(Received for publication, September 18, 1995; and in revised form, October 20, 1995)
From the
A variety of protease inhibitors have been used to study ubiquitin-dependent proteolysis by the 26 S protease. However, these inhibitors lack complete specificity and thus affect ubiquitin-independent pathways as well. We recently identified an Arabidopsis protein, MBP1, that is homologous to subunit 5a (S5a) of the human 26 S protease complex. MBP1 and S5a bind multiubiquitin chains with high affinity and presumably facilitate the recognition of ubiquitin conjugates by the 26 S protease. We show here that free MBP1 can be a potent inhibitor of ubiquitin-dependent proteolysis in several cell-free systems. When added to reticulocyte lysates or to Xenopus egg extracts, the plant protein effectively blocked the degradation of multiubiquitinated lysozyme and cyclin B, respectively. MBP1 did not enhance the removal of ubiquitin from lysozyme or affect the ability of the 26 S complex to hydrolyze fluorogenic peptides. These data suggest that the plant protein specifically interferes with the recognition of ubiquitin conjugates by the 26 S protease. Thus MBP1, human S5a, and their homologs should prove to be valuable reagents for investigating cellular events mediated by ubiquitin-dependent proteolysis.
A major proteolytic pathway in eukaryotes involves covalent attachment of ubiquitin to protein substrates. Although attachment of ubiquitin monomers may reversibly affect the structure or function of a protein (e.g. histone H2A), in most cases conjugation of multiubiquitin chains to a protein promotes its rapid degradation (1, 2, 3, 4, 5, 6) . For example, in Xenopus egg extracts, cyclin B accumulates during interphase and is degraded within a few minutes at metaphase(7, 8) . Attachment of ubiquitin chains to cyclin B is a prerequisite for its rapid destruction(9, 10, 11) .
The 26 S protease
is the only enzyme identified so far that is able to degrade
ubiquitinated proteins in an ATP-dependent reaction (12) . This
large, multisubunit enzyme is composed of a regulatory complex and the
multicatalytic protease or 20 S proteasome. The regulatory complex is
comprised of 15 or more different subunits (13) whereas the
multicatalytic protease has at least 14 unique subunits (14) .
We previously identified a 50-kDa subunit of the regulatory complex
that binds ubiquitin-lysozyme conjugates and free multiubiquitin
chains(15) . In an effort to isolate a cDNA encoding this
protein we discovered that two subunits of the regulatory complex have
apparent molecular masses of 50 kDa(16) . Two-dimensional
polyacrylamide gel electrophoresis (PAGE) ()revealed that
the more acidic of these subunits (S5a) binds multiubiquitin chains
whereas the function of the more basic subunit (S5b) is currently
unknown(16) .
In addition to binding free multiubiquitin
chains, S5a exhibits markedly increased affinity for longer
chains(15) . These properties are consistent with the
preference of the 26 S protease for substrates modified with long
multiubiquitin chains(17) . Further evidence that S5a selects
ubiquitin conjugates for proteolysis comes from binding studies using
chains synthesized from mutant ubiquitins. Binding of these chains to
S5a correlates with their ability to support ubiquitin-mediated
proteolysis. ()
We recently isolated an Arabidopsis
thaliana cDNA that encodes a multiubiquitin binding protein (MBP1)
homologous to subunit 5a of the 26 S protease. ()Two-dimensional PAGE of the recombinant MBP1 demonstrated
that it has a molecular mass and pI virtually identical to S5a from the
human 26 S protease. Like S5a, MBP1 binds free multiubiquitin chains
and has a preference for longer chains. Because MBP1 has a high
affinity for multiubiquitin chains and large quantities of recombinant
protein can be expressed in Escherichia coli, we examined the
effects of adding it to in vitro systems competent for
ubiquitin-mediated proteolysis. Here we show that the Arabidopsis MBP1 protein can be used as a specific inhibitor of
ubiquitin-mediated proteolysis.
Figure 1:
MBP1
inhibits ubiquitin-lysozyme conjugate degradation in reticulocyte
lysate. Purified high molecular weight
ubiquitin-I-lysozyme conjugates, 1 mM ATP, and
MBP1 were incubated in 100 µl of a reticulocyte lysate at 37
°C. Aliquots were removed at the indicated times, and proteolysis
was measured by quantitation of soluble radioactivity following acid
precipitation. A shows the effects of adding 5 or 50 µg of
histidine-tagged MBP1 to the degradation assay. In B,
conjugate degradation rates were measured in the presence of 5 µg
of MBP1 and several histidine-tagged proteins expressed and purified
similar to MBP1. A non-histidine-tagged version of MBP1 gave similar
results in these assays. TCA, trichloroacetic acid; HIV, human immunodeficiency virus; BP, binding
protein.
Figure 2:
Stabilization of high molecular weight
ubiquitinated I-lysozyme by MBP1. A, SDS-PAGE
and autoradiography of samples withdrawn from the degradation assays
shown in Fig. 1. In A the four lanes at the left are samples removed from the assay lacking MBP1 and the four lanes at the right are from a degradation assay
containing 5 µg of MBP1. The radiolabeled molecules that migrate to
the bottom of the gel are
I-lysozyme and its degradation
products. Each 15-min sample contains approximately 20% less total
radioactivity due to pipetting error. B, graph of the high
molecular weight (>60,000) ubiquitin-
I-lysozyme
conjugates (arrowhead in A) as a function of time.
The relative amounts of conjugates with molecular weights greater than
60,000 were quantitated by phosphorimage
analysis.
Figure 3:
MBP1 inhibition of cyclin B degradation in Xenopus egg extracts. Electrically activated Xenopus extract was prepared as described (7) and treated with 100
µg/ml cycloheximide. Cyclin 90 protein was added to 100
µg/ml and incubated for 40 min at 23 °C to induce mitosis.
Either histidine-tagged MBP1 or buffer alone (50 mM Tris, 50
mM NaCl, pH 7.5) was added to the
90 arrested extract and
incubated for 10 min. The degradation assay was initiated by adding
S-labeled cyclin, and at designated times aliquots were
removed, quenched in SDS sample buffer, and subjected to SDS-PAGE. Gels
were fixed (40% methanol, 10% acetic acid), dried, and exposed to Kodak
XAR film for 24 days at -80 °C.
Figure 4:
Effect of MBP1 on hydrolysis of a
fluorogenic peptide by the 26 S protease. Partially purified 26 S
protease from HeLa cells was incubated at 37 °C with
ubiquitin-I-lysozyme conjugates (left) or
sLLVY-MCA (right) in the presence or absence of 1 mM ATP and 50 µg/ml MBP1. The degradation of
ubiquitin-
I-lysozyme conjugates was measured by
quantitation of soluble radioactivity following acid precipitation.
Hydrolysis of sLLVY-MCA was measured by fluorescence spectroscopy. MCA
fluorescence observed in the presence of ATP and the absence of MBP1 is
defined as 100% hydrolysis. TCA, trichloroacetic
acid.
Mutations in ubiquitin pathway enzymes and 26 S protease subunits have been used to investigate ubiquitin-mediated proteolysis in mammals, yeasts, and plants(3, 6, 24) . For example, mutations in yeast 26 S protease subunits and ubiquitin-conjugating enzymes (E2s) have provided valuable information on natural substrates of the ubiquitin pathway and its involvement in cell cycle regulation(25, 26, 27, 28, 29, 30, 31, 32) . Similar genetic approaches in higher eukaryotic cells have been less successful. Mammalian cells containing a thermolabile ubiquitin-activating enzyme (E1) have been used to provide evidence for the involvement of ubiquitin-mediated proteolysis in a variety of cellular processes including autophagy, antigen presentation, and p53 degradation(33, 34, 35, 36) . However, these temperature-sensitive mammalian cell lines continue to transfer ubiquitin, albeit slowly, to cellular proteins at the restrictive temperature(37) . In addition, high molecular weight ubiquitin conjugates accumulate at elevated temperatures, a result not readily explained by the inactivation of E1(37) . Therefore, considerable caution must be used in interpreting results from mammalian cell lines containing temperature-sensitive E1s(37, 38) .
Chemical inhibitors have also been used to investigate the ubiquitin
pathway. Early experiments by Haas and Rose (39) demonstrated
that hemin inhibits the degradation, but not the formation, of
ubiquitin conjugates. This finding provided the foundation for
identification of the 26 S protease(19) . It was subsequently
found that hemin inhibits proteolysis by the multicatalytic protease or
20 S proteasome(20) , the proteolytic core of the 26 S
protease, and affects other aspects of the ubiquitin pathway, including
conjugation and disassembly(40) . Thus, the oxidized heme
derivative cannot be used to selectively inhibit ubiquitin-mediated
proteolysis by the 26 S protease. The same is true for peptide
aldehydes, compounds recently used to implicate the proteasome pathway
in the processing of antigens(41, 42) ,
NFB(43) , and the cyclin-dependent kinase inhibitor
p27(44) . However, peptide aldehydes inhibit a wide variety of
cysteine and serine proteases(45, 46) , making them
unsuitable as strict diagnostic reagents for the 26 S protease or the
20 S proteasome. More recently, it has been reported that amyloid
protein inhibits ubiquitin-dependent proteolysis in
vitro(47) . Because the amyloid protein inhibited cleavage
of fluorogenic peptides by the 20 S proteasome as well, it is doubtful
that amyloid
protein will permit clear distinction between
ubiquitin-dependent and ubiquitin-independent proteolysis by the 26 S
enzyme.
Here, we have shown that recombinant MBP1, human S5a, and
their homologs may be ideal reagents for specifically inhibiting
ubiquitin-mediated proteolysis. MBP1 inhibited degradation of
ubiquitin-lysozyme conjugates and cyclin ( Fig. 1and Fig. 3) but had little effect on the ATP-dependent hydrolysis of
fluorogenic peptides (Fig. 4). Although substantial levels of
MBP1 were required to significantly inhibit degradation of cyclin B,
this is expected if inhibition requires that the added MBP1 molecules
bind and sequester multiubiquitin chains attached to protein
substrates. For example, the steady state concentration of
multiubiquitin chains in mammalian cells is approximately 5-10
µM as estimated from immunological assays (48) and
microinjection experiments(49) . Therefore, it is not
surprising that 8 µM MBP1 was needed to block cyclin B
degradation in Xenopus egg extract. Moreover, previous
estimates of total multiubiquitin chains in our preparation of I-ubiquitin-lysozyme conjugates (15) allow us to
calculate that in the experiment shown in Fig. 4conjugate
degradation was eliminated by adding one MBP1 molecule per ubiquitin
tetramer. This ratio also supports the idea that sequestration of
multiubiquitin chains is the mechanism by which MBP1 inhibits conjugate
degradation.
The ability of MBP1 to reduce markedly cyclin degradation in Xenopus egg extracts is encouraging for those who would use it as an inhibitor. Crushed Xenopus eggs yield a very concentrated extract that supports a variety of complicated biological processes such as mitosis and nuclear envelope reconstitution. Inhibition of cyclin degradation in Xenopus egg extract makes it likely that ectopic expression of MBP1, human S5a, and their homologs will provide a useful method for inhibiting ubiquitin-dependent proteolysis within living cells.