©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Random Substitution of Large Parts of the Propeptide of Yeast Proteinase A (*)

H. Bart van den Hazel , Morten C. Kielland-Brandt , Jakob R. Winther (§)

From the (1) Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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),() 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.

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-2376) 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.


EXPERIMENTAL PROCEDURES

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 pep4-2376-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.

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.

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]HSO, and cultures were chased by the addition of 5 µl of 1 M NaSOand 5 µl of 5 mg/ml methionine.


RESULTS

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-2376. 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.

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 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).

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/(0.4514,000)) 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/(0.456500)) 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-2376 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.

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.


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-2376-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-2376, thus assisting formation of active enzyme.


DISCUSSION

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 -lytic protease, on the other hand, the noncovalently bound propeptide is strongly inhibitory and forms a stable complex with the enzyme region (34) .

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 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.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 45-33-27-52-82; Fax: 45-33-27-47-66.

The abbreviations used are: ER, endoplasmic reticulum; CPY, carboxypeptidase Y; PCR, polymerase chain reaction; PrA, proteinase A; PrB, proteinase B; proPrA, pro-proteinase A.

V. Westphal, B. van den Hazel, and J. Winther, unpublished results.


ACKNOWLEDGEMENTS

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.


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