(Received for publication, June 5, 1995; and in revised form, July 31, 1995)
From the
The 26 S protease is a multisubunit enzyme required for ubiquitin-dependent proteolysis. Recently, we identified a 50-kDa subunit (S5) of this enzyme that binds ubiquitin polymers (Deveraux, Q., Ustrell, V., Pickart, C., and Rechsteiner, M. (1994) J. Biol. Chem. 269, 7059-7061). We have now isolated, sequenced, and expressed a cDNA encoding a novel 50-kDa subunit of the 26 S protease. The recombinant protein does not bind ubiquitin polymers. Two-dimensional electrophoresis reveals that two subunits of the 26 S protease have apparent molecular masses of 50 kDa. Antibodies specific for the recombinant protein recognize the more basic of the two subunits (S5b), whereas the more acidic subunit (S5a) binds ubiquitin chains. Thus, the 26 S protease contains at least two distinct subunits with apparent molecular masses of 50 kDa.
Ubiquitin has been implicated in diverse cellular phenomena
including proteolysis of key regulatory molecules such as GCN4 and some
cyclins(1, 2, 3, 4, 5) .
Although ubiquitin may have nonproteolytic roles, covalent attachment
of ubiquitin to proteins clearly produces substrates for the 26 S
ATP-dependent protease. This enzyme is composed of the multicatalytic
protease or proteasome and a regulatory ATPase complex(6) .
Both multicatalytic protease and the regulatory complex are
multisubunit structures that associate in the presence of ATP to form
the 26 S enzyme(7) . Although the multicatalytic protease can
degrade a variety of peptides, few intact proteins have been shown to
be substrates. Subunits of the regulatory complex are, therefore,
thought to confer the ability to recognize and degrade intact proteins,
especially those conjugated to ubiquitin chains. In support of this
idea, we identified a 50-kDa subunit of the regulatory complex that
binds ubiquitinated lysozyme as well as free polymers of
ubiquitin(8) . We called this protein subunit 5 (S5) based upon
its relative mobility on SDS-polyacrylamide gels(9) . S5
efficiently binds tetrameric ubiquitin and selects for longer ubiquitin
polymers(8) . This property is consistent with characteristics
expected of a component that selects ubiquitin conjugates for
proteolysis since it has been shown that -galactosidase molecules
attached to long ubiquitin chains are degraded more efficiently than
those conjugated to shorter chains(10) . As a further
characterization of the ubiquitin-binding subunit, we have employed
standard procedures to obtain a cDNA encoding the 50-kDa subunit of the
26 S protease. We have found that two distinct proteins comprise the
band previously designated as S5 on SDS gels. Here we distinguish
between the two 50-kDa proteins and describe the cloning, sequence, and
expression of a cDNA encoding the more basic subunit, S5b.
Subunit 5 was identified as a ubiquitin recognition component of the 26 S protease by its ability to bind ubiquitin-lysozyme conjugates (8) . Subsequent analyses using a mixture of ubiquitin polymers of different lengths revealed that S5 selects for longer ubiquitin polymers even after SDS denaturation of the subunit(8) . In addition, our calculations of subunit stoichiometry suggest that only one S5 molecule is present per regulatory complex. In view of these findings, we hypothesize that repeated sequences in S5 might bind multiple sites present in ubiquitin chains. This idea can be tested by obtaining the subunit's sequence. Consequently, we used a combination of polymerase chain reaction, cDNA library screening, and 5`-RACE to obtain a composite full-length nucleotide sequence encoding the S5 subunit (see ``Experimental Procedures''). This sequence contains an open reading frame that encodes all three peptides recovered from CNBr digestion of S5 (Fig. 1). Both initiation and polyadenylation sequences present in the S5 cDNA conform to those known for eukaryotic organisms. The predicted protein, however, does not have the repeated regions that we hypothesized would be present in the ubiquitin conjugate-binding subunit. It does have numerous leucines, many of which occur as dileucine repeats. Comparison of the S5 amino acid sequence with entries in current protein databanks did not reveal significant similarities with other proteins. However, the nucleotide sequence is identical to a partial cDNA obtained by direct sequencing of random cDNAs (see ``Experimental Procedures''). Using the 5`-RACE procedure, we identified four more nucleotides at the 5` end of the S5 cDNA that encode a methionine. The resulting open reading frame predicts a 56-kDa protein.
Figure 1: Nucleotide and deduced amino acid sequences for subunit 5b of the human red blood cell 26 S protease. The predicted amino acids are shown in single-letter code with the encoded stop codon denoted with an asterisk. Sequences of CNBr-derived peptides are shown white on black. The dileucine repeats and flanking residues are boxed.
Expression of the S5 sequence in E. coli resulted in a protein that co-migrates on SDS gels with S5 of the 26 S protease (Fig. 2). Western blots show that antibodies raised against the expressed protein react with a 50-kDa subunit of the protease. In addition, antibodies specific for the human red blood cell 26 S protease recognized the recombinant S5 protein (data not shown). Taken together, these data indicate that the cDNA presented in Fig. 1encodes the entire S5 protein.
Figure 2:
Expression of S5b in E. coli. The
S5b cDNA was subcloned into the pET 16b or pAED4 vector, transformed
into BL21 cells, and induced for expression by
isopropyl-1-thio--galactopyranoside (see ``Experimental
Procedures''). Proteins were separated by SDS-polyacrylamide gel
electrophoresis and stained with Coomassie Brilliant Blue (leftpanel) or transferred to nitrocellulose and incubated
with S5b antibodies (rightpanel). Shown are the
subunits of the regulatory complex (RC), an insoluble fraction
from cells expressing S5b (I), and purified S5b expressed as a
histidine fusion protein (P). S5 of the 26 S protease is
denoted by an arrow. The migration of molecular weight markers
is shown to the left. The induced protein present in the
insoluble fraction was expressed as a nonfusion product. This protein
was excised and subjected to sequence analysis confirming the S5b
amino-terminal sequence. The purified His
-S5b fusion
protein was used for antibody production. Two faster migrating species
in the 26 S cross-react with anti-S5b antibodies. These may be
fragments of S5b or possibly subunits 9 and 11 as indicated by
two-dimensional gel analysis (see Fig. 3).
Figure 3:
Western blot analysis of 26 S subunits
separated on two-dimensional SDS-polyacrylamide gels. 26 S protease
subunits were separated by two-dimensional gel electrophoresis as
described under ``Experimental Procedures.'' The separated
proteins were stained with Coomassie Brilliant Blue (panelA) or transferred to nitrocellulose and incubated with I-labeled ubiquitin polymers (panelB)
or antibodies raised against S5b (panelC). Arrows denote S5a (white) and S5b (black)
subunits. For panelsB and C, the position
of each subunit was determined by Ponceau S staining prior to
incubations with
I-labeled ubiquitin chains or anti-S5b
antibodies, respectively. Regulatory complexes were loaded in the far
left lane of each second dimension gel shown and resolved based upon
molecular weight only. The migration of molecular weight markers is
pictured to the left of panelA. In panelC, the immune-reactive protein migrating faster
than S5b and just to the right of pI 6.0 appears to be subunit
9.
The expressed protein,
however, does not bind ubiquitin polymers (data not shown). This
finding raised the possibility that more than one 26 S protease subunit
comprises the band designated as S5 on SDS gels. This was tested by
resolving components of the 26 S protease on two-dimensional gels.
Proteins were transferred to nitrocellulose and incubated with either I-labeled ubiquitin polymers or antibodies specific for
the recombinant S5 protein. As shown in Fig. 3, the labeled
ubiquitin polymers bound to a 50-kDa subunit with a pI of 4.6 (panelB, whitearrow), and the
anti-S5 antibodies recognized a protein focusing at pH 5.3 (panelC, blackarrow). These results
demonstrate that there are two distinct subunits of the 26 S protease
in the band previously called S5. We have termed the two 50 kDa
proteins S5a (acidic) and S5b (basic). The cDNA sequence shown in Fig. 1codes for S5b.
The three CNBr peptides initially
sequenced from the S5 band are present in the cDNA encoding S5b.
However, the data in Fig. 3clearly reveal two distinct 50-kDa
proteins at the S5 position. For this reason, we separated large
amounts of the regulatory complex subunits on SDS-gels and asked
whether peptides other than those present in S5b could be identified.
By excising proteins that migrated slightly above the major S5 band, we
obtained the sequences of eight endoproteinase Lys-C digested peptides,
three of which are not encoded by S5b. These three peptides
(RIIAFVGSPVELDTK, VNVDIINFGEEEVNTE, and AGTGSHLVTVPPG) are homologous
to a recently identified multiubiquitin-binding protein from Arabidopsis thaliana. ()The identification of
peptides encoded by two distinct cDNAs confirms the presence of two
50-kDa subunits in the 26 S protease.
When 26 S protease and
regulatory complexes were electrophoresed on native gels and
transferred to nitrocellulose, antibodies raised against S5b recognized
both native particles. Similarly, these antibodies identified a 50-kDa
protein after subunits of each native complex were resolved by SDS-PAGE
(data not shown). Therefore, S5b is an integral component of the 26 S
protease. The function of subunit 5b, however, is not currently
understood. Its sequence is enriched in leucine residues; 26 of the
first 100 amino-terminal residues are leucines. In addition, 9
dileucine repeats can be found throughout S5b (see Table 1).
Similar repeats have been implicated in protein sorting to Golgi
cisternae, lysosomes, and in the internalization of certain
transmembrane proteins(15) . Haft and co-workers (15) suggested that at least two motifs contribute in
internalization and/or targeting to lysosomes. The first motif,
referred to as the tyrosine-based motif (GPLY and NPEY), contains an
essential aromatic residue, usually a tyrosine. The S5b sequence also
contains a similar tyrosine-based motif (NPNY, residues 476-479).
The second motif, the dileucine repeat, has been implicated in
trafficking of a variety of transmembrane proteins including
degradation of the insulin receptor. Residues flanking dileucine
repeats are often charged (15) , and interestingly, the
residues surrounding the dileucine pairs in S5b are also highly
enriched in charged and polar side chains (Table 1). Conceivably,
the presence of dileucine motifs in S5b localizes some 26 S protease
complexes to membranes where they may function to down-regulate
receptors or other transmembrane proteins. A transmembrane protein
could be degraded through two separate pathways, the cytoplasmic domain
by the 26 S protease and the extracellular domain within lysosomes. In
fact, ubiquitin has been implicated in the negative regulation of the
platelet-derived growth factor -receptor, possibly by promoting
the degradation of the ligand-activated receptor (16) . In
addition, the apparent role of the 26 S protease in antigen
presentation (17) might require its direct association with the
endoplasmic reticulum. Peptides generated by the protease would then be
immediately available for transport across the endoplasmic reticulum
membrane.
In summary, we have isolated a cDNA that encodes a 50-kDa subunit of the 26 S protease. Although we cannot currently assign a function to S5b, the availability of both antibodies to S5b and a histidine-tagged version of the recombinant protein should facilitate future characterization of this 26 S protease component.