The prosequence of Rhizopus niveus aspartic proteinase-I supports correct folding and secretion of its mature part in Saccharomyces cerevisiae.

Extracellular Rhizopus niveus aspartic proteinase-I (RNAP-I) was secreted effectively by Saccharomyces cerevisiae when RNAP-I with its preprosequence was synthesized in this organism (Horiuchi, H., Ashikari, T., Amachi, T., Yoshizumi, H., Takagi, M., and Yano, K. (1990) Agric. Biol. Chem. 54, 1771-1779). Certain deletions (delta pro, delta 1, delta 2), and amino acid substitutions (M1) in the prosequence blocked secretion of RNAP-I, although the protease protection assay revealed that even delta pro could be translocated across the membrane of the endoplasmic reticulum. When delta pro or M1 was synthesized simultaneously with the wild-type preprosequence in S. cerevisiae, secretion of RNAP-I was recovered. Therefore, the physical linkage of the prosequence to the mature region is not a prerequisite for secretion of active RNAP-I. Purified RNAP-I with the prosequence once denatured in 6 M guanidine HCl could be renatured and activated to have its enzymatic activity by removing guanidine HCl in vitro, but RNAP-I without the prosequence could not. Furthermore, the wild-type prosequence helped the recovery of the activity of the denatured RNAP-I in trans, but the prosequences of M1 with which secretion of RNAP-I was not observed in vivo, did not. From these results we concluded that the prosequence of RNAP-I supports correct folding of RNAP-I in the endoplasmic reticulum lumen and its subsequent secretion in S. cerevisiae. The functional role of the prosequence of an aspartic proteinase was elucidated.

thermore, it is reported that the prosequence of preprosomatostatin can protect a-globin from degradation in the ER' and enables intracellular transport and secretion when it is fused to the N terminus of a-globin and expressed in mammalian cells (Stoller and Shields, 1989).
Most of proteases have prosequences at the N termini, C termini, or both of mature enzymes. Prosequences of proteases are thought t o be involved in the inactivation of mature enzymes after synthesis until localization in the destined places either in or out of the cells (Neurath, 1989). However some of the prosequences seem to have other functions. The prosequences of some serine proteases such as subtilisin E of Bacillus subtilis (Zhu et al., 19891, a-lytic protease of Lysobacter enzymogenes (Baker et al., 19921, and carboxypeptidase Y of Saccharomyces cereuisiae (Winther and Sorensen, 1991) are shown to have the function to help refolding of the denatured mature parts in vitro. In addition, the vacuolar sorting signal of carboxypeptidase Y is within its prosequence (Valls et al., 1990). Moreover, it is proposed that the prosequence at the C terminus of serine protease of Serratia marcescens and that of IgA protease of Neisseria gonorrhoeae form pores in the outer membrane through which the mature parts are translocated (Wandersman, 1992). In comparison with these cases, little is known about the functional role of prosequences of aspartic proteinases. It is presumed that the prosequence of proteinase A of S. cereuisiae promote folding of the mature part (van den Hazel et al., 19931, but clear evidence is not obtained. Rhizopus niueus, a filamentous fungus, secretes large amounts of glucoamylases, aspartic proteinases, etc. extracellularly. We have cloned and sequenced genes encoding aspartic proteinase-I (RNAP-I) (Horiuchi et at., 1988a), aspartic proteinase-11, -111, -N, and -V (RNAP-I1 to -V) (Sakaguchi et al., 1992), and ribonuclease Rh (Horiuchi et al., 1988b). RNAP-I is synthesized as a precursor form with a presequence (21 amino acid residues) and a prosequence (45 amino acid residues) at the N terminus of the mature part (323 amino acid residues). R N A P -I is also secreted extracellularly with high efficiency in S. cereuisiae when the prepro-RNAP-I gene is expressed under the control of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene promoter (Horiuchi et al., 1990) or GAL1 gene promoter of S. cereuisiae (about 150 mg/liter under the optimum condition).' This situation opens a possibility of analyzing the functional role of the prosequence of RNAP-I taking advantage of the refined S. cereuisiae gene-manipulating system.
In this paper, we analyzed the function of the prosequence of RNAP-I and found that the prosequence of RNGP-I was essential for both secretion of mature part in S. cereuisiae i n vivo and The abbreviations used are: ER, endoplasmic reticulum; RNAP-I, R. niueus aspartic proteinase-I; PAGE, polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GST, glutathione S-transferase; kb, kilobase pair(s). refolding of denatured mature RNAP-I in vitro. We therefore propose that the prosequence of RNA€'-I guides correct folding of RNAP-I in vivo probably in the ER lumen, thereby making it possible for the enzyme to go through the secretory pathway.
S. cerevisiae strain R27-7C-1C (MATa his3 leu2 ura3 trpl) and EH13-15 (MATa t r p l ) were used a s hosts for secretion and purification of mature RNAP-I and pro-RNAP-I. Yeast cells were cultivated aerobically at 30 "C. For selection of yeast transformants YNBD medium (0.67% yeast nitrogen base without amino acids, 2% glucose) were used with appropriate supplements. RNAP-I secretion in liquid medium was tested in YNBDCU medium (YNBD containing 2% casamino acids and 0.05% uracil). Enzymes a n d Reagents-Nucleic acid modification enzymes were purchased from Takara Shuzo Co. (Kyoto, Japan) and used under conditions suggested by the manufacturer. Oligonucleotides for site-directed mutagenesis were synthesized with DNA Synthesizer 391 (Applied Biosystems).
PZasrnid Construction-Recombinant DNA manipulations were done by the standard methods (Maniatis et al., 1982). Yeast transformation was carried out using the lithium acetate procedure described by Ito et al. (1983).
Plasmid pYPR2841, in which the whole prosequence of RNAP-I gene was deleted, was constructed as follows. A 0.5-kb EcoRI-Sal1 fragment containing the coding region for the preprosequence and a part of the mature part of RNAP-I was isolated from pYPR2831 (Horiuchi et al., 19901, and it was cloned between EcoRI and Sal1 sites of M13mp19. Then an XhoI site and a BamHI site were introduced before and after the prosequence of RNAP-I gene, respectively, by site-directed mutagenesis using a 21-mer synthetic oligonucleotide "5'-GAAGCTGCTC-GAGACGGCAAG-3'" for the former site and a 20-mer synthetic oligonucleotide "5'-CAACCGAGGCCAGTGGCTCT-3'" for the latter site. Then the recombinant double-stranded phage DNA was cut with XhoI and BamHI, digested with mung bean nuclease, and fused. A 0.3-kb EcoRI-Sal1 fragment from the resultant phage DNA was ligated with 9.4-kb EcoRI-Sal1 fragment of pYPR2831S (a modified pYPR2831, in which the Sal1 site of the GAPDH terminator was eliminated) to form the plasmid pYPR2841.
Plasmids pYPR2842 and pYPR2843 encode RNAP-I derivative A 1 and A2, in which the region from LysZ6 to and from Ala5' to ThF5, respectively, was deleted. They were constructed from plasmid cleotide "5'-GCTGCACCCAACGGATCCGCCMAAATGCAC'M-3'," a pYPR2831S in a similar manner using a 33-mer synthetic oligonu-33-mer synthetic oligonucleotide "5'-GCACTTAATAAGGCTCTCGAG-GCCAGTGGCTCT-3'," respectively. Plasmid pYPR2844 encodes an RNAP-I derivative M1, in which Ala4'-Leu5' was replaced with Asp4'-Pro". It was constructed by site-directed mutagenesis using a 22-mer synthetic oligonucleotide %'-CTTAATAAGGATCCCGCCAAGT-3'." Plasmid pYGpro in which the DNA region encoding preprosequence of RNAP-I was downstream of GALl promoter was constructed a s follows. A 0.5-kb EcoRI-Sal1 fragment from pYPR2831 was cloned between EcoRI and Sal1 sites of pUC119 (resulting pUC28311, and a termination codon and a BamHI site were introduced just after the prosequence of RNAP-I gene by site-directed mutagenesis with a 39-mer synthetic oligonucleotide "5'-GGAATTACAACCGAGTAAG-GATCCTCTGTTCCTATGGTT-3'." The resultant double-stranded DNA with introduced BamHI site was cut with EcoRI and BamHI, and a 0.2-kb fragment was ligated with a n 8.1-kb EcoRI-Sal1 fragment of pYPR3831 (a modified pYPR2831, in which the GAPDH promoter was replaced by the GALl promoter) to form pYPR38pro. To obtain pYGpro, a 1.5-kb BamHI-Hind111 fragment of pYPR38pr0, which contained GALl promoter, the preprosequence of RNAP-I gene and terminator of GAPDH gene was transferred into PuuII site of YEp24.
Plasmid pGEX-31 and pGEX-44, which encoded prosequences of the wild-type RNAP-I and M1, respectively, that were fused to the C terminus of glutathione S-transferase. They were constructed as follows. A BamHI site was introduced just before the prosequence, and a termination codon and an EcoRI site were simultaneously introduced just after it in pUC2831 by site-directed mutagenesis using a 30-mer synthetic oligonucleotide '?i'-ACACTTGCTGTCGGATCCGCACCCAACG-GC-3"' for former mutation and a 38-mer synthetic oligonucleotide "5'-GGAA'MACAACCGAGTAAGAA'MCTCTG'MCCTATGGT-3'" for latter mutation, respectively. In the case of pGEX-44, the amino acid substitution mutations in the prosequence were introduced with the same synthetic oligonucleotide as used to prepare pYPR2844. A 0.14-kb BamHI-EcoRI fragments with or without the mutation was ligated with a 4.9-kb BamHI-EcoRI fragment of pGEX-ST (Pharmacia).
Accurate construction of all plasmids were confirmed by nucleotide sequencing.
Measurement ofProteinase Activity-Proteinase activity was assayed by the method previously described by Horiuchi et al. (1990).
Western Blotting Analysis-SDS-PAGE was done by the method of Laemmli (1970). Transfer of proteins to nitrocellulose filters (Hybond-C, Amersham Corp.) was performed as described in the manufacture's protocol. Detection of filter-bound antibodies was performed according to the enhanced chemiluminescence method with ECL detection reagents (Amersham Corp.).
Preparation of Microsomes and Protease Protection Assay-S. cereuisiae spheroplasts were prepared a s described by Franzusoff et al. (1990).
(1991), and microsomes were prepared by the method of Baker et al.
Protease protection assay was done as follows. One pl of 0.5 mg/ml of trypsin was added to 30 1. 11 of the microsomal fraction in the presence or absence of 1% Triton X-100 and incubated for 30 min a t 4 "C. APro was detected by Western blotting analysis with anti-RNAP-I antisera.
Purification of Mature and Pro-RNAP-Z-Mature RNAP-I was purified from the culture supernatant of S. cereuisiae EH13-15 cells harboring the plasmid pYPR2831. Those cells were cultivated in YNBDC medium at 30 "C for 72 h. The pH of the culture supernatant was adjusted to 5.3, and the prosequence was processed in this step. The solution was concentrated with ultrafiltration and dialyzed against 10 m~ NaOAc-AcOH (pH 5.3). The solution was applied on a CM-Sephadex (2-50 column (5.1 x 46 cm) which had been equilibrated with 10 m~ NaOAc-AcOH (pH 5.3). The column was washed with 0.1 M NaCl in the same buffer and then with a 600-ml linear gradient (0.1-0.4 M NaCl in the starting buffer) at a flow rate of 15 myh. Mature RNAP-I was monitored with SDS-PAGE.
Pro-RNAP-I was purified from the culture supernatant of EH13-15 cells harboring the plasmid pYPR3831. Those cells were cultivated in YEPG medium (2% polypeptone, 1% yeast extract, 2% galactose) at 30 "C for 24 h. The pH of the culture supematant was adjusted to 6.2, and the solution was concentrated with ultrafiltration and dialyzed against 20 nm sodium phosphate (pH 6.2). The solution was applied on a CM-Sephadex C-50 column (5.1 x 46 cm) which had been equilibrated with 20 mM sodium phosphate (pH 6.2). The column was washed with the same buffer and then with a 600-ml linear gradient (0 to 0.1 M NaCl in the starting buffer) a t a flow rate of 15 ml/h. The pooled fractions were further purified by the same method described above with CM-Sephadex C-50 column. Pro-RNAP-I was monitored with SDS-PAGE.
The buffer of mature and pro-RNAP-I was changed to 20 m~ sodium phosphate (pH 6.2) by Sephadex G-25 column (NAP column, Pharmacia), and these proteins were used for in vitro renaturation assay.
Part of mature RNAP-I thus purified was used to prepare rabbit polyclonal antisera.
Production a n d Purification of the Prosequences of RNAP-I-The prosequences of RNAP-I were produced and purified as a fusion proteins with glutathione S-transferase.
E. coli MV1190 cells harboring the plasmid pGEX-31 or pGEX-44 were cultivated in 15 ml of LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaC1) containing 50 mg/ml of ampicillin at 37 "C overnight. The culture was inoculated to 300 ml of the same medium, and it was cultivated a t 37 "C for 6 h. Then isopropyl-1-thio-P-D-galactopyanoside was added to a final concentration of 0.5 m~ into the culture, and it was further cultivated for 3-5 h. Cells were collected, resuspended in purification buffer (20 mM sodium phosphate (pH 7.3), 150 m~ NaCI, 100 m EDTA, 10 m~ EGTA, 100 mg/ml phenylmethylsulfonyl fluoride, 0.5 mg/ml leupeptin) and homogenized with Braun homogenizer for 1 min. Triton X-100 was added to a final concentration of 1%, and the solution was left on ice for 30 min. Then it was centrifuged at 10,000 x g for 10 min at 4 "C. The supernatant was loaded on a glutathione Sepharose 4B column (1.6 x 10 cm) which had been equilibrated with the purification buffer plus 1% Triton X-100. After being washed with the purification buffer, the column was eluted with 5 m~ glutathione in the same buffer.
The buffer was exchanged to 20 m sodium phosphate (pH 6.2) by Sephadex G-25 column (NAP column, Pharmacia), and these proteins were used for in vitro renaturation assay.
In Vitro Renaturation Assay-In vitro cis renaturation assay was done as follows. Purified mature RNAP-I (125 p1, 0.114 mg/ml) and Structures of RNAF"1s with the wild-type and mutated preprosequences. A, RNAP-I with the wild-type preprosequence contains a presequence (dark stippled box), a prosequence (striped box), and a mature region (light stippled box). Deleted regions in Apro (from Se?' to Glu=), A 1 (from LysZ6 to and A2 (from Ala51 to T h P ) are indicated by the open boxes. B, the amino acid sequence of a part of the wild-type prosequences is shown, together with the amino acid substitutions in M1. The Lys52-%53, which is suggested to interact with 2 active site aspartic acid residues Asp'" and Aspzs3, is indicated by the stippled box.
pro-RNAP-I(l25 pl, 0.239 mg/ml), both in 20 m~ sodium phosphate (pH 6.2), were mixed with 375 pl of 8 M guanidine HC1 in the same buffer and incubated for 1 h a t room temperature. These samples were put on Sephadex G-25 columns (NAP column) which had been equilibrated with 20 m~ sodium phosphate (pH 6.2) and eluted with the same buffer. Fractions (1 ml) containing mature RNAP-I or pro-RNAP-I were collected and incubated for various periods a t 4 "C. Samples (100 pl) were mixed with equal volume of 0.2 M lactate (pH 3.0) and incubated for 1 h a t 4 "C. Then proteinase activities and protein concentrations were measured.
In vitro trans renaturation assay was done as follows. Denatured mature RNAP-I (1 ml, 0.281 mg/ml) was prepared as described above. Aliquots (209 pl each) were mixed with equal molar of GST, GST-pro, or GST-M1 (final volume, 1.045 pl) and incubated a t 4 "C for various periods. Then proteinase activities were measured.

Effects of the Mutated Prosequences of RNAP-Z on Secretion of the Mature Enzyme-The yeast cells harboring prepro-RNAP-I
gene under the control of the GAPDH gene promoter of S. cerevisiae on the multicopy plasmid pYPR2831 secreted RNAP-I with high efficiency (Horiuchi et al., 1990). To analyze the role of the prosequence of RNAP-I in extracellular secretion of mature R N A P -I , we have carried out deletion analysis of the prosequence of RNAP-I in S. cerevisiae. The multicopy plasmids, pYPR2841, pYPR2842, and pYPR2843, encoding RNAP-Is without or with partially deleted prosequences were constructed. The whole prosequence (from S e P to G W ) , a region from LysZ6 t o Pro40, and another region from Ala51 to T h P were deleted to make Apro, Al, and A2, respectively (numbering is from the initiation codon Met) (Fig. 1).
We constructed another plasmid pYPR2844 that encoded an RNAP-I with a mutated prosequence designated as M1, in which Ala4'-Leu5" near Lys5'-TyP3 that is conserved among the prosequences of many aspartic proteinases (Horiuchi et al., 1988a;James and Sielecki, 1986;Sogawa et al., 1983;Tonouchi et al., 1986;Hayano et al., 1988;Chen et al., 1991;Razanamparany et al., 1992,) was changed to Asp-Pro (Fig. 1). These plasmids were transferred to S. cerevisiae strain R27-7C-1C and extracellular secretion of mature RNAF"1 in the transformants was examined. Any transformants that were expected to produce A l , A2, or M1 did not show a halo due to the extracellular RNAP-I activity on casein-containing plates (data not shown). The extracellular proteinase activity in each of these transformants expressing Apro, Al, A2, and M1 was less than 1% of that of RNAP-I expressed with the wild-type prosequence and not different from that of the transformant harboring the control vector pYE209 (Table I). The culture supernatants were also subjected to SDS-PAGE, and the bands corresponding to the pro-and mature RNAP-Is were not found except for the case of production of RNA€' -I with the wild-type prosequence (data not shown). Thus the complete or partial deletion of the prosequence and the replacement of 2 amino acid residues in it blocked secretion of RNAP-I. In Western blotting analysis of the cell extracts of these transformants using anti-RNAP-I antisera. The bands corresponding to Apro, Al, A2, and M1 were observed in the cell extracts (data not shown).
To test whether Apro was present in the cytoplasm or translocated across the ER membrane, the microsomal fraction was prepared from the cells expressing Apro, and then, the existence of trypsin-resistant Apro was investigated by protease protection assay. Fig. 2 shows that Apro in intact microsomal fraction was resistant to proteolysis (Fig. 2, lane 2 ) , whereas it was sensitive when microsomal membrane was solubilized with 1% Triton X-100 (Fig. 2, lane 3 ).
Thus, even Apro could most likely be translocated across the ER membrane into the ER lumen.

Secretion of RNAP-I with Mutated Prosequences by trans-Complementation of the Wild-type Prosequence in
Vivo-We examined whether the prosequence of RNAP-I that was synthesized as a peptide could recover the secretion of Apro or M1. The plasmid pYGpro encoding the wild-type preprosequence of RNAP-I under the control of GAL1 promoter was constructed.
This plasmid was introduced with pYPR2841 (encoding Apro) or pYPR2844 (encoding M1) into S. cerevisiae R27-7C-1C. These plasmids were designed to produce only Apro or M1 in the glucose medium but the preprosequence of RNAP-I, too, in the galactose medium.
These transformants were cultured first in YNBDC (glucosecontaining) liquid medium for 24 h at 30 "C and then in YN-BDC or YNBGC (galactose-containing) liquid medium for additional 24 h. The extracellular proteinase activities of the culture supernatants were determined. In the presence of galactose, the activity of the transformants that synthesized Apro or M1 together with the prosequence was 19 or 30% of the wild type (Fig. 3). On the contrary, little activity was detected with these transformants in glucose medium. Furthermore, in SDS-PAGE analysis of the supernatant having proteinase activity, the band corresponding to the authentic RNAP-I was detected (data not shown).

FIG. 3. Secretion of RNAP-Is by trans-complementation of the wild-type prosequence.
After 24-h culture in the medium containing glucose ( IGlc) or galactose ( / G a l ), the proteinase activities in the culture supernatants of the cells harboring the plasmids pYE209 and pYGpro ( a ) , pYPR2831 and YEp24 ( h ) , pYPR2841 and pYGpro ( c ) , and pYPR2844 and pYGpro ( d ) were measured. Proteinase activity in each of the supernatants is shown as percent of that in the culture supernatant of the cells harboring the plasmids ( h ) in the same medium.

Prosequence in
Vitro-The function of the prosequence of RNAP-I was analyzed in vitro. Mature RNAP-I and pro-RNAP-I of which the prosequence was not processed were purified from the culture supernatant of S. cerevisiae EH13-15 expressing the wild-type RNAP-I gene (see "Materials and Methods"). These mature RNAP-I and pro-RNAP-I in phosphate buffer (20 mM sodium phosphate (pH 6.2)) were denatured by adding 3 volumes of denaturing buffer (20 mM sodium phosphate (pH 6.2), 8 M guanidine HCI) and incubating for 1 h at room temperature. These denatured proteins were desalted with Sephadex G-25 (NAP column, Pharmacia) and incubated at 4 "C in 20 mM sodium phosphate (pH 6.2) for various periods to induce proper folding and then after an addition of 0.2 M lactate (pH 3.0), incubated at 4 "C for 1 h to induce autocatalytic processing to produce active mature form. The activity of mature RNAP-I and pro-RNAP-I, without denaturing process but with activating process, was taken as 100% (control).
Mature RNAP-I thus treated did not have proteinase activity even after 8-h incubation for folding (Fig. 4, circles). On the contrary, pro-RNAP-I treated in the same manner had 60% of the proteinase activity with 1-h incubation and 75% with 8-h incubation for folding (Fig. 4, squares). Pro-RNAP-I had 20% of activity even without incubation for folding. This was not due to incomplete denaturation, since mature RNAP-I was completely denatured under the same condition, but due to very rapid correct folding during the process of desalting; it took After the removal of the denaturant (0 h), they were incubated at 4 "C for various periods. The activity is shown as percent of that of the nondenatured one for both protein preparations.
several minutes. Alternatively, it might obtain proteinase activity during acid activation or incubation with the substrate. The prosequence of RNAP-I could trans-complement secretion of Apro in vivo (Fig. 3). We, therefore, examined whether it could also renature the denatured mature RNAP-I in trans in vitro. The prosequence was supplied as a in-frame fusion to glutathione S-transferase (GST-pro). The denatured mature RNAP-I was incubated at 4 "C with an equal molar of either GST-pro or glutathione S-transferase (GST) alone. The addition of GST-pro to denatured RNAP-I resulted in recovery of 70% of activity after 6-h incubation, whereas GST had no effect (Fig. 5).
Next, we examined whether the mutated prosequence of M1, with which secretion of the mature part was not observed in vivo, could renature the denatured mature RNAP-I in vitro. Prosequence of M1 was also supplied as a in-frame fusion with glutathione S-transferase (GST-M1) and used in in vitro experiment as described above. The result is shown in Fig.  5. Denatured RNAP-I did not regain activity in the presence of GST-M1, even after 48-h incubation (data not shown).

DISCUSSION
We analyzed the effect of the prosequence of RNAP-I on secretion of its mature part in the expression system of S. cerevisiae as a host. Secretion of RNAP-I was blocked by the complete or partial deletion of the prosequence or by the substitution of 2 amino acid residues in it (Table I). Even without the prosequence, Apro was proved by protease protection assay to be translocated across the ER membrane in the presence of the presequence (Fig. 2).
The preprosequence synthesized as a peptide could complement in trans the defective secretion of Apro. In addition, it rescued in trans the secretion of M1 having a mutated prosequence (Fig. 3). Therefore, the prosequence of RNAP-I is essential for secretion of the mature part and probably works after translocation of RNAP-I protein across the ER membrane into the lumen.
At the same time, the prosequence of RNAP-I had a function to activate, probably by helping correct folding, once denatured mature part in vitro. That is, pro-RNAP-I could be renatured and activated with high efficiency after denaturation in vitro, whereas mature RNAP-I itself could not (Fig. 4). Furthermore, the wild-type prosequence synthesized a s a peptide in and purified from E. coli helped renaturation of the denatured mature RNAP-I in trans, but the prosequence of M1 did not (Fig. 5). These results suggest that the prosequence of RNAP-I is necessary for correct folding of RNAP-I protein, thereby supporting secretion of the mature part in vivo.
The prosequences of some serine proteases are shown to help the renaturation of the denatured mature parts in vitro (Zhu et