration
studies have revealed that Rad23 exists primarily as a
homodimer [63]. Although the functional significance of
this interaction is unknown, one could speculate that the
dimerization of the UBA/UBL-domain-containing proteins
could be involved in regulating interaction with the 26S
proteasome or ubiquitin chains. In this simple model the
UBL proteasome-interacting domain is hidden and is only
exposed after the dimer is bound to a multi-ubiquitinated
substrate, allowing interaction with the 26S proteasome.
Concluding remarks
From the evidence presented in this review we propose
that there are many different ways to transport substrates
to the 26S proteasome for destruction (see Fig. 2). We
envisage that all these different mechanisms for substrate
transport act upstream of S5a/Pus1/Rpn10 and the newly
identified intrinsic 26S proteasome receptor encoded by
the S60 /Rpt5 19S base subunit. Further investigations into
the ubiquitin-binding proteins and their cooperation are
needed before we can fully understand the molecular
mechanisms that regulate the transfer of ubiquitinated
substrates to the 26S proteasome.
Acknowledgements
We thank Klavs B. Hendil and Nick Hastie for helpful
discussions and comments on the manuscript and apologize
to those authors whose work we were not able to cite due to
space constraints. R.H.-P. is funded by the Lundbeck
Foundation, M.S. by the Deutsche Forschungsgemeinschaft and C.G. by the Medical Research Council.
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