Tm JOURNAL OF B I O ~ I C A L C I W A I ~ Y Q 1994 by The Americao Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 1, Issue of January 7, pp. 588-592, 1994 Printed in U S A .

Autocatalytic Maturation of the Prohormone Convertase PC2* (Received for publication, March 19, 1993, and in revised form, September 9, 1993)

Glenn Matthew&, Kathleen I. J. ShennanB, Andrew J. Seal§, Neil k Taylor§, Alan ColmanS, and Kevin Dochertygn From the $School of Biochemistry, University of Birmingham, I! 0. Box 363, Birmingham B15 2TI: United Kingdom and the §Department of Medicine, University of Birmingham, Birmingham, B15 2TH, United Kingdom

PC2 is a member of the eukaryotic family of subtilisin- like proteases, which is thought to participate in the processing of prohormones and proneuropeptides in neuroendocrine cells. PC2 is synthesized as a 69-kDa prepropolypeptide. The NHa-terminal signal sequence is removed during segregation within the endoplasmic re- ticulum, where glycosylation occurs to generate a 75- kDa propolypeptide. A combination of site-directed mu- tagenesis and a cell-free translatiodtranslocation system from Xenopus eggs was used to investigate the processing of the pro-PC2 precursor. The 75-kDa poly- peptide underwent slow cleavage after the sequence A r g - b s - L y s - e to generate a W-kDa mature enzyme. Cleavage was blocked when the tetrabasic sequence was deleted (PC2M3) or when the active site Asp14a was changed to Asn (PC2M4). This latter observation sug- gested that cleavage of the 75-kDa propolypeptide to the mature 68-kDa enzyme was autocatalytic. Incubation of the PC2M4 mutant with the wild type PC2 precursor resulted in cleavage of both the wild type polypeptide and the catalytically inactive PC2M4 mutant. This indi- cates that cleavage could occur through an intermolecu- lar reaction. The results also demonstrate that the novel Xenopus egg extract translatiodtranslocation system represents a powerful cell-free method for studying pro- teolytic processing of propolypeptides.

PC2 is a member of the eukaryotic family of subtilisin-like proteases (Smeekens and Steiner, 1990; Seidah et al., 1990). Other members of the family include the yeast protease Kex2 (Mizuno et al., 1989; Fuller et al., 19891, fiuin (Van de Ven et al., 1990), PACE4 (Kiefer et al., 19911, PC3 (also known as PC11 (Smeekens et al., 1991; Seidah et al., 19911, and PC4 (Na- kayama et al., 1992; Seidah et al., 1992).

PC2 and PC3 are thought to be involved in the intracellular proteolytic processing of prohormones and proneuropeptides during their transit through the secretory pathway (Hutton, 1990; Barr, 1991; Steiner, 1991). Thus PC2 and PC3 are selec- tively expressed in neuroendocrine cells (Smeekens et al., 1991; Seidah et al., 1991), exhibit an acidic pH optimum and Ca2+ dependence (Shennan et al., 1991a; Baiyles et al., 1992) which is in keeping with the known properties of prohormone proc- essing endopeptidases (Hutton, 1990), and cleave prohormones such as proopiomelanocortin and proinsulin at pairs of basic amino acids (Benjannet et al., 1991; Thomas et al., 1991; Bai-

*This work was supported by grants from the Medical Research

costa of publication of this article were defrayed in part by the payment Council, the British Diabetic Association, and the Wellcome Trust. The

of page charges. This article must therefore be hereby marked "adver- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Queen Elizabeth Hospital, Birmingham B15 2TH, United Kingdom. 1 To whom correspondence should be addressed: Dept. of Medicine,

yles et al., 1992), a recognized processing site in prohormones. Further evidence supporting a role for these endoproteases in prohormone processing comes from studies showing coordinate regulation of PC2 and PC3 with proopiomelanocortin mRNA in the intermediate lobe of the pituitary (Birch et al., 1991; Bloom- quist et al., 1991), and from the observation that deletion of PC3 mRNA in cultured pituitary cells resulted in disruption of normal proopiomelanocortin processing (Bloomquist et al., 1991).

Furin and PACE4 are expressed in a wide variety of tissues (Hatsuzawa et al., 1990; Kiefer et al., 1991). Like Kex2 (Fuller et al., 19891, furin exhibits a neutral pH optimum and Ca2+ dependence (Hatsuzawa et al., 1992; Bresnahan et al., 1990; Molloy et al., 1992). Furin requires an Arg at the -4 position upstream of the cleavage site, i.e. the sequence Arg-X-(Lys/ Argl-Arg (Hosaka et al., 1991; Watanabe et al., 1992; Molloy et al., 19921, a consensus cleavage site commonly found in pre- cursor proteins for growth factors, membrane receptors, serum proteins, and viral envelope proteins. Thus the tissue distribu- tion, enzymatic properties, and substrate specificity (Wise et al., 1990; Bresnahan et al., 1990) suggest that furin is respon- sible for the processing of these constitutively secreted and membrane precursor proteins (Barr, 1991). PC4 is expressed in the testes where its role and physiological substrate(s) have yet to be characterized (Nakayama et al., 1992; Seidah et al., 1992).

cDNA sequences for all these proteins are known and certain common structural features are apparent (Steiner et al., 1992). They all contain an NHz-terminal hydrophobic signal se- quence, a pro-region, a subtilisin-like domain, and a COOH- terminal region, which in the case of Kex2 and furin contains a hydrophobic transmembrane domain. Maturation of Kex2 in- volves signal peptide cleavage, addition of N-linked and 0- linked oligosaccharide, and proteolytic removal of the NH2- terminal pro-region (Wilcox and Fuller, 1991). The mature enzyme is then concentrated within a late compartment of the Golgi complex (Ftedding et al., 1991). Maturation of furin has been studied in transfected monkey kidney cells (Ftehemtulla et al., 1992). Following removal of the signal peptide, cleavage of the NHz-terminal pro-sequence occurs within the Golgi. Fur- ther cleavage at the COOH terminus removes the membrane anchor and results in secretion of the enzyme.

The biosynthesis of PC2 has been studied in microinjected Xenopus oocytes (Shennan et al., 1991b). The enzyme first ap- pears in the oocytes as a 75-kDa glycosylated protein which undergoes intracellular cleavage to generate a 71-kDa protein. This protein is secreted from the oocytes and undergoes further cleavage to generate the 68-kDa mature enzyme. The identities of the enzymes involved in the activation of PC2 are not known. The present study was undertaken to investigate the proteo- lytic processing of the PC2 precursor. Here we show, using a combination of in vitro mutagenesis and a cell-free translation/ translocation system prepared from Xenopus eggs, that pro- PC2 can undergo autocatalytic cleavage to generate the mature enzyme.

588

Maturation of PC2 589

EXPERIMENTAL PROCEDURES

Chemrcals and Reagents--l.-[,‘“SlMethlonine (1000 Wmmol), I.-[“Hlleucme (130 Wmmol), and rainbow “C-methylated protem mo- lecular weight markers (molecular weight range, 14,30&200,000) were obtained from Amersham InternatIonal, Llttle Chalfont, Bucks, Umted Kmgdom Endoglycosidase H was from Boehnnger Mannhelm, Lewes, East Susex, UK. Rabbit retxulocyte lysates were purchased from Pro- mega (Promega, Southampton, Hamps, UK). Full-length cDNAs encod- mg human PC2 and mouse PC3 were provided by Dr. D. F. Sterner, Howard Hughes Medical Institute, University of Chlcago.

Site-dvected Mutagenesis-The mutant PC2 constructs, PC2M2, PC2M3, and PC2M4, have been prewously described (Sherman et al., 1991b). In PC2M2 the tetrabaslc sequence Lys-Arg-Arg-Arg at positlon 53-56 (where posltlon 1 is the first amino acid in the propolypeptlde) was changed to Lys-Val-Arg-Leu, m PC2M3 the tetrabaslc sequence at posltlon 81-84 was deleted, and m PC2M4 the actwe site aspartate at position 142 was changed to asparagme.

In V&o ?Zunscrcptcon-PC2 mRNA was syntheszed from BamHI lmearized pPC264T m a reactlon contaming 40 rnM Tris (pH 8.0), 15 my MgCl,, 4 rnM spermldme, 5 rnM dithlothreitol, 1 rnM each of ATP, CTP, and UTP, 0.1 m.v GTP, 0.5 rnM m7G(5’)ppp(5’)G, 0.25 mg/ml bovine serum albumin (RNase and DNase free), 10 umts of RNaseguard, 60 umts of SP6 RNA polymerase. and 1 pg of plasmid DNA m a final volume of 50 pl. The mixture was Incubated at 37 “C for 60 mm, and then for a further 10 mm wth 200 units/ml DNase (RNase free) The mixture was then extracted once with phenol,chloroform:lsoamyl alco- hol (25:24:1), and once wth chloroformxoamyl alcohol (24 I), precxpi- tated with 0.1 volume of 7 Y ammomum acetate and 2 5 volumes of ethanol, and resuspended m H,O at a final concentration of 0.5 mg/ml.

Xenopus Egg Extract-The preparation of the Xenopus egg extract was as prewously described (Matthews and Colman, 1991) Transla- tlons were performed by addltlon of mRNA to an aliquot of extract containing 10% (v/v) nuclease-treated rabbit reticulocyte lysate. 10 FM( creatme phosphate, 0.2 p.v spendme, and either 1 m&/ml I’%lme- thlonme or 2 mWm1 [‘Hlleucme.

Protease Protectron-PC2 mRNA was translated in the egg extract containing [:‘“Slmethionine for 1 h at 21 “C. 5 pl of translation mixture was added to 5 pl of protemase K (200 pg/ml m phosphate-buffered salme, 10% sucrose which had been previously incubated at 37 “C for 15 min to remove lipase activity) and incubated on Ice for 30 mm either m the presence or absence of 1% (v/v) Tnton X-100 Phenylmethylsulfonyl fluoride was then added to a final concentration of 5 rnM and incubation continued for a further 10 nun on ice. 12.5 pl of 2-fold concentrated SDS-PAGE’ sample buffer was added and the samples boiled for 3 mm prior to SDS-PAGE and fluorography

Endoglycoscdase H Digestcons-PC2 mRNAwas translated m the egg extract containing [Yjlmethlonine for 1 h at 21 “C. 9 pl of translation mixture was added to 11.11 of 10% SDS and bolled for 5 min. After cooling on ice the translation mixture was added to 90 pl of 50 my MES, pH 6.0, 1 rnM phenylmethylsulfonyl fluoride, and 50 milliunits/ml endoglycosl- dase H (Boehnnger Mannheim) and incubated at 37 “C for 18 h Samples were then analyzed by SDS-PAGE and fluorography.

NH,-termrnal Rad~osequenccng-In vitro translations were per- formed using a mixture of I.-[“Hlleucine (2 mCl/ml) and I.-[‘%lmethio- rime (1 m&/ml). Products were subJected to SDS-PAGE, and the pro- teins were transferred to polyvmyhdine difluonde membrane using a semi-dry blotter. The posItIon of the deswed protems was ldentlfied by overnight autoradiography, and the corresponding sections of the paper excised wth a scalpel blade. Sequencing was performed in an Applied Biosystems ABI 473A Protein Sequencer and fractions from the first 20 cycles collected. 150 pl of each fraction was added to 5 ml of OptiPhase X (LKB Scmtillation Products, Loughborough, Leics., U.K.) and the radioactivity measured simultaneously on ‘H and ‘% channels of the scintillation counter.

RESULTS

In order to study processing events in the biosynthesis of PC2, mRNA was synthesized in an SP6 polymerase-catalyzed in vitro transcription reaction using a human PC2 cDNA as template. The mRNA was first translated in a rabbit reticulo- cyte cell-free system. The primary translation product, which exhibited an M, of 69,000, was identified by NHz-terminal se- quencing as prepro-PC2. When the translation was performed

1 The abbreviations used are’ PAGE, polyacrylamide gel electropho- resis; MES, 4-morpholmeethanesulfonx acid.

30- 1

End0 t1 - - + - - -

Prot K - - - - f + TX-100 - - - - - +

FIG. 1. Translation of PC2 mRNA in a Xenopus egg extract. PC2 mRNA was translated in the egg extract containing [?“Slmethionme. After a l-h translation period, samples of the translation mixture were either untreated (lanes 1 and 41, Incubated at 37 “C for 18 h m the absence (lane 2) or presence (lane 3) of endoglycosldase H, or treated wth protemase K in the absence (lane 5 ) or presence (lane 6) of Tnton X-100. Samples were then analyzed by SDS-PAGE and fluorography Molecular weight markers are m lane M, wth their molecular masses in kDa as shown.

A) PC2 B) PC3

75k t - - - 68k t -

87k i 60k I

------

Incubation time (h) 1 3 16 Incubation time (h) 1 3 16

FIG. 2. Translation of PC2 and PC3 mRNAs in a Xenopue egg extract. PC2 and PC3 mRNAs were translated in the egg extract con- taining [““Slmethionine. At 1, 3, and 18 h samples were removed and analyzed by SDS-PAGE and fluorography. Arrows in A point to unproc- essed pro-PC2, with a molecular mass of 75 kDa, and the processed PC2 with a molecular mass of 68 kDa. Arrows in B point to unprocessed pro-PC3 and processed PC3 with masses of 87 and 80 kDa, respectively.

in the presence of dog pancreas microsomes, a 25amino acid signal peptide was removed during segregation of the nascent polypeptide chain. Glycosylation of the polypeptide then oc- curred to generate a 75kDa propolypeptide (data not shown).

To investigate further the processing of the 75kDa propoly- peptide, a cell-free translation/translocation system prepared from Xenopus eggs was used (Matthews and Colman, 1991). This system has been shown to cleave signal peptides, segre- gate proteins into membranes, and to assemble polypeptide chains of multisubunit proteins. It can also support mannose- phosphorylation and O-linked glycosylation of precursor pro- teins, two events that take place beyond the endoplasmic re- ticulum within the secretory pathway. Using PC2 mRNA, a major polypeptide with an apparent M, of 75,000 was synthe- sized (Fig. 1, lane 1). This polypeptide was glycosylated as shown by its sensitivity to endoglycosidase H digestion (Fig. 1, lanes 2 and 3 ). The polypeptide was also efficiently segregated within microsomal membranes, since it was protected from digestion with proteinase K in the absence of detergent but not in the presence of detergent (Fig. 1, lanes 4-6).

Over longer periods of incubation the 75kDa PC2 precursor was slowly processed to a 68-kDa polypeptide (Fig. 2A ). After a 3-h incubation there was no detectable processing of the 75- kDa polypeptide, while at 16 h processing was approximately 50% complete. NHz-terminal radiosequencing of the 75- and the 68-kDa polypeptides demonstrated that processing of the 75-kDa PC2 precursor involved cleavage at the sequence Arg- Lys-Lys-Arg (amino acids 81-84). Thus the 75-kDa polypeptide

590 Maturation of PC2

A) 75 kD

E '9 500

z3Lm Y

z m

r

A400 0)

l o o

1 3 5 7 9 11 13 15 17 19

Cycle

C) 68 kD 700

~~

" 1 3 5 7 9 11 13 15 17 19

Cycle - 1 L 5

6) 75 kD 500,

' 1 3 5 7 9 11 13 15 17 19

Cycle

D) 68 kD - 1 I

" 1 3 5 7 9 11 13 I 5 17 19

Cycle

10 15 m 75 kD Ala Ser Ai'Glu Arg Pro Val Phe Thr Asn His Phe Leu Val Glu Leu His L G Gly Gly Glu Asp Lys

-1 t.1 5 10 15 20 68 kD Arg Lys Lys Arg Gly Tyr Arg Asp Ile Asn Glu Ile Asp Ile Asn Met Asn Asp Pro Leu Phe Thr Lys Gln

FIG. 3. Peptide sequence analysis of the mqjor 75- and WkDa PC2-related polypeptides synthesized in the Xenopus egg extract. Translations were performed in the presence of IRHlleucine and ["Slmethionine for 16 h, and the producta separated by SDS-PAGE prior to amino acid sequencing. The amino acid sequence of PC2 with the deduced cleavage sites generating the 75- and 68-kDa polypeptides indicated by the arrows is shown.

contained leucine at positions 10 and 13 and no methionines (Fig. 3, panels A and B ), confirming that signal peptide cleav- age had occurred after Ala25 in the nascent polypeptide chain, while the 68-kDa polypeptide contained leucine at position 16 and methionine at position 12 (Fig. 3, panels C and D ) . In a similar system the PC3 precusor (molecular mass 87,000) was processed much more rapidly to an 80-kDa polypeptide; proc- essing was 40% complete in 1 h, 80% in 3 h, and 100% after 16 h (Fig. 2 B ) .

To investigate mechanisms in the processing of pro-PC2, mRNAs encoding mutant PC2 precursors were translated using the Xenopus egg extract. The mutant PC2M2 precursor, in which the tetrabasic sequence Lys-Arg-Arg-Arg (amino acids 53-56) was changed to Lys-Val-Arg-Leu, was cleaved at the same rate as the wild type PC2 precursor (Fig. 4). The mutant precursor PC2M3, in which the tetrabasic sequence Arg-Lys- Lys-Arg (amino acids 81-84) was deleted, was not processed (Fig. 41, confirming that this tetrabasic sequence is a recogni- tion site for the activity involved in processing the 75-kDa polypeptide. No cleavage of the precursor was observed when the active site Asp'42 was changed to Asn in PC2M4 (Fig. 5 ) , suggesting that the observed processing event was autocata- lytic.

The mutant PC2M4 propolypeptide migrated with a slightly faster mobility than the wild type PC2 propolypeptide (Fig. 5, compare lanes 1 and 2 with lanes 3 and 4 ). This effect of chang- ing the active site Asp to Asn on the gel mobility of subtilisin precursors ha$been previously noted (Power et al., 1986), and probably results from secondary structure changes induced by changing the catalytic pocket. When the wild type PC2 and the mutant PC2M4 mRNAs were co-translated, after 1 h the two precursor polypeptides could be clearly resolved (Fig. 5, lane 5). After 16 h two processed products were observed, consistent

PC2M2 PC2M3 PC2M4

Incubation time(h) 1 3 16 I 3 16 1 3 16

FTG. 4. Translation of mutant PC2 mRNh in a Xenopru egg extract. Reactions were performed for the indicated period of time in the presence of [35S]methionine. The products were subjected to SDS- PAGE and fluorography. The mutants are: PC2M2, Lys-Arg-Arg-Arg"6 to Lys-Val-Arg-Leu"6; PC2M3, a deletion of the tetrabasic sequence Arg-L~s-Lys-ArgR~; and PC2M4, the active site Asp'''* to AS^'^^.

with processing of both the wild type and the catalytically inactive mutant propolypeptides (Fig. 5, lane 6). This result demonstrated that the autocatalytic maturation of the PC2 precursor could involve intermolecular cleavage.

DISCUSSION In this study we have examined the post-translational proc-

essing of pro-PC2 in an in vitro translatiodtranslocation sys- tem prepared from Xenopus eggs. The 75-kDa pro-PC2 precur- sor was shown to undergo autocatalytic processing to the 68- kDa mature enzyme. Autocatalysis involved an intermolecular reaction. Our data do not, however, rule out the possibility that another protease of the secretory pathway might also be in- volved in the maturation of PC2. Furin, which recognizes the sequence Arg-X-(Lys/Arg)-Arg might be expected to recognize the Arg-Lys-Lys-Arg processing site within pro-PC2, and is

Maturation of PC2 591 M~ M I 2 3 4 5 6 200-

9 2 . 5 ~

69D -- . - """

46, .

30, - Time(h) I 16 1 16 1 16

RNA WTT M4 WiT+M4

FIG. 5. 'Runslation of wild type and mutant PC2 mRNAs in a Xenopus egg extract. Wild type PC2 mRNA ( W / 2') and X 2 M 4 mRNA (M4) were translated either separately or together in the egg extract containing ["%lmethionine. At 1 and 16 h, samples were removed and analyzed by SDS-PAGE and fluorography. Protein molecular mass markers are shown in lane M and their size in kDa indicated on the side of the gel.

thus a potential candidate for involvement in the maturation of pro-PC2.

That these processing events represent the situation in neu- roendocrine cells which normally express PC2, is supported by the characterization of a similar mature form of PC2 in bovine chromafin granules (Christie et al., 19911, insulin secretory granules isolated from islets of Langerhans (Guest et al., 1992), and in the sommatomammotrophin cell line GH4 or the African green monkey kidney cell line BSC40 infected with recombi- nant vaccinia constructs expressing a full-length PC2 cDNA (Benjannet et al., 1992).

In a previous study we demonstrated that, when expressed in Xenopus oocytes, maturation of PC2 involved the processing of the 75-kDa precursor to the 68-kDa form by way of a 71-kDa intermediate (Shennan et al., 1991b). In the present study the 75-kDa polypeptide was processed directly to the 68-kDa form. A possible explanation for this discrepancy is that the 75- to 71-kDa reaction is catalyzed by an endogenous oocyte protease, and that this activity is absent from, or markedly reduced in, the egg extract. The precise composition of the Xenopus egg extract in terms of cellular organelles is not known (Matthews and Colman, 1991). The extract will carry out mannose 6-phos- phate addition and 0-glycosylation, reactions normally per- formed in cellular compartments distal to the endoplasmic re- ticulum. However, the extract will not confer resistance to endoglycosidase H, suggesting that transfer to a functional medial-Golgi compartment does not occur. The 75- to 71-kDa processing may therefore be catalyzed by an endogenous oocyte protease within the Golgi or a post-Golgi compartment. Alter- natively, the protease responsible for this cleavage may not be active under the conditions used.

In addition to PC2 and PC3, Xenopus egg extract will also support processing ofXenopus furin.2 This emphasizes the gen- erality of the use of the Xenopus egg extract in the study of propolypeptide processing in vitro.

Processing of the PC3 precursor was much more rapid than that of the PC2 precursor. If pro-PC3 processing is also auto- catalytic, then the more rapid processing of pro-PC3 may be due to PC3 exhibiting a near neutral pH optimum close to that of the Xenopus extract, while PC2 has a more acidic pH opti- mum and higher Ca2+ requirement (Shennan et al., 1991a). In fact processing of pro-PC2 by the egg extract is enhanced when the reaction is performed in the presence of 5 mM calcium (data not shown). This difference in the rate of activation of PC3 and PC2 may explain the sequential processing of proinsulin in the

G. Matthews and A. Colman, unpublished results.

islets of Langerhans (Rhodes et al., 1992), first by PC3, which may become active at the near neutral pH of the cis-Golgi, and then by PC2, which is activated a t a later stage in the secretory pathway as the pH decreases and the Ca2+ concentration in- creases.

The 80-kDa processed form of PC3 was shown previously to be secreted from Xenopus oocytes, and to exhibit proteolytic activity against the peptide substrate proinsulin (Baiyles et al., 1992). This polypeptide may be similar to the 80-kDa form of PC3 secreted from BSC4O or GH4 cells infected with vaccinia recombinants expressing PC3 (mPC1) (Benjannet et al., 1992). Partial amino acid sequencing has shown that the 80-kDa poly- peptide is generated by cleavage of pro-PC3 after the sequence Arg-Ser-Lys-ArgR3 in BSC40 and GH4 cells.

Autocatalysis is a common feature in the activation of sub- tilisin-like proteases. Thus, the Kex-2 precursor (Wilcox and Fuller, 1992; Germain et al., 19921, the furin precursor (Rehe- mtulla et al., 19921, pro-subtilisin (Power et al., 1986; Ikemura et al., 19871, the proteinase B precursor of the yeast Saccharo- myces cerevisiae (Nebes and Jones, 19911, the precursor of the alkaline extracellular protease of Yawonia lipolytica (Fabre et al., 1991), and the precursor of the a-lytic protease of the bac- terium Lysobacter enzymogenes (Silen et al., 1989) all undergo autocatalytic processing. In some instances autocatalytic proc- essing has been shown to involve an intramolecular reaction (Carter and Wells, 1987; Ikemura and Inouye, 1988). Our pre- sent data demonstrate that the autocatalytic processing of pro- PC2 can involve an inter- molecular reaction.

The role of the propeptide of the subtilisin-like proteases has been the subject of intense interest. I t may serve a similar role to that of the pro-sequence of trypsinogen, which is to maintain the stable zymogen in an inactive state (Wong et al., 1984). In addition, the pro-sequence of the PC2 precursor may serve as a membrane anchor which is involved in the intracellular sorting of the precursor. Thus the 75-kDa form of pro-PC2 is mem- brane-associated, while the 68-kDa polypeptide is soluble (Ben- jannet et al., 1992).3 Another possible function of the pro-se- quence is to act as a template which stabilizes the protease domain allowing correct folding to occur (Silen et al., 1988). This proposal is supported by a series of elegant experiments demonstrating that the pro-sequences of the precursors of sub- tilisin, a-lytic protease, and alkaline extracellular protease, can activate the correct folding of an unfolded protease domain in an intermolecular reaction (Zhu et al., 1989; Silen and Agard, 1989; Fabre et al., 1991). I t remains to be seen whether the pro-sequence of PC2 or PC3 fulfills a similar role in guiding the correct folding of the protease domain. I t is clear, however, that the Xenopus egg extract translatiodtranslocation system will prove useful in addressing this problem and other aspects of propolypeptide processing.

Acknowledgments-We thank Don Steiner and Steve Smeekens for proriding cDNAs.

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