MOLECULAR AND CELLULAR BIOLOGY,0270-7306/98/$04.0010

Jan. 1998, p. 400–408 Vol. 18, No. 1

Copyright © 1998, American Society for Microbiology

Activation of the Kexin from Schizosaccharomyces pombe RequiresInternal Cleavage of Its Initially Cleaved Prosequence

DALE POWNER AND JOHN DAVEY*

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom

Received 15 July 1997/Returned for modification 4 September 1997/Accepted 15 October 1997

Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteinsduring their transport through the secretory pathway. The enzymes themselves are made as inactive precur-sors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fissionyeast Schizosaccharomyces pombe. Using a cell-free translation-translocation system prepared from Xenopuseggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into theendoplasmic reticulum. The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probablyintramolecular and is inhibited by disruption of the P domain. Prosequence cleavage normally occurs atArg-Tyr-Lys-Arg1022 (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site. Cleavage of the prosequence is necessary butnot sufficient for activation, and Krp1 is initially unable to process substrates presented in trans. Full activationis achieved after further incubation in the extract and is coincident with the addition of O-linked sugars. Oglycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initiallycleaved prosequence at the internal site. Our results are consistent with a model in which the cleavedprosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of theenzyme. Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence. Furthersupport for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavableinternal site dramatically reduced the growth rate of otherwise wild-type S. pombe cells, an effect that was notseen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack theLys-Arg102 residues.

The maturation of prohormones and propolypeptides ofteninvolves cleavage at pairs of basic residues as the precursor istransported through the secretory pathway. In many cases,cleavage is performed by a member of the prohormone con-vertase or kexin family of endopeptidases (the Kex2 proteasefrom Saccharomyces cerevisiae was the first member of thefamily to be characterized) (for a review, see reference 45).The kexins themselves are also made as inactive precursors,and there has been considerable interest in defining the mech-anism by which they are activated. The process has been stud-ied in systems that include mammalian cells, yeast, Xenopusoocytes, and cell extracts, and the activation mechanism isthought to be broadly similar for each member of the family.All are synthesized with an amino-terminal presequence, aprosequence, a subtilisin-like catalytic domain, a P domain (12,15), and a carboxy-terminal region that may (e.g., Krp1, Kex2,and furin) or may not contain a hydrophobic transmembranedomain (Fig. 1). The presequence directs the precursor to thesecretory pathway and is removed during segregation into theendoplasmic reticulum (ER). The prosequence is presumed toplay a role in the correct folding of the catalytic domain and isusually removed in the ER (7, 31, 49) in a reaction that isbelieved to be autocatalytic and probably intramolecular (6, 16,29), although intermolecular cleavage can occur (29). The ma-ture enzyme is then transported along the secretory pathway toits site of action.

Prosequence cleavage at the primary cleavage site is neces-sary for the kexin to become active but is not sufficient for

activation, and additional steps are required before the enzymeis able to cleave substrates presented in trans (7, 31). The firstinsights into the nature of these additional steps came not fromkexins but from studies of the bacterial serine proteinasessubtilisin and a-lytic protease. These enzymes are evolution-arily related to the eukaryotic kexins and are also synthesizedwith an N-terminal prosequence that facilitates correct foldingof the protein before being removed by intramolecular cleav-age (18, 35, 42, 52). The cleaved prosequence is not releasedfrom the enzyme but remains noncovalently associated withthe catalytic domain and acts as an autoinhibitor (4, 13, 25, 26).The enzyme is able to become fully active only when theprosequence is degraded (18). A recent study of furin matu-ration suggests that the prosequence plays a similar inhibitoryrole in the activation of kexins (1). To investigate whether thecleaved prosequence plays a role in the activation of otherkexins, we studied the maturation of Krp1, a kexin from thefission yeast Schizosaccharomyces pombe (8). Because Krp1 isan essential enzyme and its overexpression is also lethal (8), ithas not been possible to perform these studies in its normalenvironment within the cell. Maturation has therefore beenstudied in vitro by using a coupled translation-translocationsystem prepared from Xenopus laevis eggs (28, 29), a systemthat has proved invaluable for the analysis of other members ofthe kexin family (29, 39). We found that prosequence cleavageis autocatalytic and probably intramolecular and that the effi-ciency of this reaction is influenced by both the sequence at thecleavage site and by the integrity of other parts of the protein.The cleaved prosequence seems to inhibit the activation ofKrp1, and the enzyme is able to become fully active only afteran additional, internal cleavage of the prosequence. Furthersupport for this model comes from in vivo analysis, showingthat overexpression of a mutant prosequence lacking the in-

* Corresponding author. Mailing address: Department of BiologicalSciences, University of Warwick, Coventry CV4 7AL, United King-dom. Phone: 01203 524204. Fax: 01203 523701. E-mail: PDJT@dna.bio.warwick.ac.uk.

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ternal cleavage site inhibited the growth of otherwise wild-typeyeast cells, presumably via inhibition of Krp1. Our results aresimilar to those reported for furin, although there are somedifferences between the two enzymes and the significance ofthese is discussed.

MATERIALS AND METHODS

Krp1 constructs. The starting template for constructing various mutants was a2,320-bp EcoRV-SnaBI fragment containing the entire coding region of the krp1gene (8). The SnaBI restriction site lies just downstream of the stop codon,whereas the EcoRV site was introduced immediately upstream of the initiatorcodon by PCR with primer JO95 (59-GGGATATCCCAGCACCATGCATCC-39[the EcoRV site is underlined, and the initiator ATG is shown in boldface]). AllPCRs used Pwo DNA polymerase (from Pyrococcus woesei; supplied by Boehr-inger Mannheim, Lewes, East Sussex, United Kingdom), as this has a 39-59exonuclease, or proofreading, activity and greatly reduces the introduction oferrors during amplification. All constructs were sequenced by the dideoxynucle-otide method with double-stranded DNA to confirm that only the appropriatechanges had been introduced.

Truncated forms of Krp1 were generated by PCR with JO95 as the senseprimer and an appropriate antisense primer that included two stop anticodons(TCATCA [shown in boldface]) and an EcoRV restriction site (underlined); JO617(59-GGGATATCATCACCAATTTTCAAACGTACC-39) for Krp1[W578*],JO616(59-GGGATATCATCACAACTGCCAATTTTCAAACG-39) for Krp1[L580*],JO618 (59-GGGATATCATCACAAAGCCAACTGCCAATTTTC-39) for Krp1[L582*], JO615 (59-GGGATATCATCACCACAAAGCCAACTGCCAA-39) forKrp1[W583*], JO516 (59-GGGATATCATCATCCCCACAAAGCCAACTG-39)for Krp1[G584*], JO517 (59-GGGATATCATCATTCTCCCCACAAAGCCAAC-39) for Krp1[E585*], JO613 (59-GGGATATCATCAAGAAGGGTTTTCAGATTCTCCC-39) for Krp1[S590*], JO518 (59-GGGATATCATCAGTATATTCCCAAGACCATC-39) for Krp1[Y611*], JO519 (59-GGGATATCATCATCTATAAGAGGGTTCCAATAC-39) for Krp1[Y667*], JO520 (59-GGGATATCATCATTTCCAAAATGCGGAAATCC-39) for Krp1[K696*], JO456 (59-GGGATATCATCATCGTTTTCGAATTGAACTTTG-39) for Krp1[R82*],JO457 (59-GGGATATCATCAGCGCCATCTGGGCGTCTGGGC-39) forKrp1[R99*], and JO458(59-GGGATATCATCACCGCTTGTAGCGCCATCTGGG-39) for Krp1[R102*].The PCR products were digested with EcoRV and cloned into pBluescript-KS(Stratagene Ltd., Cambridge, United Kingdom).

To change Ser371 to Ala, we took advantage of a KpnI restriction site imme-diately upstream of the appropriate codon in the krp1 gene and designed a sense

primer that incorporated this restriction site and the necessary TCA-to-GCAchange (JO459 is 59-GGTGGTACCGCAGCGGCTGC-39, where the KpnI siteis underlined and the T-to-G mutation is shown in boldface). JO459 and JO66(an antisense primer that lies downstream of the SnaBI site) were used to amplifythe C terminus of krp1, and this was then digested with KpnI and SnaBI. Themutated fragment was exchanged for the equivalent region from the wild-typekrp1 gene to produce krp1S371A.

Changes to the basic residues at the primary and/or internal cleavage siteswere achieved by exploiting the EcoRV site that we had previously introducedimmediately upstream of the initiator ATG (with JO95) and a unique BamHIsite that corresponds to codons 122 to 124 of the krp1 open reading frame. Thisregion can be amplified with JO95 and JO58 (antisense to a region just down-stream of the BamHI site). The mutation of Arg102 to Ala was thereforeachieved by first amplifying upstream regions with JO95 and JO420 (59-ACTCGCATCCGCCTTGTAGCG-39; antisense primer that changes the Arg102 anti-codon from CCG to CGC [shown in bold] for Ala) and then amplifying C-terminal regions with JO419 (59-CGCTACAAGGCGGATGCGAGT-39; senseprimer that changes the Arg102 codon from CGG to GCG [shown in bold] forAla) and JO58. The products from these individual reactions were mixed andamplified with JO95 and JO58 to generate the full-length EcoRV-BamHI frag-ment containing the Arg102-to-Ala mutation. This was then exchanged for theequivalent fragment from the wild-type krp1 gene to give krp1[KR82][KA102].The Arg102-to-Lys change was made in the same way by using JO349 (59-CGCTACAAGAAGGATGCGAGT-39; changes the CGG codon to AAG [shown inboldface]) as the sense primer and JO350 (59-ACTCGCATCCTTCTTGTAGCG-39; changes the CCG anticodon to CTT [shown in boldface]) as the antisenseprimer.

Mutations at the internal site took advantage of a unique BstBI site immedi-ately upstream of the appropriate region in the krp1 gene. We therefore designedsense primers that incorporated the BstBI site and made the appropriate changeat the internal cleavage site. A PCR with the relevant primer and JO58 (seeabove) therefore amplifies a mutated BstBI-BamHI fragment that can then beexchanged for the equivalent fragment of the krp1 gene. By using constructsalready containing appropriate mutations at the primary cleavage site, we wereable to prepare constructs mutated at both sites. The following two sense primerswere used to make the changes at the internal site (the changed codons [Lys81is normally AAA, and Arg82 is normally CGA] are shown in boldface, and theBstBI site is underlined): JO426 (59-GTTCAATTCGAAAAGCCGGCATTGATGCC-39) for Krp1[KA82] and JO427 (59-GTTCAATTCGAATTGCCGGCATTGATGCC-39) for Krp1[IA82].

All oligonucleotides were synthesized on an automatic synthesizer (modelBT8510; Biotech Instruments) by using the materials and conditions recom-

FIG. 1. Schematic of Krp1. Krp1 is a type I membrane protein with an N-terminal presequence (diagonal lines), a single transmembrane domain (wavy lines), anda short cytoplasmic domain (residues 696 to 709). The residues at the end of the prosequence are shown to highlight the location of the primary (Lys-Arg102) andinternal (Lys-Arg82) cleavage sites. The locations of residues used to truncate Krp1 are also indicated (Y611, R667, and K696). There are five potential sites for Nglycosylation (lollipops) and a Ser/Thr-rich region (light shading just N terminal of the transmembrane domain) with several potential sites for O glycosylation. Thecatalytic domain contains the active-site residues (Asp, His, Asn, and Ser) and is followed by the P domain (dark shading), a region of high sequence homology betweenmembers of the kexin family (15); residues near the predicted C terminus of the P domain are shown for comparison. S. pombe (Sp) Krp1 (8), Saccharomyces cerevisiae(Sc) Kex2 (10), Kluyveromyces lactis (Kl) Kex1 (46), Mus musculus (Mm) PC6 (33), Homo sapiens (Hs) PACE4 (20), Drosophila melanogaster (Dm) fur1 (36), Dm fur2(37), Hs PC2 (43), Hs PC3 (5 PC1) (44), Hs PC7 (5 PC8) (3), Hs fur1 (48), X. laevis (Xl) fur1 (21), and Mm PC4 (34) sequences are shown. AAs, amino acids.

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mended by the manufacturer. (This work was performed by Alta Bioscience atThe University of Birmingham, Birmingham, United Kingdom.)

In vitro transcription. mRNA was synthesized in an in vitro transcriptionreaction with SP6 RNA polymerase and templates produced by subcloning therequired Krp1 constructs into vector pSP64T (22). Transcription reactions wereperformed as described previously (28). The construction of pSP64Tmap2 hasalready been described (8); the map2 gene encodes the precursor of the S. pombeP-factor mating pheromone (19).

Xenopus egg extract. Xenopus egg extract was prepared as described previously(28). Translations were initiated by adding mRNA (final concentration, 100mg/ml) to an aliquot of extract containing 10% (vol/vol) nuclease-treated rabbitreticulocyte lysate (Promega, Southampton, United Kingdom), 10 mM creatinephosphate, 0.2 mM spermidine, and 2 mCi of [3H]leucine (130 Ci/mmol) (Am-ersham International, Little Chalfont, Buckinghamshire, United Kingdom) perml. All samples were incubated at 21°C for 1 h. When required, cycloheximidewas added to a final concentration of 2 mM to inhibit further translation, and theincubation was continued at 21°C. The analysis of catalytic fragments by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performedas described previously (28, 29), and cleaved prosequences were separated on a15% peptide gel by using an SDS-Tris-tricine buffer system (38). Quantitativeanalysis of the amount of Krp1-related material was made by using a Phosphor-Imager (Molecular Dynamics) with ImageQuant software. N-terminal radiose-quencing was performed as described previously (8). Briefly, proteins were trans-ferred to a polyvinylidene difluoride (PVDF) membrane with a semidry blotterand the appropriate section of the membrane (identified by autoradiography)was excised and subjected to cycle sequencing in an ABI 473A protein sequencer(Applied Biosystems) (this was performed by Alta Bioscience, University ofBirmingham). The injection of Xenopus laevis oocytes with Krp1 mRNA or acontrol mRNA (Map2 mRNA) and the preparation of membrane extracts forincubation with Krp1S371A were performed as described previously (8).

Krp1 activity assays. Samples of extract containing translated Krp1 werediluted by the addition of ice-cold 100 mM HEPES (pH 6.5)–2 mM CaCl2–1 mMphenylmethylsulfonyl fluoride–1 mM tosyl-L-phenylalanine chloromethyl ketoneto osmotically lyse membrane vesicles. These were collected by centrifugation atfull speed in a microcentrifuge at 4°C, and the pellets were resuspended in assaybuffer (100 mM HEPES [pH 6.5], 2 mM CaCl2, 0.5% Triton X-100, 1 mMphenylmethylsulfonyl fluoride, and 1 mM tosyl-L-phenylalanine chloromethylketone). Reaction mixtures were assembled in two halves, with one half contain-ing membranes from the extract and the other containing the fluorogenic peptideBoc.Arg-Thr-Lys-Arg–4-methyl-comaryl-7-amide (MCA) (Peptides InstituteInc., Louisville, Ky.) at 100 mM. All reactions were performed at 29°C, and bothhalves were preincubated at this temperature for 5 min before mixing. Theincrease in fluorescence was monitored with a luminescence spectrometer (mod-el LS-5; Perkin-Elmer) at an excitation wavelength of 365 nm and an emissionwavelength of 460 nm.

Expressing prosequence constructs in S. pombe. The prosequence regions ofrelevant Krp1 constructs were amplified by a PCR approach similar to thatdescribed above for generating truncated forms of Krp1. Briefly, sense primerJO638 introduced a BamHI restriction site just upstream of the initiator ATG(59-GGGGGATCCAGCACCATGCATCCTGCTTTGC-39 [the BamHI site isunderlined, and the initiator ATG is shown in bold]) and a stop anticodon (TCA[shown in bold]) and a BamHI restriction site (underlined) were introduced atthe required positions with the appropriate antisense primer, JO458 (59-GGGGATCCTCACCGCTTGTAGCGCCATCTG-39) for prosequences that termi-nate immediately after Arg102 (Krp1[R102*]) and JO451 (59-CCGGATCCTCAGTAGCGCCATCTGGGCG-39) for prosequences that terminate after Tyr100(Krp1[Y100*]). By using different Krp1 constructs as templates for PCR, wewere able to generate prosequences with different motifs at the internal cleavagesite ([KR82] and [KA82]). The PCR products were cloned into the BamHI siteof expression vector pREP3X (30), sequenced to confirm that only the appro-priate changes had been introduced, and transformed into S. pombe JY330 (aheterothallic haploid strain of the Plus mating type). Transformants were cul-tured in either minimal medium or minimal medium lacking thiamine to induceexpression from the nmt1 promoter (30). All yeast procedures were performedby standard methods (32).

RESULTS

Primary cleavage of the prosequence is autocatalytic. A 1-htranslation of Krp1 mRNA in Xenopus egg extract generatedtwo products (Fig. 2, lane 1). We had previously used radio-sequencing to show that the smaller polypeptide (apparent Mrof ;76,000) is the C-terminal portion of Krp1 generated bycleavage after Lys-Arg102 (8), and we believe that the otherproduct (apparent Mr of ;90,000) is proKrp1 (i.e., Krp1 thatlacks the presequence but still contains its prosequence). Toinvestigate whether cleavage of the prosequence is autocata-lytic, we used site-directed mutagenesis to change Ser371 toAla (a product referred to as Krp1S371A). A sequence com-

parison with other related enzymes indicated that Ser371 ofKrp1 is likely to form an essential part of the active site, and ifso, Krp1S371A would be expected to be catalytically inactive(14, 15, 40). Therefore, the fact that translation of Krp1S371AmRNA in Xenopus egg extract produced only proKrp1 (Fig. 2,lane 2) suggests that prosequence cleavage at the primary siteis an autocatalytic event. To confirm that Krp1 can cleavethe prosequence from proKrp1, we mixed the product fromKrp1S371A with membranes prepared from Xenopus oocytesthat had been injected with Krp1 mRNA (Fig. 2, lane 3). Thesemembranes provide a source of active Krp1 that is relativelyfree from related proteases (8), and the conversion of proKrp1to Krp1 demonstrates that Krp1 can cleave its own prose-quence through an intermolecular interaction.

We took advantage of the inability of Krp1S371A to removeits own prosequence to identify the site of presequence cleav-age. Krp1S371A mRNA was translated in egg extract, andproKrp1S371A was transferred to a PVDF membrane beforebeing subjected to amino acid sequencing (8). Peaks of [3H]leucine were observed in cycles 4, 9, and 11 (not shown), whichsuggests a presequence that is cleaved after Ser23 in the se-quence Val-Ser-Ser23-Cys-Ser-Pro.

N-terminal requirements for prosequence cleavage. A strik-ing feature of the prosequences of most kexins, including Krp1,is the presence of two distinct clusters of basic amino acids(41). One cluster (Arg-XXX-Lys-Arg) is the position that wehave identified as the site of initial cleavage of the Krp1 pro-sequence (8) and others have shown to be the site of prose-quence cleavage in furin (7, 24, 31, 49), Kex2 (2, 51), PC2 (29),and PC3 (16, 27, 50). This is often referred to as the primarycleavage site. The second cluster of basic residues has beenstudied less extensively, and until recently (see below), therehas been no convincing evidence of cleavage at this internalsite. We therefore decided to investigate cleavage in moredetail by generating mutant forms of Krp1 that lack the normalbasic motifs at either or both of these sites and analyzing theeffects in Xenopus egg extract (Fig. 3).

Mutations that disrupted the internal cluster of basic resi-

FIG. 2. Prosequence cleavage is autocatalytic. Krp1S371A or Krp1[IA82][KK102]S371A mRNA was translated in Xenopus egg extract for 1 h in thepresence of [3H]leucine and was then mixed in the presence of 1% Triton X-100with membrane extracts prepared from Xenopus oocytes that had been injectedwith either a control mRNA (lanes 2 and 5) or Krp1 mRNA (lanes 3 and 6). Thedigestion products were separated by SDS-PAGE. Lane 1 is included to aidinterpretation. It contained the products obtained from a 1-h translation of Krp1mRNA in egg extract; the products are proKrp1 (upper band) and Krp1 (lowerband). Lane 4 was from a 1-h translation of Krp1[IA82][KK102] mRNA anddemonstrates that the Lys-Lys102 motif at the primary cleavage site is cleaved inan active form of Krp1. The positions of molecular weight markers (in thou-sands) are shown on the left.

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dues (without affecting the primary cleavage site) did not pre-vent cleavage of the prosequence at the primary site (Fig. 3A,lanes 2 and 3). This is not unexpected, but it indicates thatcleavage of the prosequence at Lys-Arg102 does not requireprior cleavage at the internal site. We confirmed this by usinga high-percentage acrylamide gel to demonstrate that thecleaved prosequence was indeed full-length and comigratedwith the product obtained by translating Krp1[R102*] in ex-tract (Fig. 3B, lane 1). Krp1[R102*] is a shortened form ofKrp1 that is truncated immediately after Arg102 to give aproduct that contains residues 24 to 102 (inclusive) of full-length Krp1 (the first 23 residues are removed as the prese-quence).

Changing the primary cleavage site from the normal Lys-Argmotif to Lys-Lys also had little effect on processing at thisposition (Fig. 3A, lanes 4 through 6) (see below), but convert-ing the dibasic motif at the primary site to Lys-Ala inhibitedcleavage at this position and allowed us to investigate process-ing at other sites within the prosequence (Fig. 3A, lanes 7through 9). A mutant possessing the normal Lys-Arg82 motifat the internal site was cleaved at this position to generate acatalytic fragment that starts at residue 83 (Fig. 3A, lane 7; theidentity of this product was confirmed by radiosequencing [notshown]) and a prosequence that corresponds to residues 24 to82 of Krp1 (Fig. 3B, lane 2). The catalytic fragment underwenta second cleavage to generate a product with an apparent Mrthat is between those of products cleaved at the primary siteand products cleaved at the internal site. This cleavage mustoccur after the cleavage at Lys-Arg82, as we see only oneproduct on the high-percentage gel (Fig. 3B, lane 2); we would

not expect to detect the small peptide produced from thecatalytic fragment by this second cleavage. The identity of thissecond product is discussed below.

Removing the dibasic motifs at both the primary and inter-nal sites greatly reduced processing of the proprotein (Fig. 3A,lanes 8 and 9), and only limited cleavage was observed afterprolonged incubation in extract (Fig. 3A, lane 10). The cata-lytic fragment produced by this cleavage comigrated with theproduct generated by the second cleavage of Krp1[KA102](Fig. 3A, lane 7), whereas the prosequence migrated betweenthose produced after digestion at the primary and internal sites(Fig. 3B, lane 3). Krp1 contains three Arg residues between theinternal and primary cleavage sites (Fig. 1), and we suspectedthat this product was generated by cleavage at one of theseresidues. Given that Kex2 can cleave substrates at Pro-Argmotifs (53), we expected this cleavage to occur after Pro-Arg97and were surprised that sequencing showed that cleavage oc-curs after Trp-Arg99 (Fig. 4). This site is clearly less accessiblein full-length proKrp1 than in the protein that has already beencleaved at Lys-Arg102.

Primary cleavage of the prosequence is intramolecular. AsKrp1 does not normally cleave a substrate that contains aLys-Lys motif (8), the apparently efficient cleavage of mutantKrp1 proteins containing Lys-Lys102 (Fig. 3A, lanes 4 through6) appeared to contradict our earlier observation that the pri-mary cleavage of the prosequence is autocatalytic. We ad-dressed this issue by combining the Lys-Lys102 change with theSer371-to-Ala mutation that inactivates the enzyme. To avoidpotential problems with cleavage at the internal site, we alsointroduced the Ile-Ala82 change that completely inhibits cleav-age at this site (Fig. 3, lanes 9 and 10). As expected, translationof Krp1[IA82][KK102]S371A mRNA in Xenopus egg extractgenerated a single product corresponding to the proenzyme(Fig. 2, lane 5). However, in contrast to the result withproKrp1S371A (Fig. 2, lanes 2 and 3), active Krp1 was unableto remove the prosequence from proKrp1[IA82][KK102]S371A when it was presented in trans (Fig. 2, lane 6). Thisdifference is consistent with our previous work showing thatKrp1 has a strong preference for substrates containing Lys-Argmotifs rather than Lys-Lys motifs (8). Therefore, the efficientcleavage of the Lys-Lys102 site when it was present as part ofan enzymatically active form of Krp1 (Fig. 2, lane 4, and 3,lanes 4 through 6) suggests that initial cleavage of the prose-quence is not an intermolecular event but, rather, an intramo-lecular event normally.

C-terminal requirements for prosequence cleavage. Therehave been several studies to investigate the role of C-terminalsequences in the maturation of kexins (15, 17, 47). Much of thiswork has focused on the role of the P domain, a conservedregion of about 150 residues that is found just C terminal of thecatalytic domain and is present only in subtilisin-like enzymesimplicated in proteolytic processing rather than degradation(12, 15). Disruption of the P domain blocks the production ofactive enzyme, and although the nature of the defect has notalways been determined, it appears that this region is requiredfor efficient cleavage of the prosequence (15). We thereforeinvestigated the role of the C terminus in the maturation ofKrp1.

Stop codons were introduced at various positions along thekrp1 open reading frame, and mRNAs corresponding to thevarious truncated enzymes were translated in egg extract (Fig.5). Truncations that successively removed the cytoplasmic tail(Krp1[K696*]), the transmembrane domain (Krp1[R667*]),and the Ser/Thr-rich domain (Krp1[Y611*]) had no effect oncleavage of the prosequence (Fig. 5, lanes 4 through 6), whichappeared to occur as efficiently as it did in the full-length

FIG. 3. N-terminal requirements for prosequence cleavage. (A) Krp1 mRNAand mRNAs from various Krp1 mutants that lack the normal basic motifs ateither the primary or internal site were translated in Xenopus egg extract for 1 hin the presence of [3H]leucine before being analyzed by SDS-PAGE on a low-percentage gel (lanes 1 to 9). Krp1[IA82][KA102] mRNA was also translated for6 h before analysis in order to investigate the slow processing of this mutant (lane10). The positions of molecular weight markers (in thousands) are shown on theleft. (B) Selected samples were also analyzed on a high-percentage acrylamidegel (38). To aid in the interpretation of prosequence fragments, we preparedversions of Krp1 with stop codons introduced immediately after Arg102 (Krp1[R102*]), Arg99 (Krp1[R99*]), and Arg82 (Krp1[R82*]). Translation of thesetruncated proteins in extract resulted in translocation into microsomes andpresequence cleavage to generate markers that are appropriate for differentprosequences.

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protein (Fig. 5, lane 1) (full-length Krp1 contains 709 resi-dues). In contrast, truncating Krp1 at or N terminal of Ser590dramatically inhibited cleavage of the prosequence (Fig. 5,lanes 2 and 3); only partial processing was observed even afterprolonged incubation (Fig. 5, lanes 7 and 8). It should be notedthat for brevity, Fig. 5 contains only the results for Krp1[W578*] and Krp1[S590*] and that we obtained very similarresults with a series of mutants truncated between these twopoints (Krp1[L580*], Krp1[L582*], Krp1[W583*], Krp1[G584*],and Krp1[E585*]). Further truncations between Ser590 andTyr611 are needed to more closely define the region requiredfor efficient cleavage of the prosequence.

Activation of Krp1 in Xenopus egg extract. Autoproteolyticcleavage of the prosequence indicates that Krp1 becomes ac-tive soon after synthesis in Xenopus egg extract. However, wewere unable to detect any proteolytic activity in such sampleswhen fluorogenic substrates were used under conditions thatsupported cleavage by Krp1 prepared from yeast membranes(not shown). The inability of these samples to process sub-strates presented in trans is reminiscent of earlier studies inwhich furin activation was shown to require events that oc-curred after the cleavage of the prosequence at the primary site(31, 49). We therefore investigated whether egg extract wouldsupport the additional steps required to activate Krp1. To sim-plify interpretation of the results, we translated Krp1 mRNAfor 1 h in the presence of [3H]leucine before adding cyclohex-imide to inhibit any further translation. Incubation was con-tinued for various periods, and samples were analyzed by SDS-PAGE and assayed for the ability to cleave an appropriatesubstrate (Fig. 6). Little or no Krp1 activity was detected inearly samples, but considerable activity was detected in sam-ples incubated for 15 and 23 h. As translation had been inhib-ited by the addition of cycloheximide, this activity was not theresult of increased production of Krp1; it was due to activationof the enzyme.

Activation of Krp1 appears to correlate with a significantdecrease in the mobility of the protein. Such heterogeneouschanges often accompany the addition of carbohydrates to

proteins. We investigated whether Krp1 underwent O glyco-sylation or whether there was further elaboration of the N-linked sugars that are added soon after translocation intomembranes (Fig. 7). The tripeptide (acetyl)-Asn-Tyr-Thr-(amide) competes with proteins for the transfer of sugar chainsfrom their dolichol lipid carrier (23) and has previously beenshown to completely inhibit N glycosylation of Krp1 in eggextract (8). It did not, however, prevent the decrease in themobility of Krp1 (Fig. 7A), which suggests that this modifica-tion is not due to the elaboration of N-linked sugars. As noequivalent inhibitor of O glycosylation was available, we soughtto investigate this indirectly by exploiting the ability of glyco-proteins to bind to concanavalin A (ConA)-Sepharose (Fig.7B). ConA preferentially binds to a-mannopyranosyl and a-glycopyranosyl residues but recognizes a somewhat widerrange of both N- and O-linked sugars; therefore, the binding ofKrp1 to ConA-Sepharose in the absence of N glycosylation isreasonable evidence that this protein is O glycosylated. In

FIG. 5. C-terminal requirements for prosequence cleavage. Krp1 mRNA andmRNAs from truncated forms of Krp1 were translated in Xenopus egg extract for1 h in the presence of [3H]leucine before being analyzed by SDS-PAGE (lanes1 to 6). mRNAs from Krp1[W578*], Krp1[S590*], and Krp1[Y611*] (included toprovide a convenient reference point between processed and unprocessed pro-teins) were also translated for 24 h before analysis (lanes 7 to 9). The positionsof molecular weight markers (in thousands) are shown on the left.

FIG. 4. Prosequence cleavage between the primary and internal sites. Krp1 (shown as Krp1[KR82][KR102] to emphasize the sequences at the primary and internalsites) and Krp1[IA82][KA102] mRNAs were translated in Xenopus egg extract for 1 h in the presence of [3H]leucine, and the products were separated by SDS-PAGEbefore being transferred to a PVDF membrane. The positions of proteins were identified by autoradiography, and the sections of the membrane corresponding to theproteins were excised and subjected to cycle sequencing. The amount of [3H]leucine in each cycle was determined by scintillation counting and compared to the proteinsequences. The cleavage sites are indicated by black arrows, and shaded arrows indicate the positions of mutated sites in Krp1[IA82][KA102].

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contrast, the Map2 protein does not become O glycosylatedand fails to bind ConA when N glycosylation is inhibited. Themost likely location for O glycosylation of Krp1 is the Ser/Thr-rich domain located just proximal to the transmembrane do-main (19 Ser or Thr residues between positions 619 and 647)(9). Such domains become O glycosylated in other transmem-brane proteins, including Kex2 (10). The suggestion that theheterogeneity we observed was due to O glycosylation of thisregion was provided by the Krp1[Y611*] mutant describedabove. This mutant is truncated just N terminal of the Ser/Thr-rich domain. Although it became N glycosylated soon aftertranslation, it did not undergo any further modifications duringextended incubation in extract (Fig. 5, lane 9). The Krp1[Y611*] mutant also demonstrated that the correlation be-tween O glycosylation and activation is not obligatory and thatlike Kex2 (11, 15), Krp1 can become active without being Oglycosylated (Table 1). It appears therefore that O glycosyla-tion merely provides a convenient marker for some other eventthat is essential for the activation of Krp1.

To discover which mutants could become active in the ex-tract, we translated the appropriate mRNAs for 1 h beforeadding cycloheximide to inhibit further translation and allow-ing the incubation to continue for a further 23 h. Equivalentamounts of Krp1 and various mutant enzymes were assayedfor activity (Table 1). As expected, the active-site mutantsKrp1S371A and Krp1[IA82][KK102]S371A were inactive, butall of the other mutants developed at least some activity. Re-moving the cytoplasmic tail, transmembrane domain, and Ser/Thr-rich region (e.g., Krp1[Y611*]) had only a small effect onthe activation process, but truncating Krp1 at or near theC-terminal end of the P domain (e.g., Krp1[S590*]) had amore dramatic effect. These mutants attained less than 10% ofthe activity associated with the full-length protein. Mutatingthe primary cleavage site from Lys-Arg102 to Lys-Ala102 pre-vented proper cleavage of the prosequence and caused at least

a fivefold reduction in activity. These mutants were cleaved atTrp-Arg99 (Fig. 4), and there appeared to be no difference inactivation between constructs which were cleaved only at thissite (e.g., Krp1[KA82][KA102]) and those which were initiallycleaved at Lys-Arg82 (e.g., Krp1[KR82][KA102]). It was per-haps not surprising to find that incorrect cleavage of the pro-sequence affected activity, but we were more surprised to dis-cover that the nature of the cleaved prosequence appears toinfluence subsequent activation of the enzyme. Indeed, onlyone mutant (Krp1[KR82][KK102]) developed activity that wassimilar to that of Krp1. We considered it to be perhaps signif-icant that this was the only construct in which the internalcleavage site was the same as that in the wild-type enzyme, andwe wondered whether cleavage at this site was required foractivation of the enzyme. To investigate this, we examined theprosequences produced from a selection of constructs andfound that activation did appear to correlate with internalcleavage of the prosequence (Fig. 8). Internal cleavage of theKrp1 prosequence appeared to be coincident with its activation(Fig. 8, lane 1), whereas the prosequence initially cleaved fromthe Krp1[IA82] mutant did not undergo secondary cleavage atthe internal site (Fig. 8, lane 2) and the enzyme did not becomeactive (Table 1). As there is no apparent difference in theinitial cleavage of the prosequence from these two enzymes(both are cleaved at the primary site of Lys-Arg102) and theycontain identical catalytic domains, we suggest that the inabil-

FIG. 6. Activation of Krp1 in Xenopus egg extract. Krp1 mRNA was trans-lated in Xenopus egg extract for 1 h in the presence of [3H]leucine. Cyclohexi-mide was added (final concentration, 2 mM) to inhibit any further translation,and incubation was continued for various times (as indicated) before sampleswere analyzed by SDS-PAGE. The amount of Krp1 in each sample was deter-mined by using a PhosphorImager (Molecular Dynamics) with ImageQuantsoftware, and equivalent amounts of protein were assayed for the ability to cleavethe fluorogenic substrate Boc-Arg-Thr-Lys-Arg-MCA. Activity was determinedunder initial rate conditions and is expressed relative to the maximum activityobserved in the experiment. The positions of molecular weight markers (inthousands) are shown to the left of the gel.

FIG. 7. Krp1 undergoes both N glycosylation and O glycosylation in Xenopusegg extract. Krp1 mRNA was translated in egg extract for 1 h in the presence of[3H]leucine before a 23-h chase (cycloheximide was added after 1 h to inhibitfurther translation). Translations were also performed in the presence of thetripeptide (acetyl)-Asn-Tyr-Thr-(amide) (NYT; final concentration, 10 mM) toinhibit N glycosylation (8, 23). (A) Samples were analyzed by SDS-PAGE. (B)Samples from extract (prepared in the presence and absence of NYT) werediluted in binding buffer (20 mM Tris [pH 7.5], 0.5 M NaCl, 0.5% Triton X-100)and allowed to bind to ConA-Sepharose in the same buffer. Unbound materialswere removed by extensive washing, and beads were resuspended in electro-phoresis sample buffer and analyzed by SDS-PAGE. T, total sample prior toincubation with ConA-Sepharose; B, material remaining bound to beads afterbeing washed. Map2, a protein that undergoes N glycosylation but not O glyco-sylation (8, 19), was included as a control for the inhibition of N glycosylation byNYT and for the specificity of beads. The positions of molecular weight markers(in thousands) are shown on the left.

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ity of the mutant to become active is due to its failure to cleavethe internal site of the initially cleaved prosequence. In theother two mutants whose results are shown in Fig. 8, mutationof the primary cleavage site leads to the production of short-ened prosequences that do not undergo subsequent cleavagebut are also unable to prevent the enzymes from becoming atleast partially active.

The prosequence interacts with Krp1 in vivo. Our studieswith Xenopus egg extract helped us to define events in thematuration of Krp1, and although we believe that our findingsreflect events in this fission yeast, we sought to confirm this ina more physiological environment. Unfortunately, such studiesare complicated by the fact that S. pombe appears to require atightly controlled level of Krp1 activity and that both the lossand overexpression of the enzyme are deleterious to cellgrowth (8). It was not possible therefore to simply repeat the invitro analysis in vivo. We took the alternative approach ofexpressing various Krp1 prosequence constructs in otherwisewild-type yeast cells (Table 2). These cells contained the nor-mal chromosomal copy of the krp1 gene, and cells transformedwith pREP3X vector alone had a doubling time of about 4 h.The expression of Krp1[KR82][KR102][R102*] (which has theLys-Arg motif at both the primary and internal cleavage sitesand terminates immediately after Arg102 to generate a prod-uct that is equivalent to the prosequence expected from theinitial cleavage of wild-type Krp1) had little effect on cellgrowth, but the expression of Krp1[KA82][KR102][R102*](same as Krp1[KR82][KR102][R102*] but with a noncleavableinternal site) caused a dramatic decrease in the rate of growth.Such a decrease in the growth rate would be expected if Krp1[KA82][KR102][R102*] inhibited Krp1. The specificity of thisinteraction was supported by our finding that Krp1[KA82][Y100*] did not affect cell growth. This construct generates aprosequence that lacks the C-terminal Lys-Arg motif of thenormal prosequence, which suggests that these residues areimportant for the apparent inhibition of Krp1 by Krp1[KA82][KR102][R102*].

DISCUSSION

Using an in vitro system prepared from Xenopus eggs, weinvestigated events in the activation of Krp1, the only memberof the kexin family of proprotein convertases present in thefission yeast S. pombe. Krp1 is a type I membrane protein thatis synthesized as a preproenzyme. The presequence is removedduring translocation into the ER, where the proKrp1 becomesN glycosylated. Cleavage of the prosequence at Lys-Arg102 isautocatalytic and can occur intermolecularly, although we sus-pect it is normally intramolecular because mutants containingLys-Lys102 are cleaved much more efficiently than would beexpected from the relative inability of Krp1 to cleave Lys-Lys-containing substrates in trans (8). Indeed, an inactive form ofKrp1 that contains the S371A mutation as well as the Lys-Lys102 motif was not cleaved by incubation with Krp1. A closeranalysis of the kinetics of this processing event will be required

TABLE 1. Relative activities of various Krp1 mutantsa

Construct Internal site Primary site Activity

Krp1 Lys-Arg Lys-Arg 100

Krp1S371A Lys-Arg Lys-Arg ,1Krp1[IA82][KK102]S371A Ile-Ala Lys-Lys ,1

Krp1[KA82][KR102] Lys-Ala Lys-Arg 1–10Krp1[IA82][KR102] Ile-Ala Lys-Arg 1–10

Krp1[KR82][KK102] Lys-Arg Lys-Lys .120Krp1[KA82][KK102] Lys-Ala Lys-Lys 1–10Krp1[IA82][KK102] Ile-Ala Lys-Lys 1–10

Krp1[KR82][KA102] Lys-Arg Lys-Ala 10–20Krp1[KA82][KA102] Lys-Ala Lys-Ala 10–20Krp1[IA82][KA102] Ile-Ala Lys-Ala 10–20

Krp1[W578*] Lys-Arg Lys-Arg 1–10Krp1[S590*] Lys-Arg Lys-Arg 1–10Krp1[Y611*] Lys-Arg Lys-Arg 80–90

a Various mRNAs were translated in Xenopus egg extract for 1 h in thepresence of [3H]leucine. Cycloheximide was added (final concentration, 2 mM)to inhibit any further translation, and incubation was continued for 23 h to allowmaturation to occur. Samples were analyzed by SDS-PAGE, and the amount ofKrp1-related material in each sample was determined by using a PhosphorIm-ager (Molecular Dynamics) with ImageQuant software (an allowance was madefor the number of leucine residues in each construct). Equivalent amounts ofprotein were assayed for the ability to cleave the fluorogenic substrate Boc-Arg-Thr-Lys-Arg-MCA under initial rate conditions. The results are expressed rela-tive to the activity of Krp1. Each sample was translated and assayed on at leastthree occasions, and each result was within the range indicated.

FIG. 8. Internal cleavage of the initially cleaved prosequence is required foractivation of Krp1. Krp1 mRNA and mRNAs from various Krp1 mutants thatlack the normal basic motifs at either the primary or internal site were translatedin Xenopus egg extract for 1 h in the presence of [3H]leucine. (Top) Sampleswere analyzed by SDS-PAGE on a high-percentage gel. (Bottom) Duplicatesamples were treated in the same way except that cycloheximide was added after1 h (to inhibit translation) and incubation was continued for a further 15 h beforesamples were analyzed. Mutant forms of Krp1 with stop codons introducedimmediately after Arg102 (Krp1[R102*]), Arg99 (Krp1[R99*]), and Arg82(Krp1[R82*]) were translated in extract to provide appropriate markers and aidin the interpretation of results.

TABLE 2. Inhibitory properties of different prosequences in vivoa

Prosequence Internalsite

Primarysite

Doubling time

Actual(h)b Relative

Krp1[KR82][KR102][R102*] Lys-Arg Lys-Arg 4.12 1.00Krp1[KA82][KR102][R102*] Lys-Ala Lys-Arg 5.55 1.35

Krp1[KR82][Y100*] Lys-Arg Absent 4.21 1.02Krp1[KA82][Y100*] Lys-Ala Absent 4.13 1.00

a Plasmids containing the indicated Krp1 prosequence constructs under thetranscriptional control of the nmt1 promoter were transformed into S. pombecells expressing wild-type Krp1 from its chromosomal allele. Transformants werecultured at 29°C in the absence of thiamine to induce expression from the nmt1promoter.

b Data are averages of five separate determinations.

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to prove that prosequence cleavage is intramolecular. Prose-quence cleavage was unaffected in mutants lacking the cyto-plasmic tail, the transmembrane domain, and even the Ser/Thr-rich region near the C terminus of the protein but wasinhibited by deletions that affect the P domain. Even though itwas initially identified as a homologous region C terminal tothe catalytic domain, mutational analyses have demonstrated afunctional role for the P domain in kexin maturation (6, 15,17). Although our results are similar to those obtained with theP domain of Kex2 (15), they do not allow us to identify the endof the P domain as accurately as was possible in the earlierstudy. The high level of sequence similarity between the twoenzymes in this region suggests that the different results areprobably due to differences in the assays used to monitor theeffects of these mutations rather than to any significant differ-ence in the precise role of the P domain.

Cleavage of the prosequence at the primary site was neces-sary but not sufficient for activation of Krp1, and despite theability of the enzyme to cleave its own prosequence, it wasinitially unable to process substrates presented in trans. Theenzyme became fully active only upon further incubation inextract. The relationship between activation and O glycosyla-tion appeared to be fortuitous, however, as a truncated form ofKrp1 that failed to become O glycosylated did become active.It seems more likely that this is simply a convenient marker foranother event that is necessary for activation. Our results donot precisely define this event but show that activation is ac-companied by internal cleavage of the initially cleaved prose-quence and, furthermore, that mutant enzymes in which thiscleavage is prevented fail to become activated. These resultsare consistent with a model in which the cleaved prosequenceremains noncovalently associated with the catalytic domain ofKrp1 and acts as an autoinhibitor. Inhibition is relieved bycleavage of the prosequence by Krp1 at the internal site, andthe enzyme is then able to process other substrates. This modelis further supported by the finding that overexpression of anoncleavable prosequence reduced the growth rate of yeastcells, presumably through inhibition of Krp1. The reduction ingrowth rate and presumably therefore the interaction withKrp1 require the presence of the two basic residues (Lys-Arg102) at the C terminus of the prosequence.

Our results are similar to those recently reported for thematuration of furin (1). The furin prosequence is removedautoproteolytically but remains associated with the maturepart of the enzyme and acts as a potent inhibitor. Inhibition isrelieved by cleavage of the prosequence at the internal site.Indeed, the only significant difference between furin and Krp1appears to be the trigger that allows cleavage of the inhibitoryprosequence. The activation of furin requires its movementfrom the ER to the Golgi complex and appears to be triggeredby the decreased pH and increased Ca21 associated with thistransport. These changes are thought to reduce the interactionbetween furin and its prosequence and allow the enzyme tocleave the inhibitory peptide at the internal site. Such com-partment-specific activation of furin may prevent inappropri-ate cleavage of substrates in the ER or may be required toallow the enzyme sufficient time to adopt its correct confor-mation. As predicted by this model, inhibiting the transport offurin to the Golgi complex by adding an ER retrieval signalprevents the enzyme from becoming active (31). This ER-retained form of furin can be activated in vitro by mimickingthe changes normally associated with transport to the Golgicomplex (1). In contrast, despite using conditions similar tothose used for furin (1), we were unable to promote the acti-vation of Krp1 by changing the pH and/or Ca21 concentration(not shown). This may suggest that the activation of the yeast

enzyme does not require its export from the ER. Indeed, wesometimes observed activation of Krp1 in Xenopus egg extractin the absence of O glycosylation (not shown), but we cannotyet discriminate between Krp1 molecules that remain in theER and those that are transported to the Golgi complex butare not O glycosylated. It is perhaps significant that the addi-tion of an ER retrieval signal to Kex2 does not prevent itsactivation (5).

A possible molecular explanation for the pH-dependent ac-tivation of furin is ionization of the histidine residues near theC terminus of the prosequence (1). These may influence theinteraction between the prosequence and the catalytic domainand may be affected by the transport of the enzyme from theER to the Golgi complex. Changes in the ionization state ofthese residues may reduce the stability of the prosequence-enzyme complex and trigger activation of the enzyme. Thepositions of these histidine residues are conserved in almost allof the kexins identified in higher eukaryotes but are absentfrom both Krp1 and Kex2, which is consistent with evidencethat activation of the yeast enzymes, like that of the bacterialenzymes, is not pH dependent.

Many members of the kexin family contain a cluster of basicresidues that can be aligned with the internal site of furin andKrp1 (41), and cleavage at this site may be a common featurein the activation of these other enzymes. However, not allkexins contain this second site. If the inhibitory prosequence istherefore a common theme in this family, it will be necessary toinvoke other mechanisms of degradation; it is perhaps signif-icant that furin can be activated by digestion with trypsin (1).

Our results have gone some way to defining the events re-quired for the activation of Krp1, but they have revealed littleabout how these events are controlled at the molecular level.For example, how does the prosequence remain associatedwith the enzyme and what events are required to trigger dis-sociation? It will also be interesting to discover whether theprosequence physically dissociates from the Krp1 before beinginactivated by cleavage at the internal site. We are using Xe-nopus egg extract to address these and other questions and arecomplementing our in vitro work with in vivo analysis.

ACKNOWLEDGMENTS

We thank Kevin Davis and Glenn Matthews for expert technicalassistance during this work.

This work was supported by a studentship from the Biotechnologyand Biological Sciences Research Council (ref. 94305555) (D.P.) andby grants from the Cancer Research Campaign (ref. Sp1972). J.D. is aLister Institute Research Fellow.

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