(Received for publication, September 1, 1995; and in revised form, November 15, 1995)
From the
Cytochrome b is not cleaved by the
tail-specific protease Tsp in vitro or in the periplasm of Escherichia coli but becomes a good substrate when the
C-terminal sequence WVAAA is added. Following randomization of the
final three residue positions of this substrate, 54 different mutants
with single residue substitutions were recovered. The steady-state
expression levels of cytochrome variants bearing these mutant tails
were similar in an E. coli strain deleted for the tsp gene but differed markedly in a strain containing Tsp. Wild-type
cytochrome b
and seven variants, displaying a
range of intracellular expression levels, were purified. These proteins
were found to have the same T
values in
thermal denaturation experiments but to be cleaved by Tsp at rates
differing by as much as 30-fold. Overall, the rates of Tsp cleavage of
these proteins in vitro correlate with their rates of cleavage in vivo as determined by pulse-chase experiments. These
results indicate that the C-terminal sequence of the
cytochrome-b
variants is important in
determining their proteolytic fate in the cell and show that this
degradation is mediated predominantly by Tsp. There are different
selectivity rules at each of the three C-terminal positions. The
identity of the C-terminal residue of the substrate, where small,
uncharged residues (Ala, Cys, Ser, Thr, Val) are preferred, is most
important in determining the rate of substrate cleavage by Tsp.
Non-polar residues are also preferred at the second and third
positions, but larger and more hydrophobic side chains are also
acceptable at these positions in good substrates.
The observed selectivity of intracellular proteolysis implies the existence of mechanisms that allow proteases to discriminate between correct and incorrect protein substrates(1, 2) . Some substrate recognition may be possible at the level of exposure of appropriate cleavage sites. For example, a protease might cleave almost any unfolded or partially folded protein in which peptide bonds flanked by the proper P1 and P1` residues are accessible. This type of mechanism may serve to rid the cell of misfolded or unfolded proteins but is unlikely to allow significant selectivity unless the local sequence determinants of cleavage site selection occur only rarely. Some protein substrates may be marked for degradation by covalent modification with molecular tags, which then serve as recognition determinants in subsequent steps. The ubiquitin-proteasome system of eukaryotic cells is currently the best understood example of this type, although the determinants that cause particular proteins to be modified by ubiquitin are still not well understood(3) . In bacteria, there are systems in which the identity of sequences at the C-terminal or N-terminal ends of proteins appear to serve as determinants of proteolytic degradation (4, 5, 6) . In these cases, such sequences may serve as secondary binding sites, which allow a protease to tether a substrate while waiting for rare unfolding events that expose the sites of primary cleavage.
Specific degradation of proteins with
non-polar C-terminal sequences was first reported for several
cytoplasmic proteins in Escherichia
coli(4, 5) , but the protease that mediates this
degradation has not been identified. Tsp (tail-specific protease) is a
periplasmic protease of E. coli that was purified based on its
ability to differentially degrade two protein substrates that differed
only in their C-terminal residues(7) . The protein substrate
cleaved by Tsp had a relatively apolar C-terminal sequence (WVAAA),
while the protein resistant to Tsp cleavage had a relatively polar
C-terminal sequence (RSEYE). Although this specificity in vitro is similar to that observed in vivo for cytoplasmic
degradation(5) , gene knockout experiments have shown that Tsp
is not involved in cytoplasmic degradation(8) . Experiments in vitro have established that Tsp is an endoprotease that
cleaves substrates at discrete sites throughout the polypeptide chain
in a reaction that depends upon the identity of the substrate's
C-terminal sequence and requires the presence of a free -carboxyl
group(9) . The precise determinants that allow some C-terminal
sequences but not others to be recognized by Tsp are currently unknown.
Moreover, it has not yet been established that C-terminal-specific
degradation of substrates by Tsp occurs in the cell. In this paper, we
address these issues by studying the susceptibility of
cytochrome-b
variants to Tsp-mediated
proteolysis in vivo and in vitro. Cytochrome b
is a periplasmic protein that can be readily
detected in cell lysates because binding of the protein to heme gives
rise to a characteristic red absorbance spectrum(10) . We show
that wild-type cytochrome b
is resistant to
Tsp-mediated cleavage but becomes a good substrate when a WVAAA
C-terminal tail is added. To investigate sequence preferences at the C
terminus, we constructed libraries of cytochrome b
-WVAAA, with each of the last three tail
positions randomized individually, and assayed for steady-state levels
of the modified variants in cells containing Tsp or deleted for Tsp.
These results reveal different preferences at each of the three
C-terminal positions and show that Tsp is the major periplasmic
protease responsible for C-terminal-specific degradation of these
substrates.
A plasmid (pCyb2)
encoding a variant of cytochrome b with the
C-terminal tail sequence WVAAA was constructed by ligating the PstI-BamHI backbone fragment from pRW-1 (the PstI site is near the 3`-end of the
cytochrome-b
gene; the BamHI site is
roughly 150 base pairs downstream) with a double-stranded
oligonucleotide cassette encoding the 3`-end of the gene, codons for
the WVAAA sequence, and the wild-type stop codon and termination
sequences. The structure of pCyb2 was confirmed by restriction mapping
and DNA sequencing. To randomize the C-terminal codons of the
cytochrome-b
-WVAAA gene, the pCyb2 construction
was repeated using an oligonucleotide cassette containing an equimolar
mixture of G, A, T, and C at the appropriate codon. These libraries
were transformed into X90 cells, single colonies were isolated, and the
C-terminal sequences of genes from 60-75 independent candidates
were determined by DNA sequencing.
Wild-type cytochrome b is not cleaved
by Tsp in vitro (Fig. 1A) and is expressed at
comparable levels in tsp
and tsp
cells as determined by heme absorbance (Fig. 1B). Neither finding is surprising. The Tsp
protease is thought to prefer non-polar tails that are accessible in
the folded protein(7, 9) , whereas cytochrome b
has a very polar C-terminal sequence (HQKYR),
which is relatively inaccessible in the crystal
structure(15, 16) . In an attempt to make cytochrome b
a substrate for Tsp, we constructed a gene
with the cytochrome coding sequence followed by codons for the
C-terminal pentapeptide WVAAA. This sequence was chosen because
purified Tsp is known to cleave variants of the N-terminal domain of
repressor and of Arc repressor, which have the WVAAA tail in
vitro(7) . (
)As shown in Fig. 1A, the purified
cytochrome-b
-WVAAA protein is also cleaved by
Tsp in vitro. The cytochrome-b
-WVAAA
protein is expressed at a much lower steady-state level in cells
containing Tsp than in cells lacking Tsp (Fig. 1B), and
pulse-chase experiments show that the reduced steady-state level is
caused by increased intracellular degradation, which is Tsp dependent (Table 1).
Figure 1:
Sensitivity of cytochrome b and a variant bearing the WVAAA C-terminal
tail to Tsp degradation in vitro and in vivo. A, cleavage of 2.5 µM of purified cytochrome b
or cytochrome b
-WVAAA
by 0.3 µM Tsp in vitro monitored by loss of heme
absorbance at 418 nm. B, steady-state levels of cytochrome b
and cytochrome b
-WVAAA
in periplasmic fractions prepared from tsp
cells (X90) or tsp
cells (KS1000)
determined by absorbance spectra.
Figure 2:
Steady-state levels of
cytochrome-b variants in the tsp
strain X90 (filled bars) and the
otherwise isogenic tsp
strain KS1000 (open bars). The error bars indicate the standard
deviation from the mean for three different X90
cultures.
Cytochrome variants with Ala, Cys, Val, Ser, and Thr at the C terminus (position 1) are expressed at the lowest steady-state levels in cells containing Tsp (Fig. 2), indicating that Tsp prefers substrates that have small, uncharged side chains at this position. Variants with polar side chains, large side chains, Pro, or Gly at the C terminus are expressed at reasonably high levels even in the presence of Tsp. In many cases, the expression levels of these variants are as high as in cells deleted for Tsp (Fig. 2). This suggests that the identity of the C-terminal residue is extremely important in determining whether a protein will be a good or poor substrate for Tsp.
At the penultimate amino acid residue (position 2), variants with
Ala, Tyr, Ile, and Trp are expressed at the lowest levels in the
presence of Tsp, and variants with Arg, Lys, and Gly are expressed at
the highest levels. In general, hydrophobic residues at position 2
appear to be preferred by Tsp relative to hydrophilic residues.
Moreover, only a few side chains at this position increase expression
to levels comparable to those seen in tsp cells. This suggests that position 2 is less important than
position 1 in determining resistance to Tsp cleavage.
At the third position from the C terminus, Tsp prefers Ala, Leu, Val, and Ile. The least preferable side chains are Asn, Gln, and Met. It is somewhat surprising that Leu and Met, which are often considered to be conservative substitutions for each other, have such different effects at this position. No side chains at position 3 increase steady-state expression to levels observed in the absence of Tsp, suggesting that this position is less important than either position 1 or 2 in determining resistance to Tsp cleavage.
Figure 3:
Pulse-chase assays of the QAA and AAA
cytochrome-b variants. Uninduced controls are
shown in the lanes marked U. Arrow indicates
the position of cytochrome-b
variants.
Figure 4:
Thermal denaturation of
cytochrome-b variants. The stability of variants
was determined by monitoring CD ellipticity as a function of
temperature. The fraction of folded protein was determined by fitting
the thermal denaturation curves to a two-state transition between
native and denatured protein. Different variants are indicated by their
three C-terminal residues.
The work presented here has established the importance of specific amino acids at each of the three C-terminal residues in determining whether a protein is efficiently cleaved by Tsp in vitro and in vivo. In previous studies based on screening of a small number of potential protein and peptide substrates, we had concluded that Tsp appeared to recognize substrates with non-polar or hydrophobic C-terminal residues and not to recognize substrates with polar C-terminal residues(7, 9) . The data summarized in Fig. 2reveal that this view is an over-simplification. While no good substrates have highly polar tails and most good substrates do have non-polar residues at the three C-terminal positions, there is considerable fine specificity. For example, small non-polar residues are preferred relative to larger hydrophobic side chains at the C-terminal position. At the other two positions, there is no simple correlation between the size of non-polar side chains and effects on Tsp cleavage. For example, alanine and tyrosine are the most destabilizing side chains at the penultimate residue, while valine has a significantly smaller effect at this position.
How do the
C-terminal residues of a substrate affect its degradation by Tsp?
Unlike systems in which sequence signals act to target substrates to
subcellular compartments specialized for degradation(17) , the
C-terminal sequences that mediate degradation by Tsp appear to be
recognized by the protease itself. This is shown most clearly by the
strong correlation between the half-lives of the
cytochrome-b variants in vivo and the
resistance of these purified variants to cleavage by purified Tsp in vitro. Since other macromolecules are not required for
Tsp-mediated degradation of substrates with non-polar tails, then Tsp
must either recognize these C-terminal sequences directly or recognize
indirect effects of these sequences on stability or structure. Several
experiments suggest that Tsp recognizes the tail sequences directly. As
shown here, C-terminal sequences that make cytochrome b
a good substrate for Tsp do not alter the
thermodynamic stability of the protein. The same is true of C-terminal
sequences that make Arc repressor and the N-terminal domain of
repressor good substrates for Tsp cleavage in
vitro(7, 9) . Moreover, it seems unlikely that
destabilizing C-terminal tails act indirectly by allowing other
sequence or structural determinants to be recognized because the same
tail sequence (e.g. WVAAA) can make Arc repressor,
repressor, and cytochrome b
(proteins that
differ in primary, secondary, tertiary, and quaternary structure) good
substrates for Tsp cleavage. We believe that Tsp directly recognizes
protein and peptide substrates by using a binding site that requires a
free
-carboxyl group and side chains of the appropriate size,
shape, and hydrophobicity at the last three residue positions of the
substrate. Such a binding site would serve to tether the substrate to
the enzyme, and binding of an appropriate C-terminal tail at this site
might also function to activate the enzyme.
Our studies have shown
that Tsp can efficiently degrade a periplasmic protein with a WVAAA
C-terminal tail. An independent protease in the cytoplasm of E.
coli is also capable of rapidly degrading proteins with WVAAA
tails(5, 8) . Why does E. coli use the Tsp
system in the periplasm and an independent system in the cytoplasm to
degrade proteins with certain C-terminal sequences? It seems unlikely
that this is a general mechanism for removing unfolded or misfolded
proteins from the cell. First, most damaged or misfolded proteins would
not be expected to have the proper C-terminal sequence to allow
degradation by a C-terminal-specific pathway. Second, Tsp and its
cytoplasmic counterpart are not limited to degrading unfolded proteins.
Both the cytochrome-b variants studied here and
the
-repressor variants used to study cytoplasmic
C-terminal-specific degradation are stably folded(5) . Recent
studies suggest that one function of tail-specific proteases is to work
in conjunction with a peptide-tagging system that marks certain
proteins in E. coli for degradation (18) . (
)In this system, proteins translated from damaged mRNA are
modified by C-terminal addition of a peptide with the sequence
AANDENYALAA. The C-terminal residues of this peptide tag then render
the tagged protein susceptible to degradation by Tsp or by its
cytoplasmic counterpart.