A matrix-located processing peptidase of plant mitochondria

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc1 complex
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  Plant Molecular Biology  36:  171–181, 1998.  171 c    1998  Kluwer Academic Publishers. Printed in Belgium. A matrix-located processing peptidase of plant mitochondria Cristina Szigyarto 1 , Patrick Dessi 2 , M. Kathleen Smith 3 , Carina Knorpp 1 ,Matthew A. Harmey 4 , David A. Day 3 , Elzbieta Glaser 1 and James Whelan 2 ;   1  Department of Biochemistry, Arrhenius laboratories for Natural Sciences, Stockholm University, Stockholm 106 91, Sweden;  2  Biochemistry Department, University of Western Australia, Nedlands 6907, Perth, Western Australia, Australia (    author for correspondence);  3  Division of Biochemistry and Molecular Biology, School of  Life Sciences, Australian National University, Canberra, ACT 0200, Australia;  4  Department of Botany, UniversityCollege Dublin, Belfield, Donnybrook, Dublin 4, Ireland  Received 1 April 1997; accepted in revised form 28 August 1997 Key words:  mitochondrial processing peptidase, mitochondrial matrix, processing specificity Abstract Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion afterimport. The general mitochondrial processing activity in plant mitochondria has been shown to be integratedinto the cytochrome bc   1  complex of the respiratory chain. Here we investigate the occurrence of an additional,matrix-locatedprocessingactivitybyincubationoftheprecursorsofthesoybeanmitochondrialproteins,alternativeoxidase,theF A dsubunitoftheATPsynthetaseandthetobaccoF 1    subunitoftheATPsynthase,withthemembraneand soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-locatedpeptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive toorthophenanthroline,a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of thematrix peptidase was illustrated by the inhibition of processing of the alternativeoxidase precursorin both soybeanand spinach matrix extracts upon altering a single amino acid residue in the targeting presequence ( ,   2 Arg toGly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix.The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The resultsdemonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to thepreviouslywell characterizedmembrane-boundMPP integratedinto the cytochrome bc   1  complexof the respiratorychain.  Abbreviations:  ATPsynthase,F o F 1 -adenosinetriphosphatesynthase;EDTA,ethylenediaminetetraaceticacid;E64,N-[N-(L-3- trans -carboxirane-2-carbonyl)-L-leucyl]-agamatine;MPP, mitochondrialprocessing peptidase; MOPS,3-(  N  -morpholino)propane sulphonic acid; NEM,  N  -ethylmaleimide; OPT, orthophenanthroline; Pefabloc, 4-(2-aminoethyl)-benzenesulphonylfluoride, hypochloride; PMSF, phenylmethylsulfphonyl fluoride; Rieske FeS, non-haem iron sulphur protein of the ubiquinol cytochrome  c  oxidoreductase complex; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis. Introduction The majority of proteins imported into mitochon-dria are synthezised as precursors containing aminoterminal presequences which are proteolytically pro-cessed by the mitochondrial general processing pepti-dase (MPP) [23, 29]. This peptidase was first isolatedfrom  Neurospora crassa  and shown to consist of twodifferent subunits, now termed    -MPP and    -MPP[24, 29]. A heterodimeric structure for the MPP hasbeen shown also for  Saccharomyces cerevisiae  and rat[35, 48]. Both    -MPP and    -MPP are required forthe processing activity [24, 37]. Analysis of the struc-ture of the MPP in  N. crassa  showed that    -MPP was  172identical to the Core 1 subunit of the cytochrome bc   1 complexofthe respiratorychain[38]. Thisbifunction-al protein is encoded by a single gene in  N. crassa  andhas two different locations in the mitochondrion: 70%of the protein being localized in the cytochrome bc   1 complex, where it plays a structural role in electrontransport, and 30% localized in the matrix where itplays a role in protein processing [38]. In yeast andmammals, the protein equivalentto    -MPP is encodedby a separate but related gene to the Core 1 subunit of the cytochrome bc   1  complex [29, 48]; both the    -MPPand    -MPP subunits and the processing activity arelocalized in the matrix.Preliminary characterization of the processingpropertiesof plant mitochondriahas shown that plantscontain a processing peptidase with similar propertiesto fungal MPP [44, 46]. Detailed studies on MPP inplantsdemonstratedthat a processingactivity was loc-atedinthemembranefractionofspinachmitochondria[14, 15], and subsequent isolation of the cytochrome bc  1  complex from potato, spinach and wheat showedthat the processing activity was integrated into thiscomplex [3, 4, 16].Despite the fact that a processing activity can beclearly located and isolated from the mitochondrialinnermembraneof plantsa processingactivity locatedin the matrix fraction was also reported from  Vicia and spinach as early as 1992 and in subsequent stud-ies, although it was not characterized in detail [14, 25,31]. The latter study showed that the matrix-locatedactivity was not due to an ATP-dependent protease asit was not dependent on ATP. Likewise, there was noevidencefornon-specificbreakdownsuch as smearingor appearance of other breakdown products on SDS-PAGE [31]. Additional evidence for the location of aprocessing peptidase in the matrix of plant mitochon-dria came from the studies in which the purified  N.crassa    -MPP subunit restored the processing activityinsolubilizedplantmitochondriawhichwereimmuno-depleted with antibodiesagainst  N. crassa    -MPP andunable to catalyze the processing activity [46]. This isin contrast to studies of the now extensively character-ized membrane-located MPP from plants which haveshown that the processing activity cannot be separatedfrom the cytochrome bc   1  complex [11].ThedifferentlocationofMPP invariousorganismsis proposed to represent divergence from a single ori-ginal evolutionary event [3]. This is in agreement witha monophyletic srcin for mitochondria [22]. It hasbeen proposed that MPP belongs to a family of endo-proteases that includes  Escherichia coli  pitrilysin anda number of other proteases [3]. It has been proposedthat a soluble protease in this family integrated intothe cytochrome bc 1  complex, thereby increasing pro-cessing specificity and or efficiency. The location of MPP in the matrix represents a secondary loss of thisproteaseintothe matrixasseenin mammalsandyeast.In  N.crassa theproteaseislocatedinthematrixbutthesame gene still codes for a subunit in the membrane,the Core I subunit of the cytochrome bc   1 -complex. Inplants examined to date, MPP has only been localizedto the membrane, integrated into the cytochrome bc   1 [3, 21 for reviews].It is noteworthy that the membrane-located MPPis the best characterized component of the plant mito-chondrial protein import apparatus. The cytochrome bc  1  complexhasbeenisolatedfrompotato,spinachandwheat and shown to contain processing activity [4, 5,6, 17,18].However,closeexaminationoftheliteratureclearly indicatesthat componentspresentin the matrixof at least some plants are capable of significant pro-cessing activity[14, 25, 31]. Ina combinedeffortfromfour laboratories, we re-investigatedthe possible pres-ence of a matrix-located processing activity in plantmitochondria,in terms of its ability to generatematuresize products from precursor proteins, its specificity,and its inhibitor sensitivity. In these studies we haveused three mitochondrial precursor proteins and twoplant species. Materials and methods  Isolation and fractionation of mitochondria Soybean mitochondria were isolated from 7-day oldseedlings according to Day  et al.  [7]. Membrane andmatrix fractions were preparedby sonication. The sol-uble fractionwill be referredto as the matrix, althoughit should be noted that this fraction also containsintermembranespace components.Mitochondriawereresuspended at 1 mg/ml protein in 10 mM Tris pH 7.5and kept on ice. The mitochondria were sonicated for10 s (five times in a Braun sonicator with a microtipsetting of three with an interval of one minute betweeneach sonication for cooling). The solution was centri-fugedat12000   g   for20mintoremoveanyremainingintact mitochondria. The supernatant was centrifugedat 130000   g   for 1 h to pellet membranes. The mat-rix fraction (supernatant) was removed and the mem-brane pellet washed twice with 10 mM Tris (pH 7.5).The volume of the supernatant was measured and the  173membranepellet was resuspended in the same volumeof 10 mM Tris pH 8.0 containing 0.25% (v/v) Tri-ton X-100, it should be noted that Triton X-100 wasadded after the fractions were separated. The matrixand membrane fractions were stored at ,   70    C.Spinach ( Spinacia oleracea  L. cv. Medania) wasgrown at 25    C under artificial light with a light peri-od of 10 h as described by Siegenthaler and Dep´esy[39]. Mitochondria were isolated from 6-week oldspinach leaves (mid vein removed)by step centrifuga-tionaccordingtoKnorpp etal.  [30].Spinachleafmito-chondriaweredilutedin0.3M sucrose,30mMMgCl 2 and 10 mM MOPS pH 7.5 to a final protein concentra-tion of 10 mg/ml. Mitochondria were disrupted usinga Branson Sonifier (equipped with a microtip, setting3), 3 times 30 s at 4    C. Membranes and matrix frac-tionswere separatedby centrifugationof the sonicatedmitochondria at 105000   g   for 45 min. The volumeof the supernatant was measured and the pellet wasresuspended in the same volume of 0.3 M sucrose,30 mM MgCl 2  and 10 mM MOPS pH 7.5. Spinachleaf matrix fraction was additionally centrifuged 2–5times at 300000   g   (60 min, 4    C) in order to removesmall membrane vesicles. The matrix and membranefractions were stored at ,   70    C.In vitro  synthesis of precursor protein and site-directed mutagenesis 35 [S]-methionine-labelledprecursorproteinswerepre-pared using a Promega TNT translation system andthe cDNA clones for the soybean alternative oxidase(  AOX1 ) [45], the F   A d   subunit of the mitochondri-al ATPsynthase [40] and the tobacco F   1    subunit[1], according to the manufacturer’sinstructions. Site-directed mutagenesis was carried out using an altered-sites invitro mutagenesissystemfromPromega.Align-ment of the alternative oxidase precursor indicated thepresence of a ,   2 arginine residue [19, 27, 36, 43], acommon processing signal for mitochondrial precurs-orproteins[41]. Thisresidue was changedto a glycineresidue as outlined previously [43]. Processing of the precursor protein with different mitochondrial fractions Processing reactions were carried out according toEriksson  et al.  [14] except that the processing reactionwith soybean mitochondrial fractions was performedwith MgCl 2  instead of MnCl 2 . An equal volume of matrix and membrane fractions was used in each pro-cessing reaction. As the membrane pellet was re-suspended in an equal volume to the matrix fraction(supernatant) the amount of each fraction added to aprocessing reaction represents the ratio found in mito-chondria. Equal volumes (20    l) of matrix and mem-branes were incubated with equal volumes of a medi-um containing 30 mM Tris-Cl pH 8.0, 0.5% v/v Tri-ton X-100, 1.0 mM MnCl 2 ;  1.0 mM methionine and[ 35 S]-labelled precursor proteins. Processing was alsomeasuredinthepresenceofproteaseinhibitors,PMSF,EDTA, orthophenanthroline, NEM, E64, as indicatedin figure legends.NEM was added to the extract for 15 min at roomtemperature and inactivated by the addition of 5 mMDTT. Other inhibitorswere preincubatedwith the pro-cessing extracts for 5 min on ice prior to the addi-tion of the labelled precursor. The processing reactionwas carried out for 60 min at 25    C and was stoppedby addition of Laemmli double-strengthsample buffer[32]andplacingonice.Sampleswereanalyzedon12%SDS-PAGE in the presenceof 4 M urea. The gelswerestained with Coomassie Brilliant Blue, impregnatedwith Amplify (Amersham) and dried. Densitometryfor the soybean processing experiments was carriedout by scanning autoradiographsand quantification of bands using NIH image. Quantification of the spin-ach processing reactions was carried out using FujixBAS1000 MacBAS Bio-imaging Analyzer system.  Immunological analysis of mitochondrial fractions Immunological characterization was performed byimmunodetection of western blots on nitrocellulosemembranes (Hybond-ECL, Amersham). Immunolo-gical cross-reactivity was detected by horseradishperoxidase-labelled secondary antibodies and aBoehringer chemilumenesence kit.  Miscellaneous Cytochrome  c  reductase and oxidase assays were car-riedoutaccordingtoNeuberger etal.  [34].Proteinwasdetermined with a BioRad protein assay reagent basedon the method of Bradford [2]. Bovine serum albuminwas used as a standard.  174 Results Processing of precursor proteins by the matrix peptidase in soybean mitochondria Incubation of alternative oxidase and F A d precursorproteinswithmatrixandmembranefractionsfromsoy-bean mitochondria resulted in processing of the pre-cursors to mature size proteins (Figures 1 and 2). The36 kDa alternative oxidase precursor protein yieldeda mature protein with an apparent molecular mass of 32 kDa upon incubation with the various fractions.The F A d precursor of 24 kDa produced a mature formof 21 kDa upon incubation with the various fractions.The  in vitro  processed products have similar molecu-lar mass as products generated upon import into intactmitochondria for both precursors [9, 43].Examinationoftheproductsproducedbyeachfrac-tion indicated that while both the membrane and mat-rix fractions processed the precursors, they displayeddifferent inhibitor sensitivity. Processing by the mem-brane fraction was only fully inhibited by orthophen-anthroline(Figures1 and 2, lane 11), being insensitiveto the protease inhibitors PMSF (Figure 1 lane 9, Fig-ure 2 lane 10), NEM (Figures 1 and 2, lane 12), andE64 (Figures 1 and 2, lane 13). This indicates that theprecursors were most likely processed by membrane-bound MPP in this fraction [10, 18, 21]. Non-specificproteolysiswas also evident in the membranefraction,since not all of the added precursor was recovered asmature or precursor forms (ie. some protein had beenfurther degraded). For the F A d and alternative oxidaseprecursors with the matrix fraction 95% of the addedprecursorwasrecoveredasprecursorormatureproductin the processing reaction that contained no inhibitor.In contrast with the membrane fraction only 30% of addedprecursorwasrecovered.Processinginthepres-ence of NEM, a sulphydryl group alkylating reagentand a powerful inhibitor of some proteases (Figures 1and2, lane 12)greatly reducedthe amountof degrada-tion. Thisincreasedrecoveryinthemembranefractionto 70% of the added precursors. This suggests thatthe non-specific proteolysis in the membrane fractionwas due to the membrane-bound,ATP-dependentpro-tease previously characterized in plant mitochondria[31]. It is clear that NEM did not inhibit processingby MPP, because more mature product was detectedin the presence of NEM (Figures 1 and 2, lane 12).The insensitivity of the membrane fraction processingactivity to NEM is in agreement with previous studieswith MPP purified from membranes [10, 18].When the matrix fraction was used in similarexperiments, specific processed products were alsoobserved (Figures 1 and 2, lanes 2–7). Two charac-teristics distinguished the matrix-locatedactivity fromthe membrane-bound activity. Firstly, processing bythe matrix fraction was completely inhibited by NEM(Figures 1 and 2, lane 6). Secondly, there was no evid-enceofnon-specificproteolysis:thebandsontheauto-radiographs were very sharp and scanning by densit-ometery indicated that all of the added protein wasrecoveredin the mature and precursor forms. Thus theNEM-sensitive proteolysis seen in the matrix fractionwas distinct from that detected in the membrane frac-tion. Characterization of a matrix-located processingactivity from spinach mitochondria The membrane-located MPP from spinach has beenextensively characterized in terms of inhibitor sensit-ivity and subunit composition [21, 42]. We comparedthe properties of the membrane-associated processingactivity integrated into the cytochrome bc   1  complexwith the properties of the matrix located activity usingthe F 1    precursor of ATP synthase from tobacco andthe alternative oxidase precursor from soybean (Fig-ures 3 and 4). It can be seen that these precursorswerealso processed to a mature form by the spinach mat-rix fraction, although the efficiency of processing inspinachwaslowerthanthat insoybean.Theamountof protein in the membrane and matrix fractions corres-ponded to the total mitochondrial protein from whichthe fractions were derived and the efficiency of pro-cessing in the matrix was calculated to be equivalentto 20% of the activity foundin the membranefraction.The 59 kDa precursor of F 1    was cleaved to themature form of 53 kDa by both membrane and mat-rix fractions (Figure 3). Two chelators, EDTA andorthophenanthroline,inhibitedboth types of activities.In agreement with the soybean studies, NEM com-pletely inhibited processing with the matrix fractionin spinach but had no effect on processing with themembrane fraction (Figure 3, lanes 6 versus 12).The processing ability of spinach leaf matrix andmembrane fractions was also tested with the soybeanalternative oxidase precursor (Figure 4). The precurs-or protein of 36 kDa was processed to a mature sizeproduct of 32 kDa by both membrane and matrix frac-tions, as found in soybean. Again, the efficiency of processing activity with alternative oxidase was foundtobelowerinthematrixthaninthemembranefraction  175 Figure 1 . Processing of the alternative oxidase precursor by matrix and membrane fractions from isolated soybean cotyledon mitochondria inthe presence of different inhibitors. Lane 1, alternative oxidase precursor protein alone (P). Lane 2, processing in the presence of the matrixfraction without the addition of any inhibitors, an additional mature form (M) is indicated. Lanes 3 to 7, alternative oxidase precursor proteinincubated with the matrix fraction in the presence of various inhibitors as indicated. Lane 8, processing in the presence of the membrane fractionwithout the addition of any inhibitors. Lanes 9 to 13, incubation of the alternative oxidase precursor with the membrane fraction in the presenceof various inhibitors as indicated. Figure 2 . Processing of the F A d subunit by soybean membrane and matrix fractions in presence of inhibitors. Lane 1, F A d precursor alone (P).Lane 2, processing in the presence of the matrix fraction without the addition of any inhibitors, a mature form(M) is indicated. Lanes 3 to 7, F A dprecursor protein incubated with the matrix fraction in the presence of various inhibitors as indicated. Lane 8, processing of the F A d precursorby the membrane fraction. Lanes 9 to 13, processing in the presence of membrane fraction in the presence of various inhibitors as indicated. Figure 3 . Processing of F 1    by spinach membrane and matrix fractions in presence of inhibitors. Lane 1, F 1    precursor alone. Lane 2,processing in the presence of the matrix fraction without the addition of any inhibitors. Lanes 3 to 7, F 1    precursor protein incubated with thematrix fraction in the presence of various inhibitors as indicated. Lane 8, processing of the F 1    precursor by the membrane fraction. Lanes 9 to13, processing in the presence of membrane fraction in the presence of various inhibitors as indicated.
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