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(Received for publication, September 1,
1995; and in revised form, November 15, 1995) From the
Cytochrome b
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
A plasmid (pCyb2)
encoding a variant of cytochrome b
Wild-type cytochrome b
Figure 1:
Sensitivity of cytochrome b
Figure 2:
Steady-state levels of
cytochrome-b
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 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
Figure 4:
Thermal denaturation of
cytochrome-b
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 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
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2589-2593
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
-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.
Strains, Plasmids, and Mutagenesis
E. coli strain X90 is ara 
(lac pro) gyrA
argE(Am) Rifthi-1/F` lacI
lacpro
; E. coli strain KS1000 is X90
tsp(prc)3::kan eda-51::Tn10(8) . Plasmid pKK101 is a
pBluescript-derived vector that encodes ampicillin resistance and the
Tsp-His
protease under control of a lac-promoter(9) . Plasmid pRW-1 (a gift from Michael
Hecht) is a pEMBL-18 derived plasmid that encodes ampicillin resistance
and the cytochrome-b gene under transcriptional
control of a lac promoter(11) .
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.
Protein Purification
Wild-type Tsp with a
six-histidine tag was purified from E. coli strain
KS1000/pKK101 using nickel-chelate chromatography and ion-exchange
chromatography as described(9) . Cytochrome b was purified from E. coli strain KS1000/pRW-1 using a
protocol adapted from Ames et al.(12) . Cells were
grown at 37 °C with gentle shaking for 12 h in 1 liter of 2X-YT
broth supplemented with 150 µg/ml ampicillin and 1 mM isopropyl-1-thio-
-D-galactopyranoside and were
harvested by centrifugation. The cell pellet was resuspended in 10 ml
of chloroform, incubated at room temperature for 15 min, and 100 ml of
10 mM Tris-HCl (pH 8.0) was added. This mixture was
centrifuged for 20 min at 4,000 rpm, and the red aqueous supernatant
containing cytochrome b and other periplasmic
proteins was recovered. 10 ml of a 0.5 M potassium citrate (pH
4.0) buffer was added, and the pH was brought to 4.0 by addition of 1 M HCl. After stirring for 30 min at 4 °C, precipitated
proteins were removed by centrifugation at 10,000 rpm for 30 min. The
supernatant was loaded onto a Mono S column (Pharmacia Biotech Inc.)
equilibrated in 25 mM potassium citrate (pH 4.0), and the
column was developed with a linear gradient from 0 to 300 mM KCl in the same buffer. Fractions containing cytochrome b
, which eluted between 75 and 100 mM KCl, were pooled, concentrated, and chromatographed on a Superose
12 column (Pharmacia) in buffer containing 50 mM Tris-HCl (pH
8.0), 0.1 mM EDTA. Fractions that contained cytochrome b
at greater than 95% purity, as assayed by
SDS-polyacrylamide gel electrophoresis, were pooled. Variant
cytochrome-b
proteins were purified in the same
manner as the wild type.
Expression Assays
The steady-state levels of
cytochrome-b variants in E. coli strain
X90 (which has a chromosomal copy of the tsp gene) or strain
KS1000 (which is deleted for the tsp gene) were determined
following 12 h growth at 37 °C after inoculating 50 µl of an
overnight culture into 5 ml of 2X-YT broth supplemented with 150
µg/ml ampicillin, 1 mM isopropyl-1-thio-
-D-galactopyranoside, and 10
µg/ml FeCl
. Cells were harvested by centrifugation, and
the cell pellets were frozen in an ethanol/dry ice bath for 15 min and
thawed in a 4 °C water bath for 15 min. This freeze-thaw protocol
releases cytochrome b(11) . After 3
freeze-thaw cycles, the cell pellets were resuspended in 1 ml of water
and incubated on ice for 30 min, and the cells were removed by
centrifugation at 4 °C. The supernatants were recovered, and the
absorbance spectra from 220 to 450 nm were recorded. Assays from three
independent cell cultures were performed for each
cytochrome-b
variant, and the absorbance values
at 426 nm (the absorbance maximum for cytochrome b
with a reduced heme group) were averaged.
Pulse-Chase Assays
The half-lives of
cytochrome-b variants in vivo were
determined by growing cells to mid-log phase in M9 minimal medium
containing no Cys or Met and inducing protein expression by addition of
1 mM isopropyl-1-thio-
-D-galactopyranoside. 20
min after induction, a labeling pulse of 100 µCi of
[S]methionine was added, and 30 s later a chase
of unlabeled L-methionine was added to a final concentration
of 1.4 mg/ml. At different times, 0.5-ml aliquots were removed,
immediately frozen in an ethanol-dry ice bath, and lysed by 3 cycles of
thawing at 4 °C followed by refreezing. Samples were
electrophoresed on SDS-polyacrylamide gels, and the radiolabeled bands
were visualized by phosphorimaging. Half-lives were determined by
fitting the pulse-chase data to an exponential decay. For these
calculations, the intensities of the induced bands were integrated
using the phosphorimager ImageQuaNT software and were normalized to the
intensities of a set of stable bands.
Cleavage Assays
The cleavage of
cytochrome-b variants by Tsp in vitro was monitored by the change in heme absorbance, which accompanies
its dissociation from the cleaved protein. For this assay, 2.5
µM of the purified cytochrome-b
variant was incubated at 22 °C with 0.3 µM Tsp-His
in 50 mM Tris-HCl (pH 8.0), 20 mM NaCl, and the loss of absorbance at 418 nm was monitored (418 nm
is the absorbance maximum for the protein with an oxidized heme group;
oxidation of the heme in the purified protein occurs spontaneously but
does not alter the structure or stability of cytochrome b). As a control, aliquots were removed from the
cuvette at various time points and assayed for cleavage of cytochrome b
by SDS gel electrophoresis. As expected, loss
of absorbance at 418 nm was found to correlate with loss of the intact
cytochrome-b
band.
Stability of Cytochrome-b
The circular dichroism spectra of
cytochrome-b Variants
variants were measured using 25
µg/ml protein in buffer containing 50 mM Tris-HCl (pH
8.0), 0.1 mM EDTA. Thermal melts were performed using the same
protein concentration and buffer composition by monitoring the
ellipticity at 222 nm as a function of temperature. Melting curves were
fit to a two-state transition between native and denatured protein by
non-linear least squares fitting using the program NONLIN for
Macintosh(13, 14) .
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).
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.
Steady-state Expression Levels Depend on the Identity of the
C-terminal Residues
Each of the three C-terminal residues of the
cytochrome-b-WVAAA substrate was individually
randomized, and from 16 to 20 different amino acids were recovered at
each position. The resulting variants were assayed for steady-state
expression levels in tsp
and tsp
cells as shown in Fig. 2. In this
figure, open bars represent expression levels in the absence
of Tsp, and closed bars represent expression levels in the
presence of Tsp. In the cells containing Tsp, there are significant
differences in expression levels depending on the chemical identity of
the three C-terminal residues. These differences are discussed in
greater detail below. In the cells lacking Tsp, all of the variants are
expressed at similar levels. This suggests that Tsp is the major
periplasmic protease responsible for C-terminal-specific protein
degradation of the cytochrome-b
substrates.
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.
cells. This suggests that position 2 is less important than
position 1 in determining resistance to Tsp cleavage.
Comparison of Cleavage Rates in Vivo and in
Vitro
We purified cytochrome-b variants
with at least one stabilizing and one destabilizing residue at each of
the three C-terminal positions and assayed rates of cleavage by Tsp in vitro (Table 1). Tsp rapidly cleaved variants with
the AAA, AAV, AYA, and LAA tails but did not cleave or only slowly
cleaved variants with the AAK, AGA, and QAA tails. Pulse-chase
experiments were used to determine half-lives for these same variants
in the cell (Fig. 3). As expected, variants that had low
steady-state expression levels (AAA, AYA, LAA) had the shortest
half-lives, and variants that had high steady-state expression levels
(AAK, AGA) had considerably longer half-lives (Fig. 2, Table 1). As shown in Table 1, the resistance of the
cytochrome-b
variants to cleavage by Tsp in
vitro correlates reasonably well (R
0.9)
with the half-lives of the variants in a tsp strain in vivo. Thus, these data support the model that
differential degradation of these proteins by Tsp in vivo is
responsible for the observed differences in their half-lives and
steady-state levels.
variants. Uninduced controls are
shown in the lanes marked U. Arrow indicates
the position of cytochrome-b
variants.
Effects of Tail Sequence on Protein Stability
In
principle, the different C-terminal sequences of the
cytochrome-b variants might affect
susceptibility to Tsp by reducing the thermodynamic stability of the
protein. To test for this possibility, the stabilities of the purified
cytochrome-b
variants were measured by
temperature denaturation monitored by CD spectroscopy (Fig. 4).
The melting curve of each variant showed a T
between 68 and 69 °C, indicating that the C-terminal
mutations do not affect the global stability 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.
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. 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.
))
-We thank Michael Hecht for the gift of pRW-1
and Cliff Robinson for helpful advice.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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