a cysteine endopeptidase with a c-terminal kdel motif isolated from castor bean endosperm is a...

Abstract. A papain-type cysteine endopeptidase with amolecular mass of 35 kDa for the mature enzyme, waspuri®ed from germinating castor bean (Ricinus co-mmunis L.) endosperm by virtue of its capacity toprocess the glyoxysomal malate dehydrogenase precur-sor protein to the mature subunit in vitro (C. Gietl et al.,1997, Plant Physiol 113: 863±871). The cDNA clonesfrom endosperm of germinating seedlings and fromdeveloping seeds were isolated and sequence analysisrevealed that a very similar or identical peptidase issynthesised in both tissues. Sequencing established apresequence for co-translational targeting into theendoplasmic reticulum, an N-terminal propeptide anda C-terminal KDEL motif for the castor bean cysteineendopeptidase precursor. The 45-kDa pro-enzyme sta-bly present in isolated organelles was enzymaticallyactive. Immunocytochemistry with antibodies raisedagainst the puri®ed cysteine endopeptidase revealedhighly speci®c labelling of ricinosomes, organelles whichco-purify with glyoxysomes from germinating Ricinusendosperm. The cysteine endopeptidase from castorbean endosperm, which represents a senescing tissue, ishomologous to cysteine endopeptidases from othersenescing tissues such as the cotyledons of germinatingmung bean (Vigna mungo) and vetch (Vicia sativa), theseed pods of maturing French bean (Phaseolus vulgaris)and the ¯owers of daylily (Hemerocallis sp.).

Key words: Cysteine endopeptidase ± Endosperm ±Microbody ± Ricinus ± Seed germination ± Senescence± targeting

Introduction

A feature of the eukaryotic cell is the compartmental-isation of di�erent metabolic processes into membrane-enclosed organelles. Some of these have doublemembrane envelopes, such as the nucleus, mi-tochrondria and chloroplast, while others have a singlemembrane envelope with no internal membranes, suchas vacuoles and microbodies (comprising peroxisomesand glyoxysomes). Glyoxysomes have been isolatedfrom fatty-acid-metabolising tissues such as the endo-sperm of castor bean (Ricinus communis; Breidenbachand Beevers 1967) and the cotyledons of Cucurbitaceae(Trelease et al. 1971). These two plant tissues representtwo major ways for higher plants to store and mobiliselipids and proteins for developing seedlings (Gietl1996). Castor beans store fat and proteins in a livingendosperm, which is laterally attached to the cotyle-dons. From days 1 to 5 of germination, glyoxysomesdevelop rapidly in the endosperm cells and mobilise thetriglycerides of the oleosomes into fatty acids, acetyl-CoA and succinate. When these reserves are depletedby days 6 to 7, the desiccated endosperm abscises;castor bean endosperm in these stages represents asenescing tissue.

Endosperm tissue from castor bean contains anadditional microbody-like organelle, only slightly largerthan glyoxysomes. This organelle was discovered inultrastructural and cytochemical studies independentlyby two groups in 1970. It was called ``dilated cisternae'',since it seemed to develop from the ER (Vigil 1970), or``ricinosome'', since it was apparently unique to castorbean (Mollenhauer and Totten 1970). Six to seven daysafter germination, all developmental changes in the ®nestructure of the endosperm during fat mobilisation canbe seen along a cross-section extending from the seedcoat inward to the cotyledons in the centre of the seed.The two single-membrane-bounded organelles, glyoxy-somes and ricinosomes, can be distinguished by mor-phology and contents. While glyoxysomes vary in shapefrom spherical to ellipsoidal with a range in diameter

The sequence reported in this paper has been deposited in theGenBank data base (accession no. AF050756)

Abbreviations: Cys-EP � cysteine endopeptidase from castorbean; gMDH � glyoxysomal malate dehydrogenase, PCR � po-lymerase chain reaction

Correspondence to: C. Gietl;E-mail: [email protected]: 49(89) 289 22167

Planta (1998) 206: 466±475

A cysteine endopeptidase with a C-terminal KDELmotif isolated from castor bean endosperm is a marker enzymefor the ricinosome, a putative lytic compartment

Markus Schmid1, David Simpson2, Frantisek Kalousek3, Christine Gietl1

1Lehrstuhl fuÈ r Botanik, Technische UniversitaÈ t MuÈ nchen, Arcisstr. 16, D-80333 MuÈ nchen, Germany2Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark3Yale University/School of Medicine, Department of Genetics, 333 Cedar Street, New Haven, CT 06510, USA

Received: 20 December 1997 /Accepted: 18 March 1998

from 0.2 to 1.7 lm, ricinosomes are spherical with adiameter averaging 0.9 lm. Glyoxysomes, but notricinosomes, can be stained for catalase by the diamino-benzidine (DAB) reaction. Ricinosomes have a rough-surfaced membrane irregularly studded with denselystaining knobs similar to ribosomes, whereas glyoxy-somes have a smooth membrane. Attempts to isolateand characterise ricinosomes have not been successful,primarily because of di�culties in recognising them afterisolation and the lack of a marker enzyme. It is almostcertain that ricinosomes were present as a contaminantin glyoxysome fractions after density gradient puri®ca-tion and observed in-vitro incorporation of proteinsmight in part be attributed to these organelles (Vigil1970).

We have puri®ed a cysteine endopeptidase fromcastor bean endosperm (Cys-EP) by virtue of its capacityto process the glyoxysomal malate dehydrogenase pre-cursor protein (pre-gMDH) to the mature subunitin vitro (Gietl et al. 1997). Protein sequence analysis ofN-terminal and internal peptides of this Cys-EP revealeda high homology to the mature papain-type cysteineendopeptidases from cotyledons of germinating mungbean (Vigna mungo) and vetch (Vicia sativa) seedlings,maturing French bean (Phaseolus vulgaris) pods anddaylily ¯owers (Hemerocallis). These endopeptidases aresynthesised with an extended pre-pro-sequence at the N-terminus in the ER and are apparently processed enroute to their ®nal destination. An interesting feature ofthis class of proteases is a C-terminal KDEL motif,which functions as a Golgi retrieval signal for solubleresidents of the ER lumen. However, for at least twomembers of this family (mung bean and vetch), theKDEL sequence is not present in the mature protease(Okamoto et al. 1994; Becker et al. 1997). Accordinglywe studied the location of the Ricinus Cys-EP byimmuno-electron microscopy with antibodies raisedagainst the puri®ed Cys-EP. Immunogold labellingoccurred exclusively in the ricinosomes while antibodiesagainst gMDH reacted exclusively with the enzyme inthe glyoxysome. The Cys-EP is thus the ®rst markerenzyme for ricinosomes, which may represent a lyticcompartment in senescing cells.

Materials and methods

Plant material. Castor bean seeds (Ricinus communis L., harvest1995; L.L. Olds Seed Co., Madison, Wis., USA) were soaked inrunning tap water overnight and grown in moist autoclavedvermiculite in the dark at 30 °C for 1±7 d (usually 5 d).

Cloning of the cDNA that encodes Cys-EP. We used two di�erenttypes of cDNA libraries: Poly(A)+ RNA was isolated from theendosperm of 4-d-old germinating castor beans and ®rst strandsynthesis of the cDNA [single-stranded (ss) cDNA] using oligo(dT)primer and random primers was carried out as described by Gietl(1990). The other cDNA library [double-stranded (ds) cDNA]exhibited expressed sequence tags from developing castor beanseeds and was prepared from stages III through V endosperm tissue(Van de Loo et al. 1995). Isolation of the cDNA clone for Cys-EPwas carried out in three steps. The central part was ampli®ed bypolymerase chain reaction (PCR) with primers of mixed oligonu-

cleotides encoding the N-terminal and internal amino acids of themature Cys-EP known from direct sequencing (Gietl et al. 1997);the Sense1 primer encoded KGAVTSVKDQG (5¢-AAA/G GGIGCI GTI ACI III GTI AAA/G GAT/C CAA/G GG-3¢;I � deoxyinosine; see Fig. 3, bases 443±474), the Antisense1primer was made based on HGVAIVGYG (5¢-CC A/GTA ICCIAC IAT IGC ITC ICC A/GTG-3¢; I � deoxyinosine; see Fig. 3,bases 896±921); the ss cDNA made from germinating castor beanwas used as template. A single PCR fragment was generated andcloned into the SrfI site of pCR-Script (Stratagene, Heidelberg,Germany). Sequence determination of the resulting plasmidshowed a 479-bp insert comprising the primers and the codingsequence for additional internal amino acids (see Fig. 3, aminoacids 272±285) known from direct sequencing (Gietl et al. 1997) inthe same reading frame. Computer analysis revealed highesthomology to cysteine endopeptidases from V. mungo, P. vulgarisand Hemerocallis in agreement with direct amino acid sequenceanalysis of the puri®ed castor bean enzyme (Gietl et al. 1997). ThisPCR product was labelled with digoxigenin (DIG; Boehringer,Mannheim, Germany) and used as a probe for screening the cDNAlibrary made from mRNA of developing endosperm. Eleven cDNAclones were isolated showing complete sequence identity as far asthey overlapped. All of them contained the 3¢ end of the clone, butdi�ered in length at the 5¢ end; the longest clone starting within thepre-sequence (see Fig. 3, base 83), so that the ®rst 14 amino acids ofthe coding region and the 5¢ untranslated region were missing. Allthe isolated cDNA clones, which represented mRNA from devel-oping seeds, showed sequence identity with the 479-bp probeampli®ed from ss cDNA of germinating endosperm. They also hadthe correct codons for the amino acids known from directsequencing of the N-terminus of the pro-enzyme (see Fig. 3, aminoacids 21±40) and the N-terminal and internal peptide of the maturesubunit (see Fig. 3, amino acids 126±145 and 272±301), which werepuri®ed from endosperm of germinating seeds. No full-length clonecould be isolated by screening the ds cDNA library. To isolate the5¢ end of the clone, poly(dG)-tailed ss cDNA was used as atemplate with the Antisense2 primer based on the nucleotidesequence for amino acids 69±77 (HNANKMDKP) of the pre-pro-enzyme as found in the isolated ds cDNA clones (see Fig. 3; bases244±270); the Sense primer included an oligo(dC) tail. After cloningthe product into the SrfI site of pCR-Script (Stratagene), a 270-bpinsert could be sequenced. It had the Cys-EP-speci®c Antisenseprimer and the adjacent coding sequence for the amino acidsalready known from the longest ds cDNA clone in the samereading frame (see Fig. 3; bases 83±243). In addition, 14 aminoacids including the start methionine, which belong to the pre-sequence, and a 5¢ untranslated region of 40 bases were found.Finally, the complete cDNA clone for Cys-EP was synthesised byPCR. The N-terminal part was synthesised with the Sense3 primerbased on the ®rst 22 bases of the coding region plus the AGpreceding the coding region (see Fig. 3; bases 39±62); a NotIrestriction endonuclease cleavage site was added for cloningpurposes: 5¢-GGCGGCCGC- AGATGCAAAAGTTTA-TACTTCTGG-3¢. The Antisense2 primer was complementary tobases 244±270 (see Fig. 3). The C-terminal part was synthesisedwith the Sense2 primer based on amino acids 15±23 (see Fig. 3;bases 83±109); the Antisense3 primer was complementary to bases1231±1254 (see Fig. 3) in front of the poly(A) tail and included aSalI site at the 5¢ end: 5¢-GGTCGAC-AGGAAAACAATCTTG-CAAACTTTA-3¢. In a third ampli®cation step, the full-lengthcDNA was produced by mixing the N-terminal and the C-terminalparts and adding an excess of NotI-sense- and SalI-antisense-primer (Horton et al. 1989). The ®nal PCR product was cut withNotI and SalI, cloned into the appropriate sites of the transcriptionvector pGEMEX-1 (Promega, Mannheim, Germany) and checkedby sequencing.

Biochemical procedures. A crude organellar pellet was preparedfrom 5-d-old castor bean endosperm. Microbodies were isolated ona discontinuous sucrose gradient and sedimented as a visible whiteband at a density of 1.24 g á mL)1 at the interface between the 50

M. Schmid et al.: Cysteine endopeptidase in ricinosomes 467

and 57% sucrose steps. These procedures, and the puri®cation ofthe 35-kDa Cys-EP, the assay for Cys-EP activity, the preparationof crude protein extracts from 1- to 7-d-old germinated endospermand western blot analysis, were carried out as previously described(Gietl et al. 1997). For sequencing the 45-kDa cysteine pro-endopeptidase, microbody-containing fractions were isolated on asucrose gradient; 12 lg protein was separated by SDS-PAGE (8%gels) and blotted on an Immobilon-P membrane (Millipore,Eschborn, Germany). The membrane was stained with CoomassieBlue R-250 (Sigma, Deisenhofen, Germany) in 50% methanol anddestained with 50% methanol. The stained 45-kDa band (seeFig. 5, lane 5) was cut out and sequenced. Antibodies against theover-expressed pre-gMDH (Gietl et al. 1996) and against thepuri®ed 35-kDa Cys-EP were raised in rabbits.

Enzymatic activity test of the 45-kDa pro-enzyme. For endopepti-dase activity of the 45-kDa Cys-EP, microbody-containing frac-tions from a sucrose gradient were diluted 1:4 with bu�er (10 mMHepes, 1 mM DTT, pH 7.4) and centrifuged (30 000 rpm, 30 min,70 Ti rotor; Beckman, MuÈ nchen, Germany). The clear supernatantwas divided in two and one part was incubated for 1, 3, 5 or 20 minat 20 °C. The reaction products were analysed by SDS-PAGE (8%gel) and the gel developed as a western blot to check the integrity ofthe 45-kDa form of the Cys-EP. The other part was diluted 10-foldor 50-fold with bu�er, and 10 lL of this protein extract wasincubated with 5 lL of in vitro translated, [35S]Met-labelled pre-gMDH (diluted 1:5 with bu�er) for 1, 3, 5 or 20 min at 20 °C. Thereaction was stopped by the addition of loading bu�er and heatingin boiling water for 2 min. The reaction products were separated bySDS-PAGE (8% gels) and the gel was developed for exposure to X-ray ®lm (Kodak X-Omat; Sigma) to check enzymatic activityagainst the substrate pre-gMDH. For comparison the same assaywas carried out with the puri®ed 35-kDa form of the Cys-EP; thepuri®ed protein was diluted 20-fold or 100-fold for assaying theenzymatic activity.

Electron microscopy. Endosperms from 4- or 5-d-old castor beanseedlings were cut into 0.5-mm sections under 3% (v/v) formalde-hyde, 3% (v/v) glutaraldehyde, 10 mMMgCl2 in 0.08 M Pipes, pH8.0 (KOH), and incubated for 1 h at room temperature (Salemaand Brandao 1973). Samples were washed with 0.2 M Pipes, pH6.8, and post-®xed in 2% (w/v) OsO4 in 0.1 M Pipes, pH 6.8, for1 h. After two washes in distilled water, samples were chemicallydehydrated in two changes of acidi®ed dimethoxypropane (25 lLof 1 N HCl + 50 mL dimethoxypropane; Dietrichs and Dosche1982). After washing in 100% ethanol, samples were in®ltrated inethanol and London Resin White (medium grade; Plano, Wetzlar,

Germany) 50% (1 h), 75% (1 h) and left overnight in 100% resin at4 °C. Samples were embedded in gelatine capsules after two further1-h incubations in 100% resin, and polymerised for 24 h at 50 °C.Thin sections were cut with a diamond knife and picked up onFormvar-coated 100 mesh nickel grids. Grids were stained with 5%(w/v) uranyl acetate (30 min at 40 °C) and Reynold's lead citrate(1 min 20 s at 20 °C; Reynolds 1963) in an Ultrostainer (LKB/Pharmacia, Freiburg, Germany) before being examined in a Zeiss109 (Oberkochen, Germany) electron microscope at 80 kV.

Immunocytochemistry. The whole procedure was carried out on10 lL drops on Para®lm. During transfer excess liquid wasremoved with ®lter paper. Thin sections on 100-mesh nickel gridswere incubated for 30 min in freshly prepared 5% (w/v) sodiummetaperiodate to remove osmium (Craig and Goodchild 1984).After 5-min washes in 0.1 N HCl and water, grids were incubatedfor 1 h in blocking solution to prevent non-speci®c binding. Theblocking solution (TBSTM) contained 2% (w/v) skimmed milk and0.05% (v/v) Tween 20 in Tris-bu�ered saline (0.5 M NaCl; TBS).The grids were then incubated overnight at 4 °C with antibodiesraised against puri®ed Cys-EP (diluted 1:100) or against glyoxyso-mal MDH (diluted 1:500) in TBSTM. After washing 3 ´ 5 min inTBSTM, the grids were incubated for 1 h at room temperature withsecondary antibody (goat anti-rabbit IgG conjugated to 5 nm or10 nm gold; Sigma) diluted 1:40 in TBSTM. The grids were washed2 ´ 5 min in water and post-stained with uranyl acetate and leadcitrate as described above. For double labelling, coated grids wereincubated as above with primary antibody, followed by a 10-nm-gold-conjugated secondary antibody. After a second blocking step,the grids were incubated with the second primary antibody,followed by a 5-nm-gold-conjugated secondary antibody. Theorder of primary antibodies was varied.

Results

The Cys-EP is a marker enzyme for ricinosomes, whereasgMDH is localised solely in glyoxysomes. Electronmicroscopy of OsO4-®xed endosperm from 5-d-oldgerminating castor beans revealed the presence of anorganelle surrounded by a single membrane, often inclose association with a glyoxysome. These were usuallyless electron dense than glyoxysomes, and some hadribosome-like bodies associated with their membranes(Figs. 1a,b and 2a). This morphology identi®ed them asricinosomes, dilations of the ER as described forgerminating Ricinus endosperm (Mollenhauer and Tot-ten 1970; Vigil 1970). Immunocytochemistry using theantibody to Cys-EP, revealed dense and speci®c labellingof the ricinosomes, with no labelling of glyoxysomes in5-d-old seedlings (Fig. 2b,c). This was the case in

Fig. 1a,b. Electron microscopy of endosperm from 5-d-old germi-nating castor beans. The endosperm was ®xed in aldehyde and post-®xed in OsO4, showing small organelles surrounded by membranes (r,ricinosome; g, glyoxysome), some of which have ribosome-likeparticles attached, (arrowheads) identifying them as ricinosomes. ´43000

468 M. Schmid et al.: Cysteine endopeptidase in ricinosomes

aldehyde-®xed materials as well as in sections contrastedwith OsO4. Quantitation of gold labelling by anti-Cys-EP gave 333 � 78 particles per lm2 for ricinosomescompared with 1.2 � 0.3 particles per lm2 for the restof the cell (Table 1A). Quantitation of gold labelling byanti-gMDH gave 136 � 29 particles per lm2 forglyoxysomes compared with 5.7 � 2.6 particles perlm2 for the rest of the cell (Table 1B). To con®rm the

localisation of Cys-EP and gMDH, double-labellingexperiments were performed on aldehyde-®xed samples,since OsO4-®xed material was not recognised by thegMDH antibody. Due to the fragile nature of thematerial, we were obliged to use coated grids. Whensections were labelled ®rst with antibody to Cys-EP/10-nm gold, followed by anti-gMDH/5-nm gold, glyoxy-somes were labelled only with 5-nm gold, while

Fig. 2a±e. Immunocytochemistry of the Cys-EP and gMDH. Endosperm tissue from 5-d-old germinating Ricinus seedlings was ®xed in aldehydealone (c±e) or post-®xed in OsO4 (a,b). Sections were incubated with antibody against Cys-EP alone (b,c) or double labelled (d,e). ´43000. aRicinosomes (r) are roughly spherical organelles, less electron dense than glyoxysomes (g), with which they are often in close contact. Thepresence of ribosome-like bodies on the membrane of ricinosomes indicates their origin from the ER. b Ricinosomes (r) are heavily labelled withthe antibody to Cys-EP with little or no labelling of glyoxysomes (g) or oleosomes (o). c As in b with aldehyde-®xed material showing labelling ofricinosomes (r) but not glyoxysomes (g). m, mitochondrion dDouble labelling, ®rst with anti-Cys-EP/10-nm gold, followed by anti-gMDH/5-nmgold, identi®es a glyoxysome (g), labelled only for gMDH. Ricinosomes (r) are labelled with 10-nm gold/Cys-EP and 5-nm gold/gMDH. eDouble labelling, ®rst with anti-gMDH/10-nm gold, followed by anti-Cys-EP/5-nm gold, showing ricinosomes labelled only with 5-nm gold.Double labelling of glyoxysomes (g) here, and ricinosomes in d presumably results from the reaction of the second (5 nm) secondary antibodywith the ®rst primary antibody. Thus there is no gMDH in ricinosomes (e) and no cysteine endopeptidase in glyoxysomes (d) in 5-d-oldgerminating endosperm, P, plastid


ricinosomes were labelled with 10-nm gold and 5-nmgold (Fig. 2d). When sections were labelled ®rst withanti-gMDH/10-nm gold, followed by antibody to Cys-EP/5-nm gold, the ricinosomes were labelled only with5-nm gold (Fig. 2e), but glyoxysomes were labelled withboth 10- and 5-nm gold. We interpret double labellingon the organelle as being due to a reaction of the 5-nm-gold-conjugated secondary antibody to the ®rst specif-ically deposited antibody.

The Cys-EP is synthesised as a pre-pro-enzyme. UsingN-terminal and internal protein sequence information,PCR primers were designed and a full-length cDNAclone of 1254 bp was isolated as detailed underMaterials and methods (Fig. 3). One open readingframe encoding a polypeptide of 360 amino acids wasidenti®ed. It codes for a cysteine endopeptidase be-longing to the papain family. Peptidases of the papainfamily are synthesised with signal peptides (pre-se-quences) and a pro-peptide intercalated between the

signal peptide and the N-terminus of the matureenzyme (Rawlings and Barrett 1994). The 20 N-terminal amino acids and the 30 internal amino acidsknown from sequencing of the mature Cys-EP (Gietlet al. 1997) were found in-frame (Fig. 3, amino acids126±145 and 272±301). Furthermore, the 45-kDa pro-enzyme was puri®ed by SDS-PAGE from isolatedorganelle fractions, as described in the next section.Sequence determination of its N-terminus revealed 20amino acids (Fig. 3, amino acids 21±40), which againwere found in-frame (Fig. 3, double underlined). Fromthese N-terminal amino acid sequence determinations,the cleavage site of the pre-sequence C-terminal toserine (position 20) and the cleavage site of the pro-peptide C-terminal to threonine (position 125) can beidenti®ed. The signal peptide exhibits a hydrophobiccore, which is the typical feature of a targetingsequence for the ER. Within the pro-sequence is theso-called ``ERFNIN motif'' (Fig. 4, boxed) which ischaracteristic of pro-peptides related to papain (Raw-lings and Barrett 1994). The catalytic residues ofpapain-type peptidases cysteine-150 and histidine-286are conserved; other residues important for catalysisinclude glutamine-144, which helps form the ``oxyanionhole'', and asparagine-307, which orientates the imida-zolium ring of histidine-286 (Figs. 3, 4). Six additionalcysteine residues are conserved, which form the threedisul®de bridges in the mature enzyme (Fig. 4). TheKDEL motif, which functions as a retention signal forthe ER, is found at the C-terminus. The 3¢ untranslatedregion contains a polyadenylation signal (AATAAA) atposition 1229.

Table 1A,B. Speci®city of labeling with antibodies raised againstthe puri®ed Cys-EP (A) or gMDH (B). Average and SE were cal-culated by counting particles over a total area of 103 lm2 (ninemicrographs) for Cys-EP and over 79.5 lm2 (eight micrographs)for gMDH

Gold particles per lm2

A Ricinosome 333.0 � 78.0Remaining part of the cell 1.2 � 0.3

B Glyoxysome 136.0 � 29.0Remaining part of the cell 5.7 � 2.6

Fig. 3. The cDNA and derived amino acidsequence of Cys-EP. The N-terminal aminoacid sequences of the puri®ed 45-kDa pro-enzyme [amino acids (aa) 21±40], of thepuri®ed 35-kDa mature enzyme (aa 126±145) and the sequence of the internalpeptide (aa 272±301) are double underlined.Primers used for PCR are marked witharrows. The cleavage sites for the prese-quence and the prosequence are indicated(accession no. AF050756)


We obtained independent and identical cDNA se-quence information for the Cys-EP from castor beanendosperm of germinating and developing seeds. Boththe expressed sequence tag from developing seeds andthe clone from germinating seedlings encoded thepeptides known from direct amino acid sequencing ofthe Cys-EP of germinating castor bean seeds (Fig. 3).Furthermore, all DNA sequences were identical as far asthey overlapped (Fig. 3; bases 83±270, 443±921). Weconclude that in the endosperm of developing andgerminating seeds the identical or homologous cysteineendopeptidase is expressed.

The 45-kDa pro-enzyme of Cys-EP is found in organellesisolated on a sucrose gradient. The 35-kDa mature formof Cys-EP has been puri®ed from organelles isolated ona sucrose gradient as well as from a crude organellarpellet (Gietl et al. 1997). We repeated the puri®cation inorder to see if the pro-enzyme of Cys-EP could beidenti®ed in a protein extract from a microbody-containing sucrose gradient fraction. The isolated or-ganelles were sonicated, centrifuged, and an aliquot ofthe supernatant was stored during the puri®cationprocedure for later analysis by SDS-PAGE (Fig. 5, lane1). The supernatant was loaded onto a column of DEAEBioGel A agarose (Bio-Rad MuÈ nchen, Germany). Thefraction containing the enzymatic activity was elutedwith 250 mM NaCl in Hepes-DTT bu�er and loadedonto a p-aminobenzamidine a�nity column (Sigma).The column was washed with 100 mM NaCl in Hepes-DTT (Fig. 5, lane 2); this fraction contained noenzymatic activity. The pure, enzymatically active 35-kDa Cys-EP was eluted with 500 mM NaCl in Hepes-DTT (Fig. 5, lane 3). However, when proteins from themicrobody-containing fraction isolated on a sucrosegradient were directly separated by SDS-PAGE withoutfurther puri®cation, no 35-kDa mature enzyme could bedetected; instead one weak (46 kDa) and one strong

(45 kDa) protein band appeared (Fig. 5, lane 5). The 45-kDa band was cut out and the 20 N-terminal aminoacids were determined. This sequence was identical toresidues 21±40 of the deduced amino acid sequence ofthe cDNA clone and is therefore the N-terminus of the

Fig. 5. Presence of the 45-kDa Cys-EP pro-enzyme and conversion tothe 35-kDa mature form in vitro during puri®cation as monitored bySDS-PAGE and Coomassie blue R-250 staining. Lane 1, proteinextract from a crude organellar pellet, stored at 4 °C during thepuri®cation procedure; lane 2, p-aminobenzamidine a�nity columneluted with 100 mM NaCl in Hepes-DTT; lane 3, p-aminobenzami-dine a�nity column eluted with 500 mMNaCl in Hepes-DTT; lane 4,protein extract from microbody-containing fractions stored overnightat 4 °C; lane 5, protein extract from microbody containing fractionsanalysed immediately. MM, molecular mass standards

Fig. 4. Alignment of the cysteine endo-peptidases from R. communis (Cys-EP;AF050756), V. mungo (SH-EP; Akasofu etal. 1989; X15732), P. vulgaris (EP-C1;Tanaka et al. 1991; X56753), V. sativa(ProtA; Becker et al. 1997; Z348959) andHemerocallis (Sen102; Valpuesta et al.1995; X74406). Identical amino acids aremarked with asterisk (*), similar aminoacids with a dot (.). The ``ERFNIN motif''within the prosequence, and amino acidsbelonging to the catalytic pocket (Cys-150and His 286) or otherwise important forcatalysis (Gln-144 and Asn-307) are boxed.Cysteine residues involved in disul®debridges are connected


propeptide (Fig. 3). When this protein fraction wasstored overnight at 4 °C or treated by freezing andthawing, and analysed again by SDS-PAGE, the 35-kDaform appeared (Fig. 5, lane 4). To con®rm that both the45-kDa and 35-kDa forms originate from the sameprotein precursor, we analysed them on a western blot(Fig. 6) with the antibodies against the puri®ed 35-kDaform of Cys-EP. Only the 35-kDa band was recognised(Fig. 6, lane 1) in a protein extract from a crudeorganelle pellet, which was stored during puri®cation(Fig. 5, lane 1). Proteins in the 100 mM NaCl-eluate ofthe p-aminobenzamidine column, which have no enzy-matic activity (Fig. 5, lane 2), did not react with theantibodies (Fig. 6, lane 4). In a protein extract frommicrobody-containing fractions stored overnight, the45-kDa and 35-kDa bands and also the 46-kDa bandwere recognised by the antibody when the same amount

(Fig. 6, lane 3) or half the amount (Fig. 6, lane 2) ofprotein was loaded as in Fig. 5 (lane 4).

Crude protein extracts from the endosperm ofgerminating castor bean seedlings on days 1±7 wereprobed with the antibody in western blots to follow theappearance of the 45-kDa pro-enzyme and its processinginto the 35-kDa mature form of Cys-EP. The radicleemerged on day 2; starting from day 4 the endospermbecame soft, and a slimy layer developed betweenendosperm and cotyledons. By days 6 and 7 theendosperm is almost exhausted and the cells collapse.This development is mirrored by the synthesis of Cys-EP(Fig. 7). In 3-d-old endosperm the 45-kDa proform wasvisible, while in 4- and 5-d-old endosperm the matureform (35 kDa) and a putative degradation product of30 kDa were seen. We do not know if processing of thepro-enzyme to the mature enzyme at days 4 and 5re¯ects the in vivo situation or whether processingoccurred during protein extract preparation.

We conclude that the 45-kDa pro-enzyme form ofCys-EP was present in the isolated organelles, but wasconverted in vitro to the mature 35-kDa form during thepuri®cation process and also during storage of theprotein extract. It remains to be established howprocessing of the pro-peptide takes place in vivo.

The 45 kDa pro-enzyme of Cys-EP has enzymaticactivity. The 45-kDa pro-protein (Fig. 5, lane 5) andthe 35-kDa mature form (Fig. 5, lane 3) were assayed asdescribed in Materials and methods for their capacity toprocess the gMDH precursor protein (pre-gMDH) invitro with the formation of the two characteristiccleavage intermediates (Fig. 8). The integrity and sta-bility of the Cys-EP pro-enzyme and mature enzymeduring incubation was followed by western blots(Fig. 9). In incubations of full-length pre-gMDH withthe puri®ed 35-kDa endopeptidase, the 37 amino acidpresequence of gMDH was consistently cleaved in three

Fig. 6. Western blot analysis of the 45-kDa and 35-kDa forms ofCys-EP during puri®cation. Lane 1, protein extract stored during thepuri®cation procedure; lanes 2 and 3, protein extract from microbody-containing fractions stored overnight, the same amount of proteinloaded as in Fig. 3, lane 4, or half the amount; lane 4, p-amino-benzamidine a�nity column eluted with 100 mM NaCl in Hepes-DTT

Fig. 7. Western blot analysis of crude protein extracts from theendosperm of germinating castor bean seedlings on days 1±7 withantibodies directed against the 35-kDa Cys-EP

Fig. 8. Enzymatic activity of Cys-EP as monitored by formation ofgMDH intermediates (i1-gMDH, i2-gMDH) and mature gMDHsubunit from pre-gMDH. The 45-kDa pro-enzyme present inmicrobody-containing fractions was diluted 10-fold (lanes 1±4) or50-fold (lanes 5±8) and incubated with [35S]Met-labelled pre-gMDH.In a similar manner, the puri®ed 35-kDa mature form was diluted 20-fold (lanes 9±12) or 100-fold (lanes 13±16) and incubated with pre-gMDH. Lane 17, in vitro translated, [35S]Met-labelled pre-MDH (TR)incubated for 20 min with bu�er instead of Cys-EP. After the timesindicated, the reactions were stopped and analysed by SDS-PAGEand ¯uorography


sequential steps, with the two intermediates i1-gMDHand i2-gMDH occurring prior to the ®nal cleavage.Using the mature endopeptidase at a 100-fold dilution,mainly i1-gMDH and i2-gMDH were seen, whereas at a20-fold dilution i2-gMDH and gMDH were produced ina time-dependent manner (Fig. 8, lanes 9±16). Nodegradation of the 35-kDa Cys-EP was detected duringthe 20-min incubation (Fig. 9, lanes 5±8). The proteinextract containing the 45-kDa pro-form of Cys-EP, butno 35-kDa mature enzyme was stable during the 20-minincubation (Fig. 9, lanes 1±4). Since the anti-Cys-EPantibody seems to recognise the 35-kDa form moree�ciently than the 45-kDa form (compare Fig. 5, lane 4with Fig. 6, lane 3), we can exclude the presence of the35-kDa form in this preparation. Incubation of this 45-kDa Cys-EP, which is only slightly contaminated byother proteins, with the full-length pre-gMDH resultedin the cleavage pattern typical of the 35-kDa Cys-EP, i.e.the appearance of i1-gMDH, i2-gMDH and gMDH in atime- and concentration-dependent manner (Fig. 8,lanes 1±8). These data indicate that removal of thepro-peptide is not necessary for activation of Cys-EP.

Discussion

The Cys-EP is synthesised as a pre-pro-enzyme leading topost-translational processing of an N-terminal and a C-terminal pro-peptide. The cysteine endopeptidase fromgerminating Ricinus endosperm is synthesised as a pre-pro-enzyme with a 20-amino-acid pre-sequence, an N-terminal pro-peptide of 105 amino acids and a ``KDELmotif'' at the C-terminus, which functions as a retentionsignal for the ER (Fig. 3). The 47-kDa precursor is co-translationally processed to a 45-kDa intermediatethrough the cleavage of the 2-kDa signal sequence.The 45-kDa pro-enzyme is found in organelle prepara-tions isolated on a sucrose gradient (Figs. 5, 6). TheRicinus enzyme, Cys-EP, is highly homologous to acysteine endopeptidase, designated SH-EP, which occursin the cotyledons of germinated seeds of V. mungo(Akasofu et al. 1989; Fig. 4). For SH-EP, a similar timecourse of post-translational processing is described: SH-EP is synthesised on membrane-bound polysomes as aninactive 45-kDa precursor, which is co-translationallyprocessed to a 43-kDa intermediate through the cleavage

of a 2-kDa signal sequence. A processing enzyme termedVmPE-1, which is involved in processing of the 43-kDaintermediate to a 39-kDa form, was isolated from V.mungo cotyledons. The processing enzyme VmPE-1 is amember of the asparaginyl endopeptidase family, whichcleaves after asparagine residues. Processing of the 39-kDa intermediate to the 36-kDa intermediate andsubsequent activation to the 33-kDa mature enzyme isautocatalytic (Okamoto and Minamikawa 1995). Inaddition to the N-terminal propeptide, the C-terminalpropeptide composed of 10 amino residues containingthe KDEL sequence is processed (Okamoto et al. 1994).In a similar way, the C-terminal peptide containing theKDEL sequence is absent in the mature proteinase A inV. sativa (Becker et al. 1997). Despite the fact that onlythe 45-kDa pro-enzyme was found in ricinosomesanalysed immediately after isolation on a sucrosegradient (Fig. 5), we puri®ed the 35-kDa mature enzymefrom such organelles (Gietl et al. 1997). The pro-peptidewas cleaved o� either autocatalytically during puri®ca-tion, or the responsible processing peptidase was alsopresent in initial steps of the puri®cation process.

The 45-kDa pro-enzyme of Cys-EP has enzymaticactivity. Since the 45-kDa pro-enzyme of the castorbean endopeptidase is stable in the organelle fraction, wewanted to know if it is enzymatically active. The 45-kDapro-enzyme, however, could not be obtained as a pureprotein, because it converts to the 35-kDa form duringpuri®cation. Therefore we demonstrated its enzymaticactivity in organelles containing the 45-kDa pro-enzymebut not the 35-kDa mature protein. Besides the active45-kDa and 35-kDa forms of Cys-EP, an immunoreac-tive 30-kDa degradation product was detected, especial-ly at days 4 and 5 (Fig. 7). A 22-kDa protease wassometimes co-puri®ed with the 35-kDa Cys-EP and hadthe same N-terminus as the 35-kDa Cys-EP (sequencedata not shown) and thus represents a C-terminallytruncated, active form of the 35-kDa Cys-EP. Proteo-lytically active pro-peptidases are not common butinclude two cysteine endopeptidases (papaya proteinaseIV and cathepsin L) and an aspartic acid protease(cathepsin D; Baker et al. 1996 and references therein).

Possible functions for Cys-EP. The 35-kDa Cys-EP waspuri®ed from organelle fractions of germinating castorbean endosperm by virtue of its capacity to process thegMDH precursor protein to the mature subunit in vitro.Immunocytochemistry, however, revealed that Cys-EPis localised in ricinosomes, whereas gMDH is found inglyoxysomes. This almost certainly rules out a role forCys-EP in processing gMDH in planta. Its involvementin protein mobilisation is also unlikely, since Cys-EPactivity (Gietl et al. 1997) correlates with the develop-ment of ricinosomes (Vigil 1970), being highest in 4- and5-d-old germinating castor bean endosperm, i.e. at adevelopmental stage when the small amount of storageprotein visible in 1- and 2-d-old endosperm is alreadydegraded.

On the other hand, the Ricinus endosperm is asenescing tissue, and its Cys-EP is highly homologous to

Fig. 9. The 45-kDa pro-enzyme and the 35-kDa mature form of Cys-EP remain undegraded during the incubation time (1, 3, 5, and 20 minat 20 °C) of the activity assay (see Fig. 6) as monitored by westernblot analysis. Lanes 1±4, 45-kDa form from microbody-containingfractions; lanes 5±8, puri®ed 35-kDa form


cysteine endopeptidases present in senescing tissues ofother plants (Figs. 4, 10). The cotyledons of the epigaeicgerminating V. mungo containing the cysteine peptidaseSH-EP do not become green; they are a senescing tissueand abscise as soon as the storage material is mobilised.The cotyledons of other species within the Fabaceae,such as soybean (Glycine max.), become green in thelight, similar to the cotyledons of the Cucurbitaceae, andsenescence is expected to occur later in development.The P. vulgaris cysteine endopeptidase (EP-C1) waspuri®ed from pods of maturing fruits of French beanplants. Pods are senescing tissue in that they ®rstaccumulate protein and then translocate it in the formof amino acids to the developing seeds (Tanaka et al.1993). Ricinus Cys-EP is 84% identical to these cysteineendopeptidases from senescing tissues (Fig. 10). Pro-teinase A is a papain-like cysteine endopeptidase whichwas ®rst detected in cotyledon extracts of seeds germi-nated for 6 d when globulin degradation has alreadyproceeded to a large extent (Becker et al. 1997). Thesedata do not support the suggested role of proteinase A intriggering storage globulin breakdown during germina-tion; a role during senescence is a possibility. As acommon denominator for the presence of the discussedcysteine endopeptidases, a function in the transfer ofmetabolites from one tissue to another emerges: fromendosperm to the cotyledons in R. communis, fromcotyledons to the primary leaves in some Fabaceae andfrom withering ¯owers back to the plant.

The Cys-EP enzyme of germinating castor bean endo-sperm is localised in ricinosomes. A papain-type cysteineendopeptidase of germinating castor bean endospermwas isolated from organelles isolated on a sucrosegradient (Gietl et al. 1997). Microbodies sediment at adensity of 1.24 g mL)1 and have little or no contami-nation from marker enzymes of other organelles such asplastids or mitochondria (Cooper and Beevers 1969).Germinating castor bean endosperm, however, containsa second microbody-like organelle, which co-sedimentswith glyoxysomes on a sucrose gradient (Vigil 1970).Immunocytochemistry using an antibody to the puri®edCys-EP, revealed a highly speci®c labelling of this

organelle ± the ricinosome (Fig. 2, Table 1). Ricino-somes seem to arise as dilations of the ER, and may ormay not pinch o� from the ER (Fig. 1; Mollenhauer andTotten 1970; Vigil 1970). Their formation resembles thatof protein bodies in the ER lumen of maize (Lendingand Larkins 1992) and in the barley mutant Nevsky(Rechinger et al. 1993), although these are clearlycontiguous with the rest of the ER. Oleosomes arecytoplasmic oil bodies surrounded by a phospholipidmonolayer, which bud o� from the ER in a processmediated by oleosins (Huang 1996). Morphologically,ricinosomes resemble most closely the aleurain-contain-ing vacuoles of barley aleurone cells (Holwerda et al.1990). These acidic vacuoles can be distinguished fromneutral protein storage vacuoles by the nature of theirtonoplast intrinsic proteins (Paris et al. 1996). Whilestorage proteins are transported via the Golgi to thevacuoles of temperate cereal endosperm (data notshown), protein storage vacuoles in vegetative cellsmay arise de novo directly from the ER (see Staehelin1997 for a summary).

The transport of Cys-EP to ricinosomes presumablydoes not go via the Golgi as long as the C-terminalKDEL sequence remains attached. This implies that thepro-peptidase is packaged into the ricinosomes as theybegin to bud o� from the ER, similar to maize proteinbodies or oleosomes. There is no direct evidencefor subsequent processing in vivo, either at the C- orN-terminus. In mung bean, when cotyledons werehomogenised, conversion of the 43-kDa form to the®nal 33-kDa form occurred over a 4-h period in vitro(Okamoto et al. 1994). In contrast, isolated ricinosomescontain only an active, unprocessed 45-kDa form, whichis converted to a 35-kDa mature form in vitro. The roleof the ricinosome in senescing cells, the presence ofsimilar organelles in other plants and the transportpathway of Cys-EP from the ER remain to be elucidat-ed. The Cys-EP is the ®rst marker enzyme for ricino-somes and opens the possibility for their puri®cation andcharacterisation. It will be interesting to see if theycontain other resident proteins of the ER lumen, andwhether N- and C-terminal processing of Cys-EP occursin planta.

We thank Professor Chris Somerville (Department of BiologicalSciences, Stanford University, Stanford, Calif., USA) for his kindgift of the cDNA library from developing castor bean seeds. Wealso thank Dana Adamcova for skillful technical help and Ann-So®Steinholz for printing the electron micrographs. Financial supportfor this work was from the Deutsche Forschungsgemeinschaft(C.G.) and National Institutes of Health (F.K.).

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a cysteine endopeptidase with a c-terminal kdel motif isolated from castor bean endosperm is a...

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