Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease

Yeast 14, 1115–1125 (1998)

Secretion and pH-Dependent Self-Processing of thePro-Form of the Yarrowia lipolytica Acid ExtracellularProtease

ROBERT K. MEWEN† AND THOMAS W. YOUNG*

School of Biochemistry, University of Birmingham, Birmingham B15 2TT, UK

The secretion and maturation of the acid extracellular protease (AXP) of the yeast Yarrowia lipolytica have beencharacterized using antiserum raised against this enzyme. A 42 kDa pro-enzyme form of AXP was identified fromlysates of radiolabelled Y. lipolytica cells and found to contain no N-linked carbohydrate moieties. Using pulse-chaseimmune precipitation it was demonstrated that the AXP precursor was secreted into the extracellular medium where,under conditions of low pH, it underwent autocatalytic activation forming the mature enzyme. Conversion of theAXP pro-form in the presence of the protease inhibitor pepstatin indicated that an intramolecularly-catalysedreaction mechanism was involved in AXP maturation. Further evidence supporting the role of autocatalyticprocessing came from the side-chain specificity of mature AXP towards the oxidized B-chain of insulin. ? 1998 JohnWiley & Sons, Ltd.

— Yarrowia lipolytica; secretion; pH; extracellular protease

INTRODUCTION

The processing and maturation of the pro-enzymesof extracellular proteases is, in general, a poorlyaddressed field of study in fungi. In particular,fungal pepsins have only been isolated in theirmature forms, and not precursor pro-forms. Thededuced amino acid sequences of the genes encod-ing extracellular aspartic proteases of Aspergillus,Mucor and Rhizopus species suggest that self-processing is the major pathway in the maturationof these enzymes. Sequences immediately upstreamof the N-terminus of the mature form of theseproteases do not contain the tandem Lys-Argdipeptides characteristic of the involvement ofprocessing enzymes similar to the Saccharomycescerevisiae KEX2 protease but consist, on thewhole, of amino acids of hydrophobic nature(Berka et al., 1990; Jarai et al., 1994; Gray et al.,1986; Tonouchi et al., 1986; Delaney et al., 1987).

Furthermore, Chen et al. (1991) and Hiramatsuet al. (1989) have demonstrated that recombinantforms of the putative zymogens of the extracellularproteases of Rhizopus chimensis and Mucor pusillusare capable of autocatalytic activation under acidicpH conditions. Thus, it has been suggested thatprocessing in vivo occurs autocatalytically at acidicpH in secretory granules of the general secretorypathway, and may represent a general pattern ofpro-enzyme maturation in fungal species (Chenet al., 1991).

In the dimorphic yeast Yarrowia lipolytica theproduction of a subtilisin-like alkaline extra-cellular protease (AEP) has been the subject ofextensive study leading to the use of this enzyme asa reporter molecule for analyses of the processesinvolved in secretion in eukaryotic cells(Ogrydziak, 1993). A strain of Y. lipolytica thatproduces, under the appropriate conditions, asingle acid protease in addition to AEP has beenisolated (Nelson and Young, 1987). The structuralgene encoding this enzyme, AXP, has been clonedand sequenced (Young et al., 1996). In this strain,designated Y. lipolytica 148, expression of AXP aswell as XPR2, the gene encoding AEP, has beenshown to be subject to regulation at the mRNA

*Correspondence to: T. W. Young, School of Biochemistry,University of Birmingham, Birmingham B15 2TT, UK. Tel:(+44) 121 414 5437; fax: (+44) 121 414 7366; e-mail:[email protected]†Current address: Centre for Clinical Research in Immunologyand Signalling, University of Birmingham, BirminghamB15 2TT, UK.

CCC 0749–503X/98/121115–11 $17.50? 1998 John Wiley & Sons, Ltd.

Received 4 March 1998Accepted 24 May 1998

level by the pH of the external medium (Younget al., 1996; Glover et al., 1997).

A common processing motif for yeast extra-cellular proteases has been proposed (Ogrydziak,1993) on the basis of the deduced amino acidsequences of the pro-regions of the acid proteasesof C. albicans (Hube et al., 1991), C. tropicalis(Togni et al., 1991) and S. fibuligera (Hirata et al.,1988). Each of these pro-peptides contains twoLys-Arg (or Arg-Arg) dipeptide cleavage sites, oneof which directly precedes the N-terminus of themature enzyme while the other is situated furtherupstream, with a consensus N-glycosylationsequence situated between them. An endo-peptidase similar to the S. cerevisiae KEX2 pro-tease (Julius et al., 1984), specific for cleavingpolypeptides after such tandem sequences, isthought to mediate the hydrolysis of these peptidebonds. These features of the putative processingmotif are also found in the same relative positionsin the pro-region of AEP (Ogrydziak, 1993). How-ever, the prepro-region of AXP does not displayany of the characteristics of this proposed model(Young et al., 1996). There are no N-glycosylationsequences or appropriately located KEX2-likeprocessing sites in the pro-peptide and the matureform of AXP results from the cleavage of a Phe-Ala peptide bond between the C-terminus of thepro-region and the N-terminus of the matureenzyme moiety (Young et al., 1996). Thus,although the mode of processing of the AXPpro-enzyme is unclear, a Kex2p-like enzyme isevidently not involved.

In this paper we report on the secretion of thepro-enzyme form of the acid extracellular proteaseof Y. lipolytica and demonstrate that maturationof this enzyme occurs extracellularly in a pH-dependent manner. Further evidence indicatingthat pro-enzyme processing involves the auto-catalysed scission of the pro-peptide is alsopresented.

MATERIALS AND METHODS

MaterialsPepstatin, phenylmethanesulphonyl fluoride

(PMSF) and tunicamycin (Sigma) were preparedin methanol, isopropanol and dimethyl sulphoxide(DMSO), respectively. Labelled nucleotides werepurchased from Amersham. Protein A–SepharoseCL-4B and oxidized B chain of bovine insulin werefrom Sigma.

Strains and mediaMaintenance media and small-scale batch cul-

tures of Y. lipolytica derepressed for AXP produc-tion were prepared as described previously (Nelsonand Young, 1987; Young et al., 1996). Media werebuffered to the appropriate pH using 50 m-citrate. Unless otherwise stated, the strain of Y.lipolytica used in these studies was the wild-typestrain 148 that secretes a single acid protease and asingle alkaline protease (Young et al., 1996).

Preparation of radiolabelled cell extracts andsupernatant media

The procedure for labelling Y. lipolytica cellswas based on that of Cheng and Ogrydziak (1987).Y. lipolytica 148 cells induced for the production ofextracellular AXP activity were harvested at a celldensity of A600 5, washed twice and resuspended inlabelling medium (0·17% (w/v) yeast nitrogen basewithout amino acids and ammonium sulphate,0·1% (w/v) casein, 1% (w/v) glycerol in 50 m-citrate buffer, pH 4·0) at a final cell density of A60010. The radioactive label -[4,5-3H]leucine wasadded following a 30-min incubation at 28)C and achase with unlabelled leucine was done, whenappropriate, by the addition of a 200-fold excess of-leucine. Protein synthesis was stopped by trans-ferring samples into a microfuge tube kept on icecontaining 20 m-sodium azide, 25 m-leucine,50 ì-pepstatin and 2 m-PMSF. The cell suspen-sion was centrifuged and supernatant samples tobe precipitated with trichloroacetic acid (TCA)were adjusted to contain 10% (v/v) TCA andincubated on ice for 5 h before being resuspendedin SDS–PAGE sample buffer.

The cell fraction was washed twice with 50 m-Tris–HCl, pH 6·8 containing 2% (w/v) SDS,20 m-sodium azide, 6 m-EDTA, 2 m-PMSFand 50 ì-pepstatin and then resuspended in thissolution. Acid-washed glass beads (425–600 ìm,Sigma) were added. Tubes were vortexed for5#60 s with intermittent 30-s intervals of coolingon ice and then centrifuged. The cell extract wasdiluted with four volumes of a solution contain-ing 60 m-Tris–HCl, pH 7·4, 190 m-sodiumchloride, 2·5% (w/v) Triton X-100, 6 m-EDTA,2 m-PMSF and 50 ì-pepstatin. Anti-AXP anti-bodies (Glover et al., 1997) were added to clarifiedcell extracts and the antibody–cell extract mixturewas incubated with gentle agitation at 4)C for 16 h.Antigen–antibody complexes were precipitatedwith protein A–Sepharose CL-4B and the pelleted

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? 1998 John Wiley & Sons, Ltd. Yeast 14, 1115–1125 (1998)

immunoadsorbent was washed three times with asolution containing 0·1% (w/v) Triton X-100,0·02% (w/v) SDS, 50 m-Tris–HCl, pH 7·4, 5 m-EDTA, 150 m-sodium chloride and once with anidentical solution lacking Triton X-100 and SDS.Immune complexes were eluted into SDS–PAGEsample buffer and, following denaturation, wereapplied to an SDS–polyacrylamide gel and treatedfor fluorography.

For immunoprecipitation of extracellular AXP,the supernatant medium from a sample of cellsuspension was adjusted to contain 2% (w/v) SDSand incubated at 95)C for 5 min. After cooling onice, the sample was adjusted to 4% (w/v) TritonX-100, and an equal volume of 1 -Tris–HCl,pH 7·4 was added to restore the pH of the sol-ution to neutrality. This buffering effect has beenverified beforehand (data not shown). Followingthe addition of anti-AXP antibodies, supernatantsamples were incubated at 4)C for 16 h. The pro-tocol for the isolation of immune complexes usingprotein A–Sepharose was identical to the pro-cedure described for the immunoprecipitation ofcell extracts (see above).

ImmunocompetitionPurified AXP (5 ìg) was adjusted to contain

50 ì-pepstatin, 25 m-sodium azide, 2% (w/v)SDS and incubated at 95)C for 5 min. After cool-ing, the sample was made 4% (w/v) in Triton X-100and anti-AXP antiserum was then added. Theantigen mixture was incubated for 16 h at 4)Cbefore it was added to the cell extract.

Tunicamycin treatmentFor tunicamycin inhibition, cells were prepared

and treated as in a standard immunoprecipitationexcept that tunicamycin was included at a finalconcentration of 10 ìg ml"1 in the labellingmedium.

Hydrolysis of the oxidized insulin B chainThe oxidized B chain of bovine insulin (2 mg

ml"1) was incubated at 28)C for 30 min withpurified AXP (final concentration 0·02 mg ml"1).The peptide fragments that resulted from hydroly-sis were separated by reversed-phase HPLC on acolumn with LC-18 packing with a gradient of0–65% (v/v) acetonitrile containing 0·1% (v/v) tri-fluoroacetic acid at a flow rate of 1 ml min"1.Eluted fragments were detected by monitoringthe absorbance of the eluate at 215 nm and the

corresponding peaks collected. Purified peptideswere subjected to amino acid analysis. The aminoacid compositions of the peptides were subse-quently matched to the known sequence of oxidizedinsulin B chain to determine the cleavage sites.

RESULTS

Immunoprecipitation of the intracellular precursorof AXP

A single 42 kDa polypeptide was specificallyimmunoprecipitated with anti-AXP antibodiesfrom Y. lipolytica 148 cells induced for AXPproduction (Figure 1, lane 3). The authenticity ofthe immunoprecipitation reaction was verified bythe ineffectiveness of control antibodies (antiserataken from the animal prior to immunization) toimmunoprecipitate any AXP-related polypeptidesand the capacity for excess unlabelled AXPto block the immunoprecipitation of labelledpolypeptides (Figure 1, lanes 1 and 5, respectively).This evidence indicated that the 42 kDa poly-peptide was antigenically related to AXP. More-over this species had a decreased electrophoreticmobility in comparison to purified mature AXP, asample of which was resolved in an adjacent laneof the gel. Taken together, these results indicatethat the 42 kDa polypeptide represents the intra-cellular precursor form of the mature enzyme. Thelow molecular weight labelled bands (Figure 1,lanes 3 and 4) probably represent the products ofthe degradation of the AXP precursor and werenot present in subsequent cell extracts preparedunder more stringent conditions (results notshown).

The Mr value for the immunoprecipitatedprecursor, as estimated by SDS–PAGE, was inreasonable agreement with a prediction of 40,000made from amino acid sequence data of the clonedAXP gene (Young et al., 1996). It is noteworthythat the predicted relative molecular mass of themature AXP molecule (37,427), like the Mr pre-dicted for the pro-enzyme, is approximately 2000lower than the estimate obtained by SDS–PAGE.

The extent to which, if at all, N-glycosidicallylinked sugar molecules contributed to the molecu-lar weight of the immunoprecipitated AXP pre-cursor was investigated using tunicamycin. Incomparison to control immunoprecipitations per-formed in the absence of tunicamycin, there wasno perceptible change in the molecular weight ofthe AXP precursor (Figure 1, lane 4, compare with

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lane 3). The presence of N-linked oligosaccharideswould have been apparent as an increase in theelectrophoretic mobility of the intracellular pre-cursor. As a further control for tunicamycin actionthe AXP precursor was immunoprecipitated fromcells radiolabelled in the presence of the solvent fortunicamycin dissolution, DMSO, with no effecton its mobility on an SDS–polyacrylamide gel(Figure 1, lane 2). Immunoprecipitation of AEPfrom Y. lipolytica cells cultured at pH 7·5 usinganti-AEP antisera served as control for tunicamy-cin action (results not shown) and demonstratedthat, as reported previously, the 55 and 52 kDaprecursors each contained about 2 kDa ofN-linked carbohydrate, while mature AEP con-tained none (Matoba et al., 1988; Matoba andOgrydziak, 1989).

Pulse-chase analysis of AXP secretionCell fractions were lysed and immunoprecipi-

tated with anti-AXP antisera (Figure 2). A singlepolypeptide species that corresponded to the

precursor of AXP was immunoprecipitated at thetime points 2, 4, 6 and 8 min after initiation ofthe chase. There was a progressive decrease in theintensity of this precursor until none was detect-able in the sample taken 12 min after the start ofthe chase. Since secreted proteases are normallytranslated as larger precursors that are subse-quently processed during intracellular trafficking,it was anticipated that a decrease in the intracellu-lar level of the AXP precursor would be concomi-tant with an increase in the intracellular level ofmature AXP. Such a process was not observed inthese immunoprecipitations, however. Immuno-precipitation of lysates of cells pulse-labelled overa narrower time scale, with shorter intervalsbetween samples, also failed to detect any intra-cellular formation of the mature form of AXPfrom its precursor (data not shown).

Extracellular maturation of AXPImmune precipitation of the extracellular

medium of pulse-labelled Y. lipolytica 148 cells

Figure 1. Immunoprecipitation of a 42 kDa intracellular precursor of AXPfrom radiolabelled extracts of Y. lipolytica 148 cells. Y. lipolytica 148 cellsinduced for AXP production were labelled for 2 min with [3H]leucine withouta chase and immunoprecipitated as described in Materials and Methods. Cellextracts immunoprecipitated with control (pre-immune) and anti-AXP anti-sera were resolved in lane 1 and lane 3, respectively. Anti-AXP antisera wasused to immunoprecipitate the cell extracts resolved in lanes 2–5. The lysatesimmunoprecipitated in lanes 2 and 4 were prepared from cells radiolabelled inthe presence of DMSO and tunicamycin (10 ìg ml"1), respectively. Cellextracts resolved in lane 5 were immunoprecipitated with anti-AXP antiserathat had been pre-incubated with AXP. The position of the putativeprecursor of AXP is indicated to the right of the figure. Numbers at the left,on all figures, indicate the molecular mass in kilodaltons (kDa) of standardproteins.

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with anti-AXP antibodies showed that the pro-enzyme, not the mature form of AXP, wassecreted into the external medium. Pulse-labelling(Figure 3) clearly demonstrates that conversion ofthe pro-enzyme to the mature form occurs extra-cellularly and is dependent upon the pH of theexternal medium. The pH values selected for theseexperiments reflected the data available regardingthe expression of the AXP structural gene and thepH optimum of AXP (Nelson and Young, 1986;Young et al., 1996). AXP is not expressed atpH 7·5 and above and expression is down-regulated above pH 3·5 (Glover et al., 1997).Under the conditions of this pulse-labelling exper-iment the majority of the AXP precursor in theextracellular medium at pH 4·0 and pH 4·6 hadbeen converted to the mature form of AXP 60 minafter the initiation of the pulse-chase (Figure 3,lanes 1–8).

At both pH 5·6 and pH 6·0 the mature form ofAXP was not detectable at levels sufficient toindicate that significant conversion of the AXPprecursor had occurred over the time course of theexperiment (Figure 3, lanes 9–16). This suggeststhat excision of the pro-peptide from the AXPprecursor is dependent upon the immediate

environmental pH of the AXP precursor. Of thepH values examined cleavage occurs at an appre-ciable rate only at pH values of pH 4·6 and below;at pH 5·6 and pH 6·0 the relative rate of matu-ration of the AXP precursor is greatly reduced incomparison.

The mature form of AXP is inhibited by pep-statin (Nelson and Young, 1986) the classicalinhibitor of aspartic proteases. Since under acidicconditions AXP represents the only extracellularprotease activity found in Y. lipolytica 148 cultures(Nelson and Young, 1986) pepstatin inhibitionfacilitated the investigation of AXP maturation.The rationale being that if AXP proenzyme-conversion was catalysed by the intermolecularaction of mature AXP molecules then pepstatin, ata concentration that is known to inhibit AXPactivity, would also prevent this process. Theextracellular media of Y. lipolytica 148 cells pulse-labelled in the presence of pepstatin were immuno-precipitated with anti-AXP antisera and subjectedto fluorography. The results of this experiment(Figure 4) would provide an explanation for theobserved absence of mature AXP from immuno-precipitated cell lysates and strongly supportthe proposal that the AXP pro-enzyme is secreted

Figure 2. The 42 kDa AXP precursor is secreted by the cell. Y. lipolytica 148 cells wereradiolabelled for 90 s with [3H]leucine and chased with a 200-fold molar excess ofunlabelled leucine. Samples were taken at the time points (min) after the initiation of thechase. Cells were lysed and immunoprecipitated with anti-AXP antisera as described inMaterials and Methods. Immunoprecipitates were resolved by SDS–PAGE and subjectedto fluorography. The position of the putative precursor of AXP is indicated to the right ofthe figure.

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Figure 3. pH-dependent processing of the 42 kDa AXP precursor. Y. lipolytica 148 cells in media buffered with 50 m-citrate buffer to pH 4·0 (lanes 1–4), pH 4·6 (lanes 5–8), pH 5·6(lanes 9–12), and pH 6·0 (lanes 13–16) were labelled for 90 s with 200 ìCi of [3H]leucine and the radiolabel was chased with an excess of unlabelled leucine. At the indicated time points(min) after the start of the chase, cells were removed from suspension and the supernatant was immunoprecipitated with anti-AXP antisera by the procedure described in Materials andMethods. Immunoprecipitates were resolved by SDS–PAGE and subjected to fluorography. The positions of the AXP precursor and the mature form of AXP are indicated to the leftof the figure.

and undergoes maturation in the extracellularenvironment. In the presence of pepstatin theactivation of the mature form of AXP from itsprecursor (Figure 4, lanes 1–3) was virtually indis-tinguishable from a control immunoprecipitationperformed in its absence (Figure 4, lanes 4–6).Methanol, the solvent for pepstatin dissolution,had no inhibitory effects upon AXP maturation(data not shown). Although there is a small dis-parity in the appearance of the AXP and pro-AXPbands in the absence and presence of pepstatin(compare lanes 2 and 5, and lanes 3 and 6,Figure 4), which suggest that some intermolecularprocessing may occur, this data indicates that, inthe main, mature AXP is produced by theintramolecularly-catalysed cleavage of the Phe-Alascissile bond between the C-terminus of the pro-peptide and the N-terminus of the mature protein.

Several lines of evidence indicate that AEP, theonly other protease secreted by Y. lipolytica 148(Nelson and Young, 1986), has no role in AXPprocessing. AEP has negligible proteolytic activityat acidic pH values (Nelson, 1986); indeed ex-pression of XPR2, the structural gene for AEP, isprogressively down-regulated at decreasing pH

values (Glover et al., 1997). Furthermore PMSF,an inhibitor of AEP activity, has no effect upon theproduction of AXP from its precursor (data notshown). Finally, AXP pro-enzyme conversionis dependent upon the pH of the extracellularmedium; the rate of activation is markedly reducedat pH values approaching neutrality where AEPactivity would be increasing.

Substrate specificity of AXPAspartic proteinases which display self-

processing are characterized by two hydrophobicamino acids at positions P1 and P1* (Koelsch et al.,1994) and, by definition, should display a specifi-city for the hydrolysis of bonds between hydro-phobic residues. The amino acid sequence of thepro-peptide of the AXP precursor, deduced fromthe nucleotide sequence of the AXP gene, indicatesthat the scissile bond in the formation of matureAXP is situated between a Phe and an Ala (Younget al., 1996); amino acids which are both highlyhydrophobic. It follows therefore that if the pre-cursor of AXP is capable of autocatalytic acti-vation the substrate specificity of the mature

Figure 4. Maturation of the 42 kDa AXP precursor is not inhibited bypepstatin. Pulse-labelling of Y. lipolytica 148 cells and immunoprecipitation ofextracellular media were done as described in Materials and Methods. Culturemedia was buffered at pH 4·0 with 50 m-citrate buffer. Where indicated in thefigure, cells were labelled in the absence (lanes 1–3) and presence of 100 ì-pepstatin (lanes 4–6), respectively and sampled and immunoprecipitated at thetime points (min) indicated after initiation of the chase. The positions of theprecursor and mature forms of AXP are indicated to the right of the figure.

1121.


enzyme should be in accord with its N-terminalcleavage site. The oxidized B chain of bovineinsulin was used as a synthetic substrate andfacilitated the determination of the specificity ofAXP. Peptide fragments produced from hydrolysisof insulin B chain by purified AXP were separatedby reversed-phase HPLC. The profile and deducedcompositions of the purified peptides are shown inFigure 5a and Figure 5b, respectively. Cleavage offour bonds of the insulin B chain were deducedwith the major cleavage site situated between Tyr-Leu (residues 16–17, Figure 5a, peaks A and B).Three additional minor cleavage sites were situatedbetween: Leu-Val (residues 11–12, Figure 5a, peak

b); Ala-Leu (residues 14–15, Figure 5a, peak c) andPhe-Tyr (residues 25–26, Figure 5a, peak a). Judg-ing by the relative sizes of the peptide peaks, theTyr-Leu site is favoured over the Phe-Tyr site.These results indicate that AXP is an endo-peptidase that, in general, favours the cleavage ofbonds between hydrophobic residues where theresidue in the P1 position is an aromatic aminoacid and the residue in the P1* position is analiphatic and hydrophobic amino acid such as Leuor Val. When viewed in the context of the sequenceof amino acids adjacent to the N-terminus ofmature AXP, since P1 is aromatic and P1* ahydrophobic aliphatic residue, these specificity

Figure 5. Determination of the substrate specificity of AXP. Oxidized insulin B chain (2 mg ml"1) wasdigested with purified AXP (final concentration 0·02 mg ml"1) at 37)C for 30 min in 0·1 -sodium formate,pH 3·2 then subjected to reversed-phase HPLC separation. The peptides were structurally identified andfitted into the insulin B chain sequence. The HPLC profile is shown in the top part of the figure (a) while thelower part summarizes the cleavage positions inferred in the insulin B chain (b). Amino acid abbreviationsfollow the alphabetical system and C* represents cysteine sulphonic acid.

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data are wholly consistent with the proposal thatan autocatalytic mechanism facilitates hydrolysisof the peptide bond between the C-terminus ofthe pro-segment and the N-terminal mature AXPmoiety. AXP displays a specificity for the insulin Bchain that shows some similarities to the asparticproteases of Mucor pusillus (Oka et al., 1973) andEndothia parasitica (Williams et al., 1972). Theseenzymes predominately favour the scission of pep-tide bonds between residues where the amino acidin the P1 position is large and of a hydrophobicnature.

DISCUSSION

Although a single acid protease is secretedby induced Y. lipolytica 148 cells (Nelson andYoung, 1986), three acid protease activities werereportedly detected in the external media of Y.lipolytica strain CX161-1B cultures (Yamada andOgrydziak, 1983). However, none of the character-istics reported for these three enzymes equate tothose of the acid protease of strain 148 (Nelsonand Young, 1986). The immunological relatednessof the extracellular acid proteases produced bystrains 148 and CX161-1B, in addition to theunrelated strain 21101-9 (Gaillardin and Ribet,1987), was confirmed using anti-AXP antisera.Both of these strains secrete a polypeptide that isantigenically related to AXP and has a molecularweight that, on an SDS–polyacrylamide gel, isindistinguishable from the extracellular acid pro-tease of strain 148 (results not shown). The con-served nature of the molecular weights and theantigenic relatedness of the proteases detected inthe extracellular medium of cultures of thesestrains strongly suggest that the acid proteaseproduced by strain 148 is common to other strainsof Y. lipolytica. Further evidence in support of thisproposal comes from the report that a restrictionfragment of the coding sequence of the AXP gene(Young et al., 1996) has been used to generate, inan unrelated Y. lipolytica strain, an Axp" mutantwhich produces no detectable extracellular acidprotease activity (Cordero Otero and Gaillardin,1996). Taken together, these data strongly suggestthat AXP is the major and probably only acidprotease secreted into the extracellular medium bystrains of Y. lipolytica. Thus, although the ques-tion of the presence of other acid proteases instrains CX161-1B still remains to be resolved, theevidence available is wholly consistent with the

secretion of a single acid protease species by thisyeast.

Although the AXP prepro-region contains noN-glycosylation sites, three such consensus sitesare found in the amino acid sequence of the matureform of the enzyme. Evidence obtained from theimmunoprecipitation of lysates of Y. lipolyticacells radiolabelled in the presence of tunicamycinindicated that none of these sites is used for thispurpose (Figure 1). In conjunction with dataobtained from analyses of the mature form of AXPusing lectins and endoglycosidase H deglycosyla-tion (data not shown) the results of tunicamycininhibition indicate that the precursor, as well as themature form, of AXP is non-glycosylated.

On the basis of DNA sequence data the AXPprepro-enzyme is predicted to contain anN-terminal signal peptide (Young et al., 1996).Although it cannot be determined from theimmunoprecipitation data whether or not the AXPprecursor contains the signal peptide, in manyeukaryotes removal of the signal sequence occurscotranslationally, and the full-length precursorwould not be detected in vivo (Rapoport andWiedemann, 1985). In Y. lipolytica excision of thesignal sequence of AEP has been demonstrated tooccur co-translationally (He et al., 1992; Yaveret al., 1992). Since several attempts to determinethe N-terminal amino acid sequence of the AXPpro-enzyme by radiosequencing were unsuccessful(data not shown), it was not possible to determinedefinitively whether or not the signal peptide hasbeen excised. However, evidence obtained from aclass of mutants that can extragenically suppressa temperature-sensitive mutation of the geneencoding the 7SL RNA of the Y. lipolyticasignal recognition particle has indicated that AXPtranslocation does occur co-translationally (C. BenMamon, personal communication).

Although it is not known whether the pro-formof AXP is enzymatically active, it has been dem-onstrated that growth under mildly acidic con-ditions (pH 6·0) result in reduced biomassconcentrations as compared to growth at valuescloser to the enzyme’s optimum pH (3·2) (Gloveret al., 1997). In conjunction with the data pre-sented here this observation is consistent withthe premise that the pro-enzyme is enzymaticallyinactive and that activation involves a pH-dependent mechanism of pro-peptide processing.As discussed earlier, AXP is the only extracellularacid protease activity detected in Y. lipolytica 148culture medium (Nelson and Young, 1986) and

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there is no detectable acid protease activity in theextracellular medium of Axp" mutants (CorderoOtero and Gaillardin, 1996), strongly excludingthe involvement of other acid protease(s) in AXPprocessing. This activation process is similar tothat observed in vitro for several other fungalaspartic proteases. Like AXP, the mature form ofthe M. pusillus aspartic protease results from thecleavage of a Phe-Ala peptide bond at itsN-terminus (Tonouchi et al., 1986) and the puta-tive pro-enzyme is capable of autocatalytic acti-vation (Hiramatsu et al., 1989). The asparticprotease of Mucor miehei has also been shown toundergo self-processing (Christensen et al., 1988).Analysis of the maturation and processing of theC. tropicalis secreted aspartic protease (Sapt1)precursor revealed that although the major path-way in vivo is mediated by a Kex2p-like endopro-tease, an alternative activation pathway involvingautocatalytic cleavage of the pro-peptide was alsopossible in vitro (Togni et al., 1996). Similar evi-dence in support of this form of processing wasobtained with a recombinant form of the zymogenof Sapt1 (Lin et al., 1993). The activation ofthe putative pro-form of aspergillopepsinogen I,the pepsin-type acid endopeptidase excreted byAspergillus niger var. macrosporus, has beenstudied in the heterologous hosts Escherichia coliand Bacillus brevis. In a manner comparable to thepro-form of AXP, auto-processing of the pro-enzyme occurred over a range of pH valuesbetween pH 2 and pH 4·5, but not at pH 5·0(Inoue et al., 1996).

It is generally recognized that the organelles ofthe exocytic pathway of eukaryotic cells becomeprogressively more acidic. The pH of the endo-plasmic reticulum is close to neutrality (Mellmanet al., 1986) while the Golgi apparatus is mildlyacidic (Schwartz et al., 1985; Anderson andPathak, 1985). Since at pH values approachingneutrality the rate of AXP maturation is relativelyslow, intracellular transit of the AXP pro-enzymeat such mildly acidic conditions would not signifi-cantly initiate activation of the mature enzyme.Thus under these conditions, AXP would not bedetectable in immunoprecipitates of cell extracts.Further evidence supporting this proposal camefrom the translation of prepro-AXP mRNA in aXenopus laevis cell-free translation/translocationsystem which has been shown to cleave signalpeptides, segregate proteins into membranes andperform core glycosylation at N-linked sites(Matthews and Colman, 1991). At the near-neutral

pH of the microsomes in this system (Matthewset al., 1994) pro-AXP was stable over an extendedtime period without detectable precursor acti-vation (data not shown).

Y. lipolytica cells have a strong tendency for acidproduction and excrete large amounts of citricand isocitric acids into the extracellular medium(Heslot, 1990) which, unless it is subject to pHcontrol, rapidly becomes highly acidic. Thus, theappropriate conditions for AXP pro-enzyme acti-vation may be facilitated by the acidification of theexternal medium by the organism. Proteolyticallyactive AXP molecules can hydrolyse extracellularprotein substrates thereby generating peptidesand amino acids for uptake and assimilation bythe cell.

ACKNOWLEDGEMENTS

R.K.M. was in receipt of a BBSRC researchstudentship. Polyclonal antiserum against AEPwas a gift from J.-M. Beckerich, INRA, Grignon,France.

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Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease