trans-complementation assay establishes the role of proregion hydrophobic amino acid residues in the...

YeastYeast 2003; 20: 397–406.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.972

Research Article

trans-Complementation assay establishes the role ofproregion hydrophobic amino acid residues in thebiosynthesis of Saccharomyces cerevisiae Kex2pendoproteaseGuillaume Lesage, Julie Guimond and Guy Boileau*Departement de Biochimie, Universite de Montreal, Montreal (Quebec) H3C 3J7, Canada

*Correspondence to:Guy Boileau, Departement deBiochimie, Universite deMontreal, C.P. 6128, Succ.Centre-Ville, Montreal, Qc,Canada H3C 3J7.E-mail:[email protected]

Received: 26 September 2002Accepted: 30 November 2002

AbstractThe proregion of Saccharomyces cerevisiae endoprotease Kex2p is essential for thebiosynthesis of an active enzyme. It has been suggested that the proregion acts inthe endoplasmic reticulum to catalyse folding of the enzyme. To identify amino acidresidues important for proregion function, we used an in vivo system in which theKex2p proregion can act in trans to activate a Kex2p enzyme synthesized withoutits proregion. Activation of Kex2p by wild-type and mutated proregions revealed theessential role of hydrophobic residues F37, V39 and F70 in enzyme activation. Furtherexploration of the role of these residues by in vitro inhibition of Kex2p activity by itsproregion indicated that they are essential to form the proregion/enzyme bimolecularcomplex. In contrast, basic residues K108 and R109, located in the C-terminus ofthe proregion, are not involved in complex formation but are necessary for thebiosynthesis of an active enzyme. Copyright 2003 John Wiley & Sons, Ltd.

Keywords: prodomain; subtilisin-like proteases; proprotein convertases

Introduction

Many secreted cellular proteins and peptides,including peptide hormones and neuropeptides,growth factors and adhesion molecules, are firstsynthesized as large protein precursors and requirelimited proteolysis to achieve activity (Bergeronet al., 2000; Seidah and Chretien, 1999). This pro-teolysis is carried out in vivo by serine endo-proteases of the subtilisin family, referred toas proprotein convertases (PCs). These enzymeshave been found in all eukaryotes, includingyeast (Julius et al., 1984; Mizuno et al., 1988;Wesolowski-Louvel et al., 1988; Davey et al.,1994; Newport and Agabian, 1997), the fruit flyDrosophila melanogaster (Roebroek et al., 1991,1992; Siekhaus and Fuller, 1999), the nema-tode Caenorhabditis elegans (Thacker and Rose,2000) and mammals (Seidah and Chretien, 1999).Like bacterial subtilisin, PCs are synthesized with

an N-terminal proregion that is cleaved in anautocatalytic manner soon after completion of theenzyme biosynthesis in the cell endoplasmic retic-ulum (ER) (Germain et al., 1992; Brenner et al.,1993; Gluschankov and Fuller, 1994; Goodman andGorman, 1994; Molloy et al., 1994; Zhou et al.,1995).

Two functions have been proposed for PC prore-gions. First, the observation that the furin proregionremains associated to the protease domain throughtransport in the secretory pathway, and is releasedonly in late compartments where calcium and pHconditions are favourable, suggested that PC prore-gions could act as specific inhibitors (Andersonet al., 1997). In vitro studies with purified activePCs and Escherichia coli-produced PC proregionsconfirmed that, while associated to the active pro-tease domain, the proregions act as competitiveinhibitors (Boudreault et al., 1998; Zhong et al.,1999; Bhattacharjya et al., 2000). Second, since

Copyright 2003 John Wiley & Sons, Ltd.

398 G. Lesage, J. Guimond and G. Boileau

work performed on yeast and mammalian PCsshowed that proregions are essential for enzymeactivity and transport out of the ER (Rehemtullaet al., 1992; Zhou et al., 1995; Lesage et al., 2000;Muller et al., 2000), it was suggested, by analogywith subtilisin, that it may help folding of the pro-tein. Because of the involvement of a domain inthe folding of the remaining part of the protein,the concept of the intramolecular chaperone wasintroduced (Inouye, 1991).

The best-studied examples of proregions arethose of bacterial secreted serine protease subtil-isin from Bacillus amyloliquefaciens and α-lyticprotease from Lysobacter enzymogenes. When pro-duced without their N-terminal prosegment, theseenzymes remained trapped in an inactive con-formation, the molten-globule (Eder et al., 1993;Anderson et al., 1999). This stable folding inter-mediate has native-like secondary structure butessentially lacks tertiary structure possessed by thenative enzyme. Upon proregion addition, the inter-mediate rapidly folds into the fully active matureprotease domain. In both cases, stabilization of asubstructure within the protease domain would actas a nucleus, from which folding propagates to theremaining parts of the enzyme (Gallagher et al.,1995; Sauter et al., 1998). In vivo, the proregionand the protease domain are linked together untilfolding of the proprotein is completed. The prore-gion is then cleaved from the protease domain in anautocatalytic and intramolecular process. This pro-teolytic event is necessary but not sufficient for pro-tease activation. Indeed, the prosegment remainsbound in a substrate-like manner with the enzyme,which it competitively inhibits. The enzyme activa-tion occurs by proregion degradation (autocatalyticprocess) concomitantly with rearrangement of theprotease N-terminus.

We have used the baker’s yeast Saccharomycescerevisiae as a model to study proregion function.This organism possesses only one PC gene, KEX2,encoding the Kex2p protein, which is essential torelease active copies of the α-mating factor froman inactive high molecular mass precursor andto activate killer toxins by limited proteolysis atpairs of basic amino acid residues (Fuller et al.,1985; Magliani et al., 1997). Kex2p was the firstmember of the eukaryotic convertase family tobe molecularly cloned (Julius et al., 1984) andsequence analysis indicated that it possesses aproregion at the N-terminus (Wilcox and Fuller,

1991). As for other PCs, the Kex2p proregionis essential for biosynthesis of an active enzyme(Lesage et al., 2000). Interestingly, the Kex2pproregion can act in trans to activate a Kex2penzyme synthesized without its proregion (Lesageet al., 2000).

In the present study, we have compared prore-gions from yeast and mammalian PCs to iden-tify conserved amino acid residues that could beimportant for active enzyme biosynthesis, and mod-ified these residues by mutagenesis. The functionof mutated proregions was assessed in vivo in atrans-complementation assay designed to monitorproduction of active α-mating factor by Kex2p(Lesage et al., 2000), and in vitro by inhibition ofenzymatic activity (Lesage et al., 2001). Our dataindicate that hydrophobic amino acid residues con-served in all eukaryotic convertase proregions areessential for proregion function in vivo and inhibi-tion of Kex2p activity in vitro.

Materials and methods

DNA manipulations

DNA manipulations were performed using stan-dard procedures (Ausubel et al., 1993). Correctsequences of PCR-generated constructs were con-firmed by DNA sequencing.

Kex2p proregion deletion and pointmutagenesis

Deletions and point mutations were performed byPCR using plasmid pAPR2 (Lesage et al., 2000)as template. The mutants used in the present studyare listed in Table 1.

Yeast strains and growth conditions

YPD, synthetic minimal, synthetic complete anddrop-out media were as described (Ausubel et al.,1993). The GLY41 strain [MATα kex2::LEU2 ura3trp1 his3 leu2 (pGL9, kex2�pro,HA, URA3 )],which expresses the kex2�pro,HA protein froma URA3-based vector (Lesage et al., 2000), wastransformed with constructs bearing various prore-gion mutants by the lithium acetate procedure(Gietz et al., 1992). Transformants were selectedon medium lacking uracil and tryptophan.

Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 397–406.

Proregion-dependent Kex2 activation 399

Table 1. Mutants used in this study

Allele DNA sequence Reference

Kex2�pro,HA Codons 24 to 109 deleteda; HA epitope Lesage et al. (2000)oligonucleotides inserted after codon 812

�10: Q24-S33b deleted Codons 24 to 33 deleted This study

�15: Q24-A38 deleted Codons 24 to 38 deleted This study�10-20: R34-E43 deleted Codons 34 to 43 deleted This studyF37G Codon 37: TTT → GGT This studyF37V Codon 37: TTT → GTT This studyV39G Codon 39: GTA → GGG This studyV39L Codon 39: GTA → CTA This studyF37G/V39G Codon 37: TTT → GGT; codon 39: GTA → GGG This studyF37V/V39L Codon 37: TTT → GTT; codon 39: GTA → CTA This studyN42Q Codon 42: AAT → CAG This studyN42A Codon 42: AAT → GCT This studyY68G Codon 68: TAT → GGA This studyY68H Codon 68: TAT → CAT This studyF70G Codon 70: TTT → GGA This studyF70L Codon 70: TTT → TTG This studyY68G/F70G Codon 68: TAT → GGA; codon 70:TTT → GGA This studyY68H/F70L Codon 68: TAT → CAT; codon 70: TTT → TTG This studyR80G Codon 80: AGA → GGA; codon 81: TCA → TCCc This studyK79G/R80G Codon 79: AAA → GGA; codon 80: AGA → GGA; This study

codon 81: TCA → TCCc

K108G/R109G Codon 108: AAG → GGG; codon 109 AGA → GGG Lesage et al. (2000)�R109: R109 deleted Codon 109 deleted Lesage et al. (2000)

a Codon numbering starts at methionine initiator codon.b Amino acid numbering includes residues from the signal peptide.c Silent mutation of S81 (TCA → TCC) introduces a BamHI site.

Halo assay for α-factor secretion

The assay for in vivo Kex2p activity was performedas previously described (Lesage et al., 2000).Briefly, exponentially growing cells (MATα) werespotted onto a lawn of M200-6CK cells (MAT a sst1sst2), hypersensitive to α-factor (Whiteway et al.,1988). Halos around tested colonies resulted fromgrowth arrest of layer cells in response to secretedactive α-pheromone.

Protein production in P. pastoris and E. coli

Production of a secreted soluble form of Kex2p,ssKex2p, in P. pastoris and production ofproregion in E. coli have been described previously(Lesage et al., 2001). Briefly, wild-type andmutated proregions were produced as GST fusionproteins and purified on glutathione-Sepharosebeads (Amersham Pharmacia Biotech, Uppsala,Sweden). The proregions were cleaved from theGST moiety by thrombin digestion, which left twoadditional amino acid residues (Gly–Ser) in theN-terminus of the purified proregions. All purified

proteins and peptides showed more than 90% purityafter separation by SDS–PAGE and silver staining(not shown).

Enzymatic assay for Kex2p activity

Assays (100 µl final volume) were performedin 50 mM Tris–acetate, pH 7.0, 2 mM CaCl2.Enzyme concentration (culture supernatant as pre-pared above) in the assay was 0.27 µg/ml. Thesubstrate (pERTKR-MCA, Peptide InternationalInc., Louisville, KY) was prepared to 2 mM inDMSO and 5 µl stock solution were added to theassay. Dilutions of wild-type and mutant proregionsranged from 10 nM to 3.5 mM in PBS, and 1 µl eachdilution was used for inhibition assays. Readingswere performed for 10 min at room temperaturein a Perkin-Elmer HTS 7000 plate reader with360 nm and 465 nm excitation and emission fil-ters, respectively. Initial velocities were determinedby calculating the slope for aligned points (reac-tions typically advanced linearly for 4–6 min) andexpressed in fluorescence units/min. For the IC50



determinations, initial velocities in the presence ofthe proregion were related to that obtained withoutthe proregion. Data were collected in duplicate andeach value is the mean of at least three independentexperiments. Finally, data were fitted using Prismsoftware (GraphPad Software Inc.).

Results

Identification of proregion target residues formutagenesis

Target residues were identified by comparisonof proregion amino acid sequences from yeast(S. cerevisiae Kex2p, Kluyveromyces lactis Kex1p

and Candida albicans Kex2p) with mammalianPCs (human furin, PC1, PC2, PACE4 and murinePC4 and PC5) (Figure 1A). Overall identity bet-ween proregions from yeasts and mammals wasvery low (12–23.2% identity between S. cerevisiaeKex2p and mammalian PCs; Figure 1A). Neverthe-less, two hydrophobic motifs, F37AV and Y68VF,in S. cerevisiae appeared to be conserved in allproregions of eukaryotic subtilase family mem-bers (Figure 1A; Siezen et al., 1995). In addition,both pairs of basic residues, K79R80 and K108R109,in S. cerevisiae, which are conserved in all fam-ily members, and a putative N-glycosylation site,N42ET, in S. cerevisiae, present in proregions fromyeast, were also identified for mutagenesis (muta-tions are depicted in Figure 1B). The function of

Figure 1. Identification of target residues and trans-complementation assay. (A) Partial sequence alignments of proregionsfrom PCs. Sequences of Kex2p homologues are available from the Swiss-Prot database. Accession Nos are: SCKex2,P13134; KLKex1, P09231; CAKex2, O13359; hFur, P09958; hPC1, P29120; hPC2, P16519; mPC4, P29121; hPACE4, P29122 ;mPC5, Q04592. Alignment was performed using the Multiple Alignment Program available from the search launcher ofBaylor College of Medicine (http://searchlauncher.bcm.tmc.edu/). The reference for calculation of sequence identity(in parentheses) is S. cerevisiae Kex2p proregion. Motifs conserved among PCs and Kex2p proregions are shown asblack boxes. Putative N-glycosylation and dibasic sites are shown as white and grey boxes, respectively. (B) Schematicrepresentation of Kex2p proregion and of target residues for site-directed mutagenesis. (C) trans-Complementation assay.In contrast to wild-type Kex2p, expression of Kex2p devoid of its proregion (kex2�pro,HA) does not restore productionof active α-mating factor in M200-6CK cells, as measured by the halo assay. However, expression of kex2�pro,HA andproregion in trans results in secretion of active α-mating factor. This trans-complementation assay is used to monitor thefunction of mutated proregions. The grey box depicts the proregion


http://searchlauncher.bcm.tmc.edu/


the mutated proregions was assessed in a trans-complementation assay that measures production ofactive α-mating factor (Figure 1C), and by in vitroinhibition of Kex2p enzymatic activity.

Hydrophobic amino acid residues at positions37, 39 and 70 are critical for proregion function

We first used deletion mutagenesis to probe theimportance of the F37AV motif in proregionfunction. Removing the proregion N-terminal 10residues (Q24 –S33, numbering includes amino acidresidues from the signal peptide) only slightlydecreased Kex2p activity (Figure 2, �10). In con-trast, deletions of amino acid residues Q24 –A38 orR34 –E43 totally inactivated the proregion (Figure 2,�15 and �11–20, respectively). Deletion �10 leftthe F37AV motif intact, while the two other con-structs deleted it either in part (�15) or in full(�11–20). The importance of this motif for prore-gion function was thus further analysed by usingsite-directed mutagenesis. The proregion was inac-tive when F37 and/or V39 were changed to G(Figure 3A, Nos 4–6). In contrast, conserving the

Figure 2. Effects of N-terminal deletions on Kex2pproregion function. Plasmids encoding deletions of theproregion N-terminus were introduced in strain GLY41and their ability to activate in trans kex2�pro,HA wasassessed by the halo assay. �10, deletion of proregionamino acid Q24 –S33;�15, deletion of proregion aminoacid Q24 –A38;�11–20, deletion of proregion amino acidR34 –E43

Figure 3. Mutational analysis of the proregion F37AVand N42ET motifs. (A) Effects of mutations on Kex2pactivity. Halo assay was performed with GLY41 expressingwild-type proregion (No. 1), no proregion (No. 2), andproregions mutated either in the F37AV (F37V/V39L, No. 3;F37G/V39G, No. 4; V39G, No. 5; F37G, No. 6; F37V, No. 7;V39L, No. 8), or N42ET motif (N42Q, No. 9; N42A, No. 10).(B) Inhibition of Kex2p by wild-type, V39L and F37G/V39Gproregions. The curves represent the progressive inhibitionof pERTKR–MCA cleavage by Kex2p in the presence ofincreasing concentrations of wild-type (open circles), V39L(closed circles), or F37G/V39G (closed squares) proregions.The relative activity was determined by dividing initialvelocities in the presence of the proregion by that obtainedfor the control without proregion. Data are the means(± standard deviation) of three independent experiments

hydrophobic character of the motif by changing F37to V (Figure 3A, No. 7) or V39 to L (Figure 3A,No. 8) or replacing both F37 and V39 by V and L,respectively (Figure 3A, No. 3) resulted in Kex2pactivity, although to a lesser level than wild-typeproregion. These results suggest a requirement forhydrophobic residues at positions 37 and 39 forproregion function.

In addition to deleting motif F37AV, mutant�11–20 destroyed a putative N -glycosylation site(N42ET) present in proregions of Kex2p from



S. cerevisiae and C. albicans (Figure 1). Theimportance of this site for proregion function wasalso analysed by site-directed mutagenesis. Chang-ing N42 to Q or A affected proregion function onlyslightly (Figure 3A, Nos 9 and 10), indicating thateven if N-glycosylation actually occurs in vivo atthis site, this modification is not essential for prore-gion function.

To better understand the role of F37AV motif inproregion function, we produced in E. coli wild-type and mutated proregions (F37G/V39G andV39L) that are, respectively, inactive or partiallyactive in the halo assay. These purified peptideswere used as inhibitors in an in vitro enzymaticassay using a soluble form of Kex2p and thefluorogenic peptide pERTKR–MCA as substrate(Lesage et al., 2001). IC50 values were determinedfor each proregion and used as relative indication ofproregion/mature protease domain interaction. TheF37G/V39G proregion, in which both hydrophobicamino acid residues were replaced by glycine,had little inhibitory effect on Kex2p (Figure 3B,IC50 > 50µM; a 200–300-fold increase over wild-type). In contrast, the V39L proregion, wherethe hydrophobic characteristic of the motif wasmaintained, had an inhibitory potency similar tothat of the wild-type proregion (Figure 3B, IC50 =130 and 160 nM for the mutant and the wild-typeproregions, respectively). These results suggest thatthe F37AV motif is important for proregion bindingto the mature protein domain.

S. cerevisiae Kex2p proregion (SCKex2p;Figure 1), harbours the sequence Y68VF thatcould correspond to the YHF sequence describedin subtilase proregions (Siezen et al., 1995). Tostudy the importance of this sequence in Kex2pproregion function, the substitutions of Y68 toH (Figure 4A, No. 3) or G (Figure 4A, No.4) were performed by site-directed mutagenesis.These mutations had no or very little effect onproregion function, as the sizes of halos observedwith mutants were comparable to that obtainedwith wild-type proregion (Figure 4A, No. 2). Insharp contrast, changing F70 to G (Figure 4A,Nos 6 and 8) abolished proregion function, whilethe conservative substitution of this residue toL (Figure 4A, No. 5) maintained function buthad a slight effect when combined with theY68H mutation (Figure 4A, No. 7). Furthermore,the F70 to G mutation significantly affected theinhibitory potency of the proregion (Figure 4B,

IC50 = 5.8µM; a 36-fold increase over wild-type),suggesting that the presence of a hydrophobicresidue is required at position 70 for efficientproregion binding to the mature protein domain.

S. cerevisiae Kex2p prodomain also exhibits aninternal pair of basic amino acid residues con-served among all proregions of PC family mem-bers (K79R80 in SCKex2p; Figure 1A). Cleavageat this site is believed to be essential for PC acti-vation (Anderson et al., 1997; Seidah et al., 1998).Changing this site to KG (Figure 4A, No. 9) orGG (Figure 4A, No. 10) resulted in growth inhi-bition zones as large as the wild-type (Figure 4A,

Figure 4. Mutational analysis of the Y68, F70 and K79R80motifs. (A) Effects of mutations on Kex2p activity. Halo assaywas performed with GLY41 alone (No. 1) or transformedwith plasmid expressing wild-type proregion (No. 2), orproregions mutated at Y68F70 (Y68H, No. 3; Y68G, No. 4;F70L, No. 5, F70G, No. 6; Y68H/F70L, No. 7; Y68G/F70G,No. 8) or internal dibasic motif (R80G, No. 9; K79G/R80G,No. 10). (B) Inhibition of Kex2p by wild-type and F70Gproregions. Kex2p activities observed in the presence ofincreasing concentrations of wild-type (open circles, samedata as in Figure 3B) or F70G (closed circles) proregionswere used to calculate relative activity as in Figure 3B



No. 2), indicating that the basic doublet K79R80 isdispensable for biosynthesis of active enzyme.

Mutation of K108R109 does not prevent bindingof proregion to mature protein domain

We have shown in previous work that the twoC-terminal basic amino acid residues (K108R109)are important for proregion function (Lesage et al.,2000). To determine whether mutations of theseresidues affected proregion binding to Kex2p,we have tested, in the in vitro enzymatic assay,the inhibitory potency of proregions in whichboth basic residues were replaced by glycine(K108G/R109G), or in which the C-terminal argi-nine was deleted (�R109). Surprisingly, none ofthe mutations had a dramatic effect on inhibitorypotency, since the IC50 of K108G/R109G and�R109 mutants were 720 and 730 nM, respectively,only a five-fold increase over wild-type (Figure 5),suggesting that the K108R109 residues are not essen-tial for proregion binding to the enzyme.

Discussion

The Kex2p endoprotease of the yeast S. cere-visiae is a subtilisin-like enzyme involved in theproteolytic processing of pro-α-mating factor andpro-killer toxins (Fuller et al., 1985; Maglianiet al., 1997). Like subtilisin and its mammalian

Figure 5. Inhibition of Kex2p by proregions mutated atK108R109. Kex2p activities observed in the presence ofincreasing concentration of wild-type (open circles, samedata as in Figure 3B), K108G/R109G (closed circles) or�R109 (closed squares) proregions were used to calculaterelative activity, as in Figure 3B

homologues, Kex2p is first synthesized with anN-terminal proregion that is rapidly removed byan autocatalytic reaction (Wilcox and Fuller, 1991;Germain et al., 1992). When produced without thisdomain, Kex2p is inactive and probably trapped inthe ER in a partially folded conformation. Activa-tion of the enzyme can be restored by expressingthe proregion in trans (Lesage et al., 2000). Fromthese observations and data obtained with subtil-isin and α-lytic protease (Ikemura et al., 1987; Zhuet al., 1989; Baker et al., 1992), it was speculatedthat Kex2p proregion interacts with the mature pro-tein domain and facilitates its folding and activa-tion (Lesage et al., 2000). To better understand themechanism of proregion-dependent Kex2p activa-tion, we have expressed in yeast cells disruptedfor the KEX2 gene a mutant Kex2p deleted of itsproregion (kex2�pro,HA mutant) and proregionsmutated at specific amino acid residues. Kex2pactivity in transformed cells was monitored in vivoby a halo assay, based on the efficiency of α-matingfactor maturation, and proregion binding to theactive protease domain was monitored in vitro byinhibition of enzymatic activity.

Three hydrophobic residues where identified asimportant for proregion binding to the proteasedomain. These are F37, V39 and F70. Interest-ingly, these residues are conserved in proregionsof eukaryote subtilisin-like enzymes. Replacementof these residues by non-hydrophobic side chainsprevented in vitro inhibition of Kex2p activity andin vivo processing of pro-α-mating factor, sug-gesting that these hydrophobic residues are criticalfor proregion–Kex2p protease domain interaction.Interestingly, crystal structure analysis revealedthat hydrophobic interactions are involved in pack-ing of the subtilisin prosegment against the proteasedomain (Gallagher et al., 1995). Our observationsare consistent with a similar role of hydrophobicresidues for the function of Kex2p proregion. Sincethese hydrophobic residues are conserved in prore-gions from all PCs, we suggest that they are alsoinvolved in proregion–PC protease domain interac-tion. However, our work (Lesage et al., 2000) andthat of others (Zhu et al., 1989; Zhong et al., 1999)has shown conclusively the specificity of interac-tion of a proregion with its cognate enzyme. Thus,other amino acid residues or structural features,yet to be identified, must be responsible for thisspecificity.



A C-terminal pair of basic amino acid residuesis also a constant feature of eukaryotic proregions(K108R109 in S. cerevisiae; Figure 1). This sequenceis also the preferred cleavage motif of Kex2p. Incontrast to our data on the hydrophobic residues,mutation or deletion of these basic amino acidresidues had little effect on the inhibition potencyof the proregion, indicating that they are dispens-able for proregion binding to the enzyme domain.In light of our previous findings that deletion ormutation of these residues severely impaired pro-α-mating factor processing in vivo (Lesage et al.,2000), we suggest that these residues may ratherbe involved in the folding catalysis per se. Similarconclusions were reached for α-lytic protease. Inthis case, deletion of the three C-terminal residuesof the proregion only slightly affected proregionbinding to the protease domain (Sauter et al., 1998)but abolished folding activity (Peters et al., 1998).In contrast, the C-terminal residues of the subtilisinproregion are critical for folding activity as wellas for inhibition (Wang et al., 1995). Furthermore,refolding of subtilisin in the presence of a mutantproregion led to a protein structurally and enzy-matically distinct from wild-type subtilisin (Shindeand Inouye, 1997). These observations suggest that:(a) proregion binding and proregion-assisted fold-ing mechanisms appear to differ from one pro-tein to another; and (b) proregion is important informing the enzyme active site. Characterization ofthe enzymatic properties of Kex2p produced withactive mutant proregions would thus be an inter-esting issue. In contrast to the C-terminal basicresidues, proregions mutated at the internal basicdoublet (K79R80) were still fully functional in trans,suggesting that these residues are not essential forKex2p proregion function.

The binding of Kex2p proregion to the catalyticdomain requires hydrophobic residues in proregionpositions 37, 39 and 70. This binding most proba-bly occurs in the ER, since the proregion is essen-tial for ER exit and transport of the enzyme tothe Golgi (Lesage et al., 2000). Work performedon mammalian furin (Anderson et al., 1997) andPCs (Seidah et al., 1998) indicates that inactivebimolecular complexes between these proteasesand their cognate proregion are transported out ofthe ER to the Golgi compartment. Activation ofthe enzymes requires dissociation of the complexes,which is triggered by cleavage of the proregion atan internal pair of basic amino acid residues, as the

enzymes enter their final subcellular compartment.A similar scenario can be proposed for Kex2p,since the fully-folded enzyme can be inhibited byits proregion (Lesage et al., 2001). However, inthis latter case, internal proteolysis is not required,since activation of the enzyme does not involve theintegrity of the K79R80 internal site. The impor-tance of this late proteolytic step for activationof kexin-like enzymes may reflect the inhibitionpotency of the proregions. In agreement with thishypothesis, proregions of furin and PC7, which arepotent inhibitors (IC50 = 4 and 0.4 nM, respectively;Zhong et al., 1999), are internally cleaved, whereasthat of Kex2p, which is less potent (160 nM; Lesageet al., 2001), is not. In the case of Kex2p, activationwould rather be triggered either by physicochemi-cal changes between ER and Golgi, such as pH orCa2+ concentration, or by other post-translationalmodification of the proregion and/or the catalyticdomain.

Taken together with our previous findings show-ing that none of the mammalian proregions couldtrans-activate kex2p�pro,HA (Lesage et al., 2000),our present results suggest that the Kex2p prore-gion acts as a specific foldase to promote biosyn-thesis of an active enzyme.

Acknowledgements

We thank Marie-Eve Lane, Melanie Tremblay and MelanieCousineau for technical assistance. We also thank Dr LuisA. Rokeach for careful manuscript reading. This work wassupported by Grant MT-10979 from the Canadian Institutefor Health Research to G. B.

References

Anderson DE, Peters RJ, Wilk B, Agard DA. 1999. α-Lyticprotease precursor: characterization of a structured foldingintermediate. Biochemistry 38: 4728–4735.

Anderson ED, Van Slyke JK, Thulin CD, Jean F, Thomas G.1997. Activation of the furin endoprotease is a multiple-stepprocess: requirements for acidification and internal propeptidecleavage. EMBO J 16: 1508–1518.

Ausubel FM, Brent R, Kingston RE, et al. 1993. CurrentProtocols in Molecular Biology. Wiley: New York.

Baker D, Sohl JL, Agard DA. 1992. A protein-folding reactionunder kinetic control. Nature 356: 263–265.

Bergeron F, Leduc R, Day R. 2000. Subtilase-like pro-protein con-vertases: from molecular specificity to therapeutic applications.J Mol Endocrinol 24: 1–22.

Bhattacharjya S, Xu P, Zhong M, Chretien M, Seidah NG, Ni F.2000. Inhibitory activity and structural characterization of a



C-terminal peptide fragment derived from the prosegment ofthe proprotein convertase PC7. Biochemistry 39: 2868–2877.

Boudreault A, Gauthier D, Lazure C. 1998. Proprotein convertasePC1/3-related peptides are potent slow tight- bindinginhibitors of murine PC1/3 and Hfurin. J Biol Chem 273:31 574–31 580.

Brenner C, Bevan A, Fuller RS. 1993. One-step site-directedmutagenesis of the Kex2 protease oxyanion hole. Curr Biol 3:498–506.

Davey J, Davis K, Imai Y, Yamamoto M, Matthews G. 1994. Iso-lation and characterization of krp, a dibasic endopeptidaserequired for cell viability in the fission yeast Schizosaccha-romyces pombe. EMBO J. 13: 5910–5921.

Eder J, Rheinnecker M, Fersht AR. 1993. Folding of subtilisinBPN′: characterization of a folding intermediate. Biochemistry32: 18–26.

Fuller RS, Brake AJ, Sterne R, et al. 1985. Post-translationalprocessing events in the maturation of yeast pheromoneprecursors. In UCLA Symposia on Molecular and CellularBiology: Yeast Cell Biology, Hicks J (ed.). Liss: New York;461–476.

Gallagher T, Gilliland G, Wang L, Bryan P. 1995. The pro-segment-subtilisin BPN′ complex: crystal structure of a specific‘foldase’. Structure 3: 907–914.

Germain D, Dumas F, Vernet T, et al. 1992. The pro-region of theKex2 endoprotease of Saccharomyces cerevisiae is removed byself-processing. FEBS Lett 299: 283–286.

Gietz D, Jean A St, Woods RA, Schiestl RH. 1992. Improvedmethod for high efficiency transformation of yeast cells. NucleicAcids Res 20: 1425.

Gluschankov P, Fuller RS. 1994. A C-terminal domain conservedin precursor processing proteases is required for intramolecularN-terminal maturation of pro-Kex2 protease. EMBO J 13:2280–2288.

Goodman LJ, Gorman CM. 1994. Autoproteolytic activation ofthe mouse prohormone convertase MPC1. Biochem Biophys ResCommun 201: 795–804.

Inouye M. 1991. Intramolecular chaperone: the role of the pro-peptide in protein folding. Enzyme 45: 314–321.

Ikemura H, Takagi H, Inouye M. 1987. Requirement of pro-sequence for the production of active subtilisin E in Escherichiacoli . J Biol Chem 262: 7859–7864.

Julius D, Brake AJ, Blair L, Kunisawa R, Thorner J. 1984.Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-αfactor. Cell 37: 1075–1089.

Lesage G, Prat A, Lacombe J, et al. 2000. The Kex2p proregion isessential for the biosynthesis of an active enzyme and requiresa C-terminal basic residue for its function. Mol Biol Cell 11:1947–1957.

Lesage G, Guimond J, Tremblay M, Boileau G. 2001. Mechanismof Kex2p inhibition by its proregion. FEBS Lett 508:332–336.

Magliani W, Conti S, Gerloni M, Bertolotti D, Polonelli L. 1997.Yeast killer systems. Clin Microbiol Rev 10: 369–400.

Mizuno K, Nakamura T, Ohshima T, Tanaka S, Matsuo H. 1988.Yeast Kex2 gene encodes an endopeptidase homologous tosubtilisin-like serine proteases. Biochem Biophys Res Commun156: 246–254.

Molloy SS, Thomas L, Van Slyke JK, Stenberg PE, Thomas G.1994. Intracellular trafficking and activation of the furin

proprotein convertase: localization to the TGN and recyclingfrom the cell surface. EMBO J 13: 18–33.

Muller L, Cameron A, Fortenberry Y, Apletalina EV, Lindberg I.2000. Processing and sorting of the prohormone convertase 2propeptide. J Biol Chem 275: 39 213–39 222.

Newport G, Agabian N. 1997. KEX2 influences Candida albicansproteinase secretion and hyphal formation. J. Biol. Chem. 272:28 954–28 961.

Peters RJ, Shiau AK, Sohl JL, et al. 1998. Pro region C-ter-minus:protease active site interactions are critical in catalyzingthe folding of α-lytic protease. Biochemistry 37: 12 058–12 067.

Rehemtulla A, Dorner AJ, Kaufman RJ. 1992. Regulation ofPACE propeptide-processing activity: requirement for apost-endoplasmic reticulum compartment and autoproteolyticactivation. Proc Natl Acad Sci USA 89: 8235–8239.

Roebroek AJ, Pauli IG, Zhang Y, Van de Ven WJ. 1991. cDNAsequence of a Drosophila melanogaster gene, Dfur1, encodinga protein structurally related to the subtilisin-like proproteinprocessing enzyme furin. FEBS Lett 289: 133–137.

Roebroek AJ, Creemers JW, Pauli IG, et al. 1992. Cloningand functional expression of Dfurin2, a subtilisin-likeproprotein processing enzyme of Drosophila melanogasterwith multiple repeats of a cysteine motif. J Biol Chem 267:17 208–17 215.

Sauter NK, Mau T, Rader SD, Agard DA. 1998. Structure ofα-lytic protease complexed with its pro-region. Nature StructBiol 5: 945–950.

Seidah NG, Mbikay M, Marcinkiewicz M, Chretien M. 1998.The mammalian precursor convertases: paralogs of thesubtilisin/kexin family of calcium-dependent serine proteinases.In Prohormone and Neuropeptide Precursor Processing,Hook VYH (ed.). Springer-Verlag: Heidelberg; 49–76.

Seidah NG, Chretien M. 1999. Proprotein and prohormoneconvertases: a family of subtilases generating diverse bioactivepolypeptides. Brain Res 848: 45–62.

Shinde U, Inouye M. 1997. Protein memory through alteredfolding mediated by intramolecular chaperones. Nature 389:520–522.

Siekhaus DE, Fuller RS. 1999. A role for amontillado, theDrosophila homolog of the neuropeptide precursor processingprotease PC2, in triggering hatching behavior. J Neurosci 19:6942–6954.

Siezen RJ, Leunissen JAM, Shinde U. 1995. Homology analysisof the propeptides of subtilisin-like serine proteases (subtilases).In Intramolecular Chaperones and Protein Folding, Shinde U,Inouye M, Austin RG (eds). Landes: Austin TX; 233–256.

Thacker C, Rose AM. 2000. A look at the Caenorhabditis elegansKex2/subtilisin-like proprotein convertase family. BioEssays 22:545–553.

Wang L, Ruvinov S, Strausberg S, et al. 1995. Prodomainmutations at the subtilisin interface: correlation of bindingenergy and the rate of catalysed folding. Biochemistry 34:15 415–15 420.

Wesolowski-Louvel M, Tanguy-Rougeau C, Fukuhara H. 1988. Anuclear gene required for the expression of the linear DNA-associated killer system in the yeast Kluyveromyces lactis . Yeast4: 71–81.

Wilcox CA, Fuller RS. 1991. Posttranslational processing of theprohormone-cleaving Kex2 protease in the Saccharomycescerevisiae secretory pathway. J Cell Biol 115: 297–307.



Whiteway M, Hougan L, Thomas DY. 1988. Expression of MFα1in MAT a cells supersensitive to α-factor leads to self-arrest. MolGen Genet 214: 85–88.

Zhong M, Munzer JS, Basak A, et al. 1999. The prosegments offurin and PC7 as potent inhibitors of proprotein convertases. Invitro and ex vivo assessment of their efficacy and selectivity. JBiol Chem 274: 33 913–33 920.

Zhou A, Paquet L, Mains RE. 1995. Structural elementsthat direct specific processing of different mammaliansubtilisin-like prohormone convertases. J Biol Chem 270:21 509–21 516.

Zhu X, Ohta Y, Jordan F, Inouye M. 1989. Pro-sequence ofsubtilisin can guide the refolding of denatured subtilisin in anintermolecular process. Nature 339: 483–484.


trans-complementation assay establishes the role of proregion hydrophobic amino acid residues in the...

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