From the
The yeast aspartic protease, proteinase A, has a 54 amino-acid
propeptide, which is removed during activation of the zymogen in the
vacuole. Apart from being involved inhibition/activation, the
propeptide has been shown to be essential for formation of a stable
active enzyme (van den Hazel, H. B., Kielland-Brandt, M. C., and
Winther, J. R. (1993) J. Biol. Chem. 268, 18002-18007).
We have investigated the sequence requirements for function of the
propeptide. The N-terminal half and the C-terminal half of the
propeptide were replaced by random sequences at the genetic level, and
collections of the mutants were subjected to a colony screen for ones
exhibiting activity. A high frequency (around 1%) of active constructs
was found, which indicates a very high tolerance for mutations in the
propeptide. Thirty-nine functional mutant forms containing random
sequence at either the N- or C-terminal half of the propeptide were
characterized. Comparison of the propeptides of the active constructs
suggests that a particular lysine residue is important for efficient
biosynthesis of proteinase A.
Secretory enzymes often undergo extensive post-translational
modifications before the mature form is generated. In many cases, these
include proteolytic removal of parts of the polypeptide as well as
glycosylation. Upon translocation of secretory proteins into the
endoplasmic reticulum (ER),
Proteinase A (PrA) of
Saccharomyces cerevisiae is a vacuolar aspartic protease,
which is synthesized as a precursor of 405 amino acids encoded by
PEP4 (Fig. 1)
(1, 2) . PrA follows the
paradigm outlined above; thus, in the ER, a signal sequence of
presumably 22 amino acids is cleaved off, carbohydrate chains are added
at two positions, and the zymogen (proPrA) folds into a
transport-competent conformation
(3) . The zymogen transits
further through the Golgi complex where the carbohydrate side chains
are modified, resulting in an increase in molecular mass of about 1
kDa. Upon arrival in the vacuole, an N-terminal propeptide of 54 amino
acids is autocatalytically removed, yielding mature PrA of 42 kDa
(4) . We have previously found that a propeptide-lacking form of
PrA (PrA-23
In order to introduce BamHI
and SmaI restriction sites, two annealed oligonucleotides, 5`
AAA GGA TCC CCC GGG CCC GCC 3` and 5` GGC GGG CCC GGG GGA TCC TTT 3`,
were subcloned into pBVH11, which had been opened with NaeI,
generating pBVH37. A SmaI- PvuII fragment from Yep24
(18) containing the tetracycline-resistance gene was subcloned
into pBVH37, which had been opened with SmaI, generating
pBVH38. A PCR was performed with pBVH17
(5) as template and the
oligonucleotides 5` ATT CGC GTC GAC CTC GAG AAG TAC TTG ACT CAA TTT GAG
3` and 5` GCA GCA GGT ACC CCG CAT CAT CGG GCT ACC CGC 3` as primers.
The 1.4-kilobase PCR product was isolated, digested with SalI
and Asp718 and subcloned into pBVH38, which had been
opened with SalI and Asp718 generating pBVH48. This
plasmid thus contains a pep4 allele in which the codons for
residues 24-52 have been replaced by a BamHI site, part
of the tetracycline-resistance gene, and a XhoI site.
Sequencing of the pep4 open reading frame of pBVH48 showed
that no unintentional mutation had been introduced.
A collection of
plasmids encoding mutant PrA propeptides in which the
propeptide's C-terminal half was replaced by random sequences
(``C-half- in- trans'' collection) was
constructed using a mixture of oligonucleotides. This mixture was
generated using two mixtures of bases: mixture ``N,''
composed of 25% of each base, and mixture ``L,'' composed of
4% A, 32% T, 32% C, 32% G. The oligonucleotides had the following
sequence: 5` G TCG ACA AGC TTA GAT ATC NNL NNL NNL NNL NNL NNL NNL
NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL CTC
GAG GGT ACC GGC 3` in which N indicates a base from mixture N,
and L indicateds a base from mixture L. The NNL combination was chosen to reduce the frequency of STOP codons. The
oligonucleotides in the mixture were made double-stranded using the
oligonucleotide 5` GCC GGT ACC CTC GAG 3` and Klenow polymerase,
digested with HindIII and XhoI and subcloned into
pBVH36, which had been opened with HindIII and XhoI.
Plasmid DNA isolated from pools of individual transformants from the
C-half- in- trans collection was digested with
SacI and EcoRV and subcloned into pBVH11, which had
been opened with SacI and NaeI. This generated new
pools of plasmids (C-half in cis collection), encoding mutant
precursors in which the C-terminal half of the propeptide was replaced
by random sequences. Plasmids numbered pBVH1201 to pBVH1336 are from
this collection. A mixture of oligonucleotides with the sequence: 5`
GGT ACC CTC GAG NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL
NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL NNL GGA TCC GCC GGC
GGG 3`, in which N and L are bases from the same base
mixtures as above, was generated. The oligonucleotides were made
double-stranded using the oligonucleotide 5` CCC GCC GGC GGA TCC 3` and
Klenow polymerase, digested with BamHI and XhoI and
subcloned into pBVH48, which had been opened with BamHI and
XhoI. This generated a collection of plasmids encoding mutant
PrA forms in which the N-terminal half of the propeptide was replaced
by random sequences (``N-half in cis'' collection).
A SacI- HindIII fragment of pBVH17, containing the
upstream part of PEP4, was subcloned into pSELECT
(19) , which had been opened with SacI and
HindIII, generating pBVH41. Plasmids pBVH42, pBVH43 and pBVH44
were generated by site-directed mutagenesis on pBVH41 using the
following oligonucleotides: O1, 5` GCT CAT TTA GGC CAA GCG TAC TTG ACT
CAA TT 3`; O2, 5` CAA CAT TTA GCT CAT TTA CTC GAT AAG TAC TTG ACT CAA
TTT 3`; and O3, 5` TTT AGG CCA AAG GTA CTT GAC TC 3`, respectively. The
site-directed mutagenesis was performed as described previously
(19) , with the modifications described by Olesen and
Kielland-Brandt
(20) . SacI- HindIII fragments
from pBVH42, pBVH43, and pBVH44 were subcloned into pBVH17, which had
been opened with SacI and HindIII, generating pBVH45,
pBVH47 and pBVH46, respectively. The part of pep4 in these
plasmids that was derived from the mutagenesis procedure was sequenced.
This verified the mutations and showed that no other mutation had been
introduced. A PCR was performed with pBVH47 as template and the M13
forward 24-mer primer and the oligonucleotide 5` AAT CTA GAT ATC GAA
GAA AGG ATG CTC CCT 3` as primers. The PCR product was isolated,
digested with SacI and EcoRV, and ligated into
pBVH11, which had been opened with SacI and NaeI,
generating pBVH63. Another PCR with pBVH47 as template, and the
oligonucleotides 5` CAA GTT GCT GCA AAA GGA TCC AAG GCT AAA ATT TAT AAA
3` and 5` GAC CAC CTT ATC AAC AGA 3` as primers was performed. The
product of this PCR was isolated, digested with BamHI and
XhoI and ligated into pBVH48, which had been opened with
BamHI and XhoI, generating pBVH64. The part of
pep4 in pBVH63 and pBVH64 that was derived from the PCR
product was sequenced. This showed that no unintentional mutations had
been introduced.
Pilot transformations of W3122 with a few plasmid
pools of the N-half in cis collection showed that the
frequency of CPY-positive transformants was substantially lower than
had been found for the C-half in cis collection. To be able to
find the most active mutants in this collection, we transformed the
In
order to determine the frequency of functional propeptides more
precisely, it was important to find out how many of the plasmids in the
collections indeed produced a full-length PrA form, i.e. did
not contain a stop codon or other mutations in the open reading frame
preventing production of PrA. Sequencing of some randomly chosen
constructs indicated that the DNA synthesizer had generated some
oligonucleotides in which one or more positions in the sequence had
been skipped. Thus, many of the plasmids from the two in cis collections (about 50-60%) did not produce PrA
because of frameshift mutations. Taking this into consideration, we
conclude that about 1.6% (98/(0.45
Pulse-chase experiments were performed to investigate the fate of
the propeptide-mutated PrA forms. The half-time of maturation of
wild-type proPrA is about 6 min
(3) , thus, immediately after a
pulse of 15 min, some of the labeled molecules are already in the
mature vacuolar form, while the others still are in the precursor form
(Fig. 4, lane 6). Upon chase, all labeled
molecules are converted into the mature form (Fig. 4, lane 7). A minor band of higher mobility is seen in all
immunoprecipitations of PrA; this band corresponds to a subpopulation
of the PrA molecules that has only received N-linked
glycosylation at one position
(28) . Some of the active mutants
isolated from the N-half in cis collection were analyzed in
this way. The mutant precursors were detected immediately after the
pulse (Fig. 4), but after a 60-min chase, they were all degraded (data
not shown). Thus, the small fraction of active PrA molecules in these
strains could not be detected in the pulse-chase experiment. Three
mutant precursors that contained an extra glycosylation site (the ones
encoded from pBVH1671, pBVH1672, and pBVH1676) showed a slower mobility
than the precursors produced from pBVH1635 and pBVH1641 and the
wild-type precursor, indicating that additional glycosylation of these
precursors had indeed taken place (Fig. 4, lanes 1-6). The most active mutants isolated from the
C-half in cis collection showed conversion of a subpopulation
of the mutant precursors to processed forms with a half-time of
maturation of more than 90 min, while the rest of the precursors were
degraded (Fig. 5). Five inactive mutants of the C-half in cis collection were similarly investigated, and the mutant precursors
were all, with varying rates, degraded upon chase (data not shown). It
should be noted that there is no linear correlation between the PrA
activity detected in the assay and the half-time of maturation of the
precursor. The activity assays were performed on cultures in stationary
phase, so most PrA molecules have been synthesized several hours before
the sample is taken. Thus, for instance, a 2-fold reduction in the
half-time of maturation from 6-12 min, would probably not give
any decrease in the level of activity detected.
In the study of in vivo protein folding,
considerable progress has been made in the identification and
characterization of auxiliary proteins, such as chaperones
(29, 30) . The role of cis-acting protein
sequences in folding is often more difficult to assess, as mutations
often affect the final folded structure. Propeptides are special
cis-acting sequences, by not being part of the mature protein,
and have in several cases been shown to promote folding of their
cognate enzymes
(6) . The present report describes an
investigation of the sequence requirements of a propeptide for its
function. The propeptide of PrA presumably covers the active site in
the zymogen, thus inhibiting the enzyme activity. Propeptide mutations
in PrA often cause degradation or a reduced rate of intracellular
transport of the zymogen, proposedly due to folding defects
(3, 5) . Here we show that precursors with propeptide
mutations retain the ER-specific molecular mass longer than the
wild-type precursor, suggesting slow ER to Golgi transport. The ER is
believed to possess a quality control system that retains proteins
until folding has been completed
(31) .
PrA is a member of
the family of aspartic proteases. These enzymes share substantial
homology, and their tertiary structures are very similar. Pepsinogen,
the zymogen of the aspartic protease pepsin, activates
autocatalytically in a low pH-dependent mechanism
(32) . In
vivo evidence indicates that proPrA activates autocatalytically
upon arrival in the vacuole
(4) . This process is not strictly
dependent on low pH, as it can occur, albeit more slowly, in yeast
mutants that have a vacuolar pH identical to that of the cytoplasm
(28) , suggesting that the activation mechanism, and thus the
nature of the interaction between the propeptide and the enzyme region
in PrA, may be quite different from that of pepsinogen. Also, no
homology in primary sequence is found between the two propeptides
(1) . While studying vacuolar targeting of PrA, Klionsky et
al. (3) constructed three small deletions in charged areas
of the propeptide, each removing 5 residues. These deletions caused no
reduction or only a limited reduction in the rate of intracellular
transport. We have previously found that the propeptide can function
without being covalently linked to the enzyme region ( in
trans). Progressive C-terminal truncations of the propeptide
showed that although the efficiency of folding was gradually reduced,
even a propeptide lacking the last 13 residues was still partially
functional
(5) . Furthermore, an autoactivation-deficient PrA
precursor can function in trans, raising the interesting
possibility that a transient interaction between the propeptide and the
enzyme region can be productive, in analogy with in vitro interactions seen for CPY
(33) . In the folding of
Since removal of several parts of the
propeptide had no effect or only a moderate effect on precursor
maturation
(3) , we chose to investigate the sequence
requirements of the propeptide using the more radical approach
described here, taking advantage of the suitability of yeast for
genetic screens of colonies. For both halves of the propeptide, many
functional sequences were found among random ones, indicating that the
propeptide has a very high tolerance for mutations. The frequency of
active constructs was higher among the C-terminal-half mutants than
among the N-terminal-half ones, even though a more stringent screening
procedure was used for the first group. On the other hand, the
mutations flanking the random sequences that were present in all
N-terminal-half constructs affected the maturation rate more strongly
than the flanking mutations in the C-terminal-half constructs.
Moreover, in the C-terminal-half mutated constructs, only 22 residues
were replaced by random sequences, while 25 residues were replaced in
the N-terminal-half mutant constructs. A higher number of active
mutants was found at lower growth temperature. Lower temperatures
probably increase the yield of folding by reducing aggregation.
Mutants containing N-linked glycosylation of the propeptide
were found among the active constructs of the N-terminal-half
collection. We cannot exclude the possibility that a small fraction of
the precursor molecules was not glycosylated in the propeptide and that
these molecules were the ones that exhibited activity. However, we find
this is unlikely as, at least in the case of the precursors encoded by
pBVH1671 and pBVH1672, the fraction of underglycosylated molecules was
not higher than in the wild-type (Fig. 4). Furthermore, if only a
putative small fraction of nonglycosylated propeptides in these mutants
was functional, these would be much more efficient in formation of
active enzyme than all of the isolated active constructs from this
collection that had no additional sites for N-linked
glycosylation. Thus, we think it is likely that some of the precursors
containing a glycosylated mutant propeptide can form an active enzyme.
The phenotypes of the mutants indicate that there is no element in
the propeptide strictly required for the formation of an active enzyme
but rather that various parts of the propeptide contribute to this
function in a somewhat additive way. However, 18 of the 20 active
constructs isolated from the C-half in cis collection
had a lysine or an arginine at position 53, and a biased distribution
was also observed at position 54, suggesting that these residues are
particularly important for correct folding. The wild-type precursor
also has a lysine at position 53 (although these positions could be
considered not to be entirely equivalent, as the propeptides of the
mutant precursors are 1 residue shorter than the wild-type). The view
that this lysine is important is supported by the observation that
Lys-53
The PrA propeptide can direct the normally secreted enzyme invertase
to the yeast vacuole, indicating that it contains information for
sorting to this compartment
(3) . The only mutant strains
described here that showed PrA activity in the medium were the ones
that had high intracellular activity. Thus, the extracellular
activities correlated with the intracellular ones, and there was no
differential degree of vacuolar sorting of the various mutant
precursors. We fused a number of our mutant PrA prosequences to the
coding sequence for invertase. No general difference in targeting
efficiency was found between propeptide-invertase fusions that were
derived from active mutant PrA constructs and ones that were derived
from inactive constructs, indicating that our screen had not selected
for activity and vacuolar sorting at the same time. These and other
results suggest that the sorting information in the PrA propeptide is
not essential for vacuolar sorting of proPrA.
All or almost all mutant precursors could form an active
enzyme in the presence of a wild-type propeptide, suggesting that few
or none of the mutant propeptides prevent productive interaction of the
enzyme region with a wild-type propeptide. Furthermore, the enzyme
activity was not inhibited in these mutants. Perhaps the mutant
propeptides are removed in spite of the drastic mutations in the
processing region. This would mean that the specificity of the
autocatalytic removal of the propeptide is low. Alternatively, mutant
precursors can be active without removal of the propeptide.
The
approach that we have undertaken has given general information about
the sequence requirements of the propeptide for function. A similar
strategy has been used by Kaiser et al. (37) to
determine the limits of variation permitted for signal sequences to
function in translocation into the ER. The high frequency of active
mutants found in this screen suggests that it is feasible to find among
random sequences some that can promote folding of other proteins that
require a propeptide for folding. Propeptide mutations do not, as a
rule, influence the final structure of their cognate enzyme. This makes
propeptides interesting targets for mutagenesis. Thus, for instance, in
production of a heterologous proenzyme in yeast, one may replace parts
of the propeptide by random sequences and be able to find some that can
improve folding efficiency.
We thank Anette Bruun for excellent technical
assistance. We also thank Bent Foltmann for critically reading the
manuscript, Cayo Ramos and BjHolst for helpful discussions,
Kirsten Lilja and Morten Meldal for synthesis of the PrA substrate, Eva
Gertman for preparation of oligonucleotides, and Ann-Sofi Steinholtz
for preparation of photographs.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
presequences are
removed. Many secretory enzymes contain additional propeptide sequences
that are not present in the mature protein. The propeptides can be
removed in various compartments of the secretory pathway, and various
functions have been attributed to propeptides, such as inhibition of
enzyme activity, folding, and sorting.
76) is completely degraded after entry into the ER. We
hypothesized that the degradation is due to misfolding and thus that
the propeptide is required for correct folding of the enzyme region
(5) .
Figure 1:
Schematic representation of the
PEP4 gene product. The prepeptide ( open bar), the propeptide ( gray bar), and
the enzyme region of PrA ( black arrow) are shown.
Lollipop- shaped symbols indicate
N-linked glycosylations. The sequence of the propeptide (amino
acids 23-76) is shown.
The role of propeptides in folding of proteases has
recently been studied in detail for several serine proteases (for
review, see Ref. 6). Few reports, however, have dealt with the sequence
requirements in the propeptides for this process
(7, 8) . Several observations suggested that there was
no strong requirement for sequence conservation in the PrA propeptide.
This prompted us to study these requirements using a more radical
approach. Large parts of the propeptide-encoding segment of PEP4 were replaced by random nucleotide sequences, and collections of
the mutants were screened for ones that exhibited activity in
vivo. Many mutants exhibiting activity were found, indicating that
many sequences can functionally replace the PrA propeptide.
Strains, Media, and Materials
Saccharomyces
cerevisiae strains W3094 ( MAT
a ura3-52
leu2-3, 112 his3-200 pep4-1137) and W3122
( MAT
a ura3-52 leu2-3, 112 his3-
200
pep4-1137 prb1::LEU2) have been described previously
(4) . Strain JHRY20-2C-
pep4 ( MAT
a ura3-52 leu2-3, 112
his3-
200 pep4::LEU2) (kindly supplied by J. H. Rothman and T.
H. Stevens, University of Oregon) was constructed by introduction of
the LEU2 gene in the HindIII restriction site of the
PEP4 gene in JHRY20-2C
(1) . A truncated form of
proPrA, containing the entire propeptide is produced in
JHRY20-2C-
pep4. All yeast strains used were
isogenic. Escherichia coli strain DH5
(9) was
used for plasmid propagation. E. coli strains JM109
(10) and BMH71-18 mutS
(11, 12) were used
in site-directed mutagenesis for preparation of single-stranded DNA and
as repair-deficient strain, respectively. Yeast was grown in YPD and SC
media
(13) . Low sulfate MV/Pro medium
(14) was used in
pulse-labeling/immunoprecipitation experiments. E. coli was
grown in LB, SOC, and 2
YT medium
(9) . Restriction
endonucleases, T4 DNA polymerase, T4 polynucleotide kinase, and T4 DNA
ligase were from Promega, Madison, WI. Klenow polymerase and
thermolysin were from Boehringer Mannheim.
[
S]Methionine for protein labeling was from
DuPont NEN. Zymolyase 100-T was from Seikagaku Kogyo, Tokyo, Japan.
Fixed Staphylococcus aureus cells were IgGsorb from The Enzyme
Center, Malden, MA. Oligonucleotides were synthesized on an Applied
Biosystems 380A DNA synthesizer, desalted on NAP-5 columns (Pharmacia
Biotech Inc.) equilibrated with 10 m
M Tris-HCl, 1 m
M EDTA, pH 8.0, and used as primers in PCR and sequencing
experiments. PCR was performed using Pfu DNA polymerase from
Stratagene, La Jolla, CA, or Taq DNA polymerase from Perkin
Elmer Corp.
Plasmid Constructions, Mutagenesis, and
Sequencing
DNA subcloning steps and transformations of E.
coli and yeast were carried out using standard procedures
(9, 15) . Sequencing was performed using custom
synthesized primers, a Taq Dye Deoxy Terminator cycle
sequencing kit, and an API 373A DNA Sequencer, both from Applied
Biosystems, Foster City, CA. Plasmid pBVH32 was constructed by
subcloning the pep4-23
76-containing
SacI- XhoI fragment from pBVH11
(5) into
pRS315
(16) , which had been opened with SacI and
XhoI. In order to introduce a SacI restriction site,
two annealed oligonucleotides, 5` GGG CCC GAG CTC CAT ATG A 3` and 5`
AGC TTC ATA TGG AGC TCG GGC CC 3`, were ligated into pSEY8
(17) , which had been opened with SmaI and
HindIII, generating pBVH30. A PCR was performed using pBVH9
(5) as template and the M13 forward 24-mer primer (Biolabs,
Beverly, MA) and the oligonucleotide 5` CGG CCG AAG CTT TAT GCC GGC CTC
GAG TAA ATG AGC TAA ATG TTG CTC 3` as primers. The 0.6-kilobase PCR
product was isolated, digested with SacI and HindIII,
and ligated into pBVH30, which had been opened with SacI and
HindIII, generating pBVH36. This plasmid thus contains a
pep4 allele, which is truncated after the codon for residue
50. This codon is followed by a XhoI site, 9 base pairs as a
spacer fragment, and a HindIII site. Sequencing of the
truncated pep4 allele of pBVH36 showed that no unintentional
mutation had been introduced.
Enzyme Assays and Pulse-Chase
Experiments
Carboxypeptidase Y (CPY) activity was determined
qualitatively by plate overlay with the substrate
N-acetyl-
DL-phenylalanyl--naphthylester as
described by Jones
(21) . PrA activity was determined using an
internally quenched fluorescent peptide substrate essentially as
described previously
(5) . In the present experiments, however,
the better substrate
2-aminobenzamide-Leu-Phe-Ala-Leu-Glu-Val-Ala-Tyr(3-NO
)-Asp,
kindly provided by Kirsten Lilja and Morten Meldal, was used. Pulse
labeling, immunoprecipitation, and gel electrophoresis were carried out
essentially as described previously
(22) using previously
described antibodies against PrA
(4) and CPY
(23) .
[
S]methionine (100 µCi) was used for
labeling instead of
[
S]H
SO
, and cultures
were chased by the addition of 5 µl of 1
M Na
SO
and 5 µl of 5 mg/ml methionine.
Propeptides Randomized in the C-terminal Half in
Trans
We constructed a collection of plasmids containing mutant
PrA prosequences in which 22 codons encoding the C-terminal half of the
propeptide were replaced with random DNA of the same length and tested
whether the mutant propeptides could function in trans. A
multicopy plasmid (pBVH36) was constructed that contained the first 50
codons of PEP4 (the presequence and the first 28 codons of the
prosequence) behind its natural promoter. The 50codon was
followed by a XhoI restriction site and, further downstream
after a spacer fragment, a HindIII restriction site. A mixture
of oligonucleotides containing random codons was generated. The
oligonucleotides in the mixture were made double-stranded, digested
with XhoI and HindIII, and inserted into pBVH36. A
collection of 17,000 independent E. coli transformants
(C-half- in- trans collection) was obtained. The
correct constructs encode mutant PrA propeptides in which residue 50 is
followed by Leu-Glu (encoded by the XhoI restriction site),
followed by 22 random residues, followed by Asp-Ile (encoded by the
EcoRV restriction site). Two thousand transformants were
collected from plates into pools of 300-350 each, plasmid DNA was
isolated from each pool and introduced into a yeast strain (W3094)
already producing PrA-23
76. About 1000 yeast transformants were
obtained from each pool, and it was tested whether they exhibited CPY
activity. PrA is required for the activation of a number of vacuolar
hydrolases, including CPY. The plate assay used for CPY activity is a
very sensitive assay for the presence of PrA activity, as strains
having as little as 2-3% of the wild-type intracellular PrA
activity are detected as CPY-positive
(5, 21) . None of
the transformants exhibited CPY activity, indicating that none of the
mutant propeptides could efficiently assist formation of an active
enzyme in trans.
Randomized Propeptides in Cis
The interaction
between the propeptide and the enzyme region in trans is not
very efficient, and even when the wild-type propeptide is overproduced
relative to the enzyme region, less than 10% of the wild-type activity
is found
(5) . Thus, the observation that none of the randomized
propeptides could interact productively with the enzyme region when
supplied in trans did not exclude that they could do so when
covalently linked to the N terminus of the enzyme region ( in
cis). To test this, we divided 15,000 E. coli transformants from the C-half- in- trans collection over 20 independent pools, isolated plasmid DNA, and
inserted the prosequence-containing SacI- EcoRV
fragments into a plasmid (pBVH11), which contained the sequence coding
for the enzyme region of PrA (Fig. 2). Twenty new pools of
transformants were obtained, each pool containing 150-600
independent E. coli transformants, corresponding to a total of
6500 (C-half in cis collection). Each desired plasmid encodes
a PrA precursor form in which residues 51-76 have been replaced
by Leu-Glu (encoded by the XhoI restriction site), followed by
22 random codons, followed by Asp (half of the EcoRV
restriction site). The mutant precursors are thus one residue shorter
than the wild-type. Furthermore, 25 codons encoding the N-terminal half
of the PrA propeptide were replaced with random DNA of the same length
to investigate the sequence requirements of this part of the propeptide
(Fig. 3). A collection of 6500 individual transformants (N-half
in cis collection) was obtained. Each desired plasmid encodes
a PrA precursor form in which residues 24-52 have been replaced
by Gly-Ser, followed by 25 random codons, followed by Leu Glu. The
transformants were divided over 59 pools, and plasmid DNA was isolated
from each pool.
Figure 2:
Propeptide sequences of plasmids isolated
from a collection in which the C-terminal half of the PEP4 prosequence was replaced by random sequences. PrA activities in
strains producing the mutant precursors. Only the mutated part of the
prosequence (residues 51-76) is shown. The top 20 plasmids
( above the dashed line) were sampled from
CPY-positive transformants of a pep4 strain. The sequence
between the dashed lines is the
corresponding wild-type sequence. Position 53 is indicated in
boldface. The propeptide sequences of 10 plasmids that could
not complement the CPY-negative phenotype of the
pep4 strain are shown below the dashed line.
Strain W3094 was transformed with the indicated plasmids, and specific
PrA activity was determined in cell extracts derived from two or three
individual transformants grown at 30 °C. The value shown is the
average activity as a percentage of the average activity detected in
extracts from strain W3094 carrying pBVH17, which produces wild-type
PrA. The indicated variation is the calculated standard deviation. The
background activity detected in a
pep4 strain (about 2%)
has been subtracted from the values. NA indicates that PrA
activity was not directly measured. The black arrow indicates the enzyme region, the open bars indicate presequences, shaded bars indicate part
of the prosequence that is wild-type, while other bars indicate
randomized regions.
Figure 3:
Propeptide-sequences of plasmids isolated
from a collection of plasmids in which the N-terminal half of the
PEP4 prosequence was replaced by random sequences. PrA
activities in strains producing the mutant precursors. Only residues 22
to 52 are shown. The top 19 plasmids ( above the dashed line) were sampled from CPY-positive transformants of a
pep4 strain. The sequence between the dashed lines is the corresponding wild-type sequence. The
propeptide sequences of six plasmids that could not complement the
CPY-negative phenotype of the
pep4 strain are shown
below the dashed line. Strain W3094 was
transformed with the indicated plasmids, and specific PrA activity was
determined in cell extracts derived from two or three individual
transformants grown at 23 °C. The value shown is the average
activity as a percentage of the average activity detected in extracts
from strain W3094 carrying pBVH17, which produces wild-type PrA. The
indicated variation is the calculated standard deviation. The
background activity detected in a
pep4 strain (about 2%)
has been subtracted from the values. The black arrow indicates the enzyme region, open bars indicate
the presequence, shaded bars indicate part of the
prosequence that is wild-type, while other bars indicate randomized
regions.
The plasmids from the C-half in cis collection were introduced into yeast to test whether any of the
mutant precursors could form active enzyme. A pep4
prb1 double mutant (W3122) was chosen as genetic background for the
screen. The vacuolar protease precursors in yeast are proteolytically
activated via a cascade with several redundancies. Initiation of the
cascade is dependent on PrA, and PrA can activate the CPY precursor
(proCPY) and the precursor of the PRB1-encoded proteinase B
(proPrB). However, PrB can also activate proPrB and proCPY and by this
way amplify the initiation of the cascade by PrA
(4, 24, 25) . Thus, in a prb1 strain,
the threshold level of PrA activity necessary for a CPY-positive
phenotype is higher than in a wild-type PRB1 strain. A
prb1 strain can therefore be used to select PEP4 mutants exhibiting relatively high PrA activity. Transformation of
strain W3122 with the 20 plasmid pools of the C-half in cis collection gave a total of 14,000 transformants. Ninety-eight
transformants were CPY-positive, indicating that many of this type of
mutant PrA precursors can form active PrA. One CPY-positive
transformant was chosen from each pool and plasmid DNA was isolated and
introduced into E. coli. From the E. coli transformants, plasmid DNA was isolated for retransformation of
yeast and for DNA sequencing. All isolated plasmids (termed pBVH1201,
1218, 1318, and 1320-1336) gave a CPY-positive phenotype upon
retransformation of W3122. Sequencing of the mutated part of the
prosequences showed that all 20 constructs had the expected restriction
sites and the expected length and were all different (Fig. 2).
Interestingly, 18 of these 20 mutant propeptides contained a positively
charged amino acid (lysine or arginine) at the most N-terminal
randomized position, position 53. Residue 53 is a lysine in the
wild-type precursor. These data suggest that this positive charge is
important for correct biosynthesis of the PrA precursor. At position 54
in the active constructs, 7 histidines, 5 arginines, 5 threonines, 2
tyrosines, and 1 valine were found, deviating significantly from a
random distribution. The wild-type precursor has a tyrosine at this
position. We see no other obvious sequence similarities between the
active constructs.
pep4 PRB1 strain W3094 with the pools and varied the
incubation temperature. More CPY-positive transformants were found on
plates that had been incubated at 23 °C than on ones that had been
at 30 °C. Twenty-two pools gave one or more CPY-positive
transformants at 23 °C. One CPY-positive transformant was chosen
from each pool and plasmid DNA was isolated and introduced into E.
coli. Plasmid DNA isolated from the E. coli transformants
was used for retransformation of yeast and for DNA sequencing. Nineteen
isolated plasmids complemented the CPY-negative phenotype upon
retransformation of W3094. Sequencing showed that 16 of the
prosequences had the expected restriction sites and the expected length
(Fig. 3). Of the three other constructs, one (pBVH1651) lacked
two of the random codons, while two others, pBVH1650 and pBVH1667,
resulted from odd ligations at the XhoI restriction site. We
could see no clear primary sequence similarities between the 19 active
constructs. Secondary structure predictions according to Rost and
Sander
(26, 27) were made for the mutant propeptides,
but no clear similarity between the predicted structures was seen.
Interestingly, four of the mutant prosequences contained an acceptor
site for N-linked glycosylation (Asn- X-Thr).
14,000)) of all mutant
precursors with a randomized C-terminal half of the propeptide can form
enough active PrA to allow complementation of the CPY-negative
phenotype of the
pep4 prb1 strain (W3122). Ten plasmids
that encoded a PrA polypeptide but did not complement the CPY-negative
phenotype of W3122 were sequenced in the randomized area to verify that
the oligonucleotide-derived sequences were indeed random
(Fig. 2). This showed that there was no general
overrepresentation neither of positively charged residues at position
53, nor of His, Arg, or Thr at position 54. For the N-half in cis collection, we conclude that around 0.5% (19/(0.45
6500)) of
all mutant precursors with a randomized N-terminal half of the
propeptide can form enough active PrA to complement the CPY-negative
phenotype of a
pep4 strain at 23 °C. Six plasmids of
this collection that produced PrA but did not complement the
CPY-negative phenotype of W3094 at 23 °C were sequenced in the
oligonucleotide-derived area (Fig. 3). Pools of plasmids from the
in cis collections were introduced into strain
JHRY20-2C-
pep4, which produces a truncated PrA form
including the entire propeptide. This was done to test whether the
randomized mutant precursors could interact with an in trans supplied propeptide to form active PrA. For both collections,
40-50% of all transformants in JHRY20-2C-
pep4 were CPY-positive. This high percentage indicates that few or none
of this type of propeptide-mutations distort the structure of the
precursor so much that an in trans supplied propeptide cannot
interact productively with the enzyme region.
PrA Activities of the Mutants and Maturation Rates of
Mutant Precursors
PrA activity assays were performed on strains
producing propeptide-mutated PrA forms. W3094 carrying pBVH17 (a
plasmid which produces wild-type PrA but is otherwise identical to the
plasmids from the two in cis collections) was used as
wild-type reference. A pep4 strain or a strain producing
PrA-23
76 had less than 2% of the reference activity. Strains
producing active mutant PrA forms isolated from the N-half in cis collection exhibited low PrA activities; the cell-associated
activities varied from 0% (not significantly more than a PrA-negative
strain) to 16% (Fig. 3). Some of the strains producing active PrA
forms isolated from the C-half in cis collection, however,
exhibited much more cell-associated activity, from 7 to 66% of the
wild-type reference (Fig. 2). A lysine residue is more favorable
than arginine at position 53, since the mutants that contained an
arginine exhibited generally less activity than the ones that had a
lysine (Fig. 2). Strains carrying plasmids that could not complement
the CPY-negative phenotype of a
pep4 strain did not
exhibit PrA activity (Figs. 2 and 3). The pBVH17-carrying reference
strain exhibited substantial PrA activity in the growth medium. This
has been observed previously and is presumably due to
overproduction-induced mislocalization of PrA. It is difficult to
determine how large a fraction of the activity is secreted, as some
intracellular PrA activity is lost during preparation of the
cell-extracts. Previous experiments, however, have suggested that about
half of all PrA activity is secreted in this strain
(5) .
Strains carrying pBVH1325 or pBVH1326 exhibited a little extracellular
activity (5-10% of the pBVH17-carrying strain). None of the other
mutant strains, however, had PrA activity in the growth medium. Thus,
the total PrA activities generated by the mutant strains are lower
percentages of the total wild-type activity than the percentages shown
in Figs. 2 and 3, which are based on intracellular activities only.
Figure 4:
Mutant PrA precursors containing a
glycosylation site in the propeptide are indeed glycosylated at this
position. Pulse labeling and immunoprecipitation of various PrA forms.
Cells were starved for sulfate, pulsed with S-labeled
methionine for 15 min, and chased for 60 min. 0 indicates no
chase; 60 indicates 60-min chase. After immunoprecipitation,
antigens were separated by SDS-polyacrylamide gel electrophoresis. The
mobility of PrA forms with two or three glycosylations is indicated. In
addition to the main bands, a minor band of increased mobility is seen,
this bands represents PrA forms that are glycosylated at one position
only. The strains were derived from strain W3094 by transformation with
plasmid pBVH1639 ( first lane), plasmid pBVH1641
( second lane), etc. Expression of PEP4 ( i.e. wild-type) was achieved by transformation with
pBVH17 ( last two lanes).
Site-directed Mutagenesis of the Wild-type
Prosequence
Site-directed mutagenesis was performed to
investigate the importance of the lysine residue at position 53 in the
wild-type propeptide. In one mutant, Lys-53 Ala-proPrA, the
lysine residue was replaced by an alanine to test the effect of removal
of the positive charge. In another mutant, the lysine was replaced by
an arginine (Lys-53
Arg-proPrA), which was expected to have less
effect as many active constructs isolated from the C-half in cis collection contained an arginine at position 53. Both precursors
matured slower than the wild-type PrA precursor; Lys-53
Arg-proPrA exhibited a half-time of maturation of about 20 min, while
Lys-53
Ala-proPrA matured with a half-time of about 40 min (Fig.
6). The mutant precursors retained the ER-specific molecular mass
longer than the wild-type precursor; thus, they are presumably more
slowly transported out of the ER (Fig. 6).
Figure 6:
Mutant PrA precursor forms that contain a
mutation of residue 53 remain longer in the ER-specific molecular
weight than the wild-type. Pulse labeling and immunoprecipitation of
various PrA forms. Cells were starved for sulfate, pulsed with
S-labeled methionine for 15 min, and chased for 0, 20, or
60 min. After immunoprecipitation, antigens were separated by
SDS-polyacrylamide gel electrophoresis. The mobilities of the ER form
of proPrA, the Golgi-form of proPrA, and the mature form are indicated.
In the first lane, a sample of extracellular antigen
from a strain producing wild-type PrA was loaded as a marker for the
molecular mass of the Golgi-modified form. These PrA molecules are
secreted as a consequence of mislocalization. They have received the
modifications that occur in the Golgi complex. Strains: W3094 carrying
pBVH45, W3094 carrying pBVH46, W3094 carrying pBVH17 ( wt or
wild-type).
We also
investigated the effect of the mutations that flanked the randomized
regions. These mutations had been introduced to allow subcloning of the
oligonucleotides into the prosequence context. In the N-half in cis collection, the random sequences were preceded by a BamHI
restriction site and followed by a XhoI site. A construct
(pBVH64) was made in which these sites were introduced, leaving the
rest of the prosequence as in the wild-type. Thus, this mutant
precursor contained the mutations Val-24 Gly, His-25
Ser,
Gly-51
Leu, and Gln-52
Glu. Similarly, for the C-half
in cis collection, a control construct (pBVH63) was made
containing the mutations Gly-51
Leu, Gln-52
Glu, and a
replacement of Thr-75 and Glu-76 by an Asp. Furthermore, a construct
(pBVH47) was made in which only the XhoI restriction site had
been introduced, thus containing Gly-51
Leu and Gln-52
Glu. Pulse-chase experiments were performed to investigate the effect
of these mutations. The mutations at positions 51 and 52 increased the
half-time of maturation to 20 min, the mutations at position 24 and 25
gave an additional increase of 20 min, while replacing Thr-Glu by Asp
at the end of the propeptide gave an additional increase in the
half-time of maturation of about 10 min. Also, these mutant precursors
retained the ER-specific molecular mass longer than the wild-type,
indicating slower exit out of the ER (data not shown).
The Propeptide Can Function in Trans When Supplied as an
Integral Part of a Full-length Precursor
We have previously
found that a propeptide-lacking form of PrA (PrA-2376) can be
rescued from degradation by a noncovalently linked propeptide ( in
trans)
(5) . We tested whether a full-length PrA precursor
could assist the formation of an active enzyme in trans.
Replacement of the PrA active-site residue Asp-294 by Glu results in
accumulation of the mutant precursor (Asp-294
Glu-proPrA), and
no CPY activity is detected in a strain producing this mutant form
(4) . A strain carrying both the Asp-294
Glu-proPrA-producing plasmid pBVH812 and the PrA-23
76-producing
plasmid pBVH32 was CPY-positive, and exhibited about 10% of the
wild-type reference PrA activity. Loss of either of the two plasmids
from the latter strain due to removal of the nutritional selection
resulted in loss of the CPY-positive phenotype. These data suggest that
the propeptide of Asp-294
Glu-proPrA can interact with
PrA-23
76, thus assisting formation of active enzyme.
-lytic protease, on the other hand, the noncovalently bound
propeptide is strongly inhibitory and forms a stable complex with the
enzyme region
(34) .
Ala-proPrA had a half-time of maturation of about 40 min;
this being the strongest reduction of the maturation rate seen for any
of the mutations affecting only one or a few codons. Porcine pepsinogen
has a lysine in the propeptide at position 36 (Lys-36p) (counting from
the N-terminus of the zymogen), and alignment of vertebrate aspartic
protease propeptides shows a clear conservation of this residue
(35) . In porcine pepsinogen, this lysine residue interacts with
the aspartic acid residues in the active site of the enzyme
(36) . Thus, it may be hypothesized that the lysine residue at
position 53 in proPrA is functionally equivalent to Lys-36p in
pepsinogen. Lys-36p of pepsinogen is followed by a tyrosine, which also
has hydrogen bonding in the active site, and Lys-53 in proPrA is
followed by a tyrosine as well. Two of the active constructs isolated
from the C-half in cis collection did not have a
positively charged residue at position 53. One precursor even had a
negatively charged residue at this position. Thus, even at this
position, introduction of a completely different amino acid does not
necessarily abolish propeptide function, but the low frequency with
which this occurred suggests that the alternative residues at position
53 dictate more stringent requirements to the rest of the sequence.
(
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.