proglobulinprocessing enzyme in vacuolesisolated from … · it hasbeenestablished thatthepumpkin i...

Plant Physiol. (1987) 85, 440-4450032-0889/87/85/0440/06/$0 1.00/0

Proglobulin Processing Enzyme in Vacuoles Isolated fromDeveloping Pumpkin Cotyledons'

Received for publication January 5, 1987 and in revised form June 8, 1987

IKUKO HARA-NISHIMURA*2 AND MIKIo NISHIMURA2Research Institutefor Biochemical Regulation, School ofAgriculture, Nagoya University,Chikusa, Nagoya 464 Japan

ABSTRACT

The enzymic conversion of proglobulin to globulin catalyzed by theextracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cvKurokawa Amakuri Nankin) cotyledons was investigated. The endo-plasmic reticulum fraction isolated from the developing cotyledons pulse-labeled with I35Slmethionine was shown to contain mainly the radiolabeledproglobulin, which was used as a substrate for assaying the proteolyticprocessing in vitro. The vacuolar extracts catalyzed the proteolyticprocessing of the proglobulin molecule to produce globulin containingtwo kinds of polypeptide chains, 'y and 5. The pH optimum for thevacuole-mediated conversion was at pH 5.0. The proteolytic processingof proglobulin by the vacuolar extracts was inhibited in the presence ofvarious thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide,iodoacetic acid, Hg2", and Cu2", but not phenylmethylsulfonyl fluoride,EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, orpumpkin trypsin inhibitor, and was activated in the presence of dithio-threitol and cysteine, indicating that the processing enzyme is a thiolprotease. The suborganellar fractionation of the vacuoles showed thatthe processing activity was localized in the matrix fraction, but not in themembrane or crystalloid fractions. During the seed development, theenzyme was shown to increase, exhibiting the maximal activity at thelate developmental stage. The matrix fraction of the protein bodiesisolated from the dry castor bean (Ricinus communis) exhibited theprocessing activity toward the pumpkin proglobulin molecules in thesame manner as that by the matrix fraction of pumpkin vacuoles.

ing of two distinct polypeptides, y and 6, linked by a disulfidebond is synthesized as precursor of a single polypeptide chain (7)and that the disulfide bond is introduced in precursor moleculesynthesized in rER (5). Several other seed proteins such as lectinswhich are composed of two distinct polypeptide chains linkedtogether by disulfide bond(s) are also known to be synthesized asa single polypeptide precursor, and both the in vivo labeling andcellular fractionation studies have clearly demonstrated that theprotein bodies are the site of the endoproteolytic cleavage of theprecursor molecules (15). However, the mechanism(s) involvedin the posttranslational proteolytic processing of the single poly-peptide precursor producing the mature form of protein consist-ing of disulfide-linked doublet polypeptide chains is not fullyunderstood. There has been only one investigation concerningthe endoproteolytic enzyme responsible for the posttranslationalcleavage of the precursor proteins by Harley and Lord (9). Theydemonstrated that whole homogenates of the developing castorbean (Ricinus communis) endosperm contained the enzymicactivities capable of converting the precursor peptides of ricinand R. communis agglutinin to their respective mature forms.

In the work reported in this communication, we have exam-ined the nature of the endoproteolytic enzyme activity in theisolated vacuoles by monitoring the ability of lysed vacuolepreparations to convert the labeled proglobulin in the presenceof specific proteinase inhibitors. The suborganellar localizationof the proteolytic processing enzyme in the vacuoles has beenalso determined.

The I 1S globulin protein is the major reserve protein of seedsof both mono- and dicotyledonous plants. Pumpkin (Cucurbitasp.) 11S globulin is structurally quite similar to other 11S glob-ulins such as glycinin and legumin. It has been demonstratedthat in the developing pumpkin cotyledons a larger precursor,preproglobulin, synthesized in the ER is cleaved cotranslationallyand assembled to make the trimeric proglobulin molecule (7),which is transported to vacuoles via dense vesicles. Proglobulinis then posttranslationally processed and assembled finally toproduce hexameric mature 1 IS globulin molecules, which aredeposited in the vacuoles as crystalloids (7). During the lastdevelopmental stage of pumpkin cotyledons, the crystalloidsbecome larger and finally bud out ofthe vacuoles to give sphericalprotein bodies (6).

It has been established that the pumpkin I IS globulin consist-

' This is paper No. 11 in a series "Pumpkin (cucurbita sp.) SeedGlobulin." Paper No. 10 is Ref. 6.

2Present address: Department of Biology, Faculty of Science, KobeUniversity, Rokkoudai, Nada, Kobe 657, Japan.

MATERIALS AND METHODS

Plant Materials. Seeds of pumpkin (Cucurbita sp. cv Kuro-kawa Amakuri Nankin) were grown during the summer seasonof 1986 at the university farm. Cotyledons of seeds freshlyharvested at middle and late developmental stages (20-40 d afteranthesis) were used as experimental materials. Dry castor bean(Ricinus communis) were donated by Dr. M. Yamada of Uni-versity of Tokyo.

Isolation of Vacuoles from Developing Pumpkin Cotyledons.Vacuoles were isolated from cotyledons at middle or late devel-opmental stage, exactly following the methods reported previ-ously (6). Isolated vacuoles were inspected by an Olympus BHSphase-contrast microscope and proved to be free from contami-nation by other subcellular components (Fig. 1). Vacuoles werealso isolated from the pulse-chased developing cotyledons asfollows. Five ,l of [35S]methionine (70 uCi) was administered tothe intact axial surface of the isolated cotyledon placed on amoistened filter paper in a Petri dish and the incubation wasperformed at 25°C. After 1 h of [35S]methionine uptake, thetissue was briefly rinsed using 50 mm unlabeled methioninesolution, followed by the further incubation for 3 h chase.

Isolation of Protein Bodies from Dry Castor Bean. Proteinbodies of dry castor bean were isolated according to the methods

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POSTTRANSLATIONAL PROCESSING ACTIVITY IN VACUOLES

FIG. 1. Microphotograph of vacuoles isolated from pumpkin cotyle-dons at the middle stage of development. Bar is 20 ,m.

described previously ( 16).Preparation of ER and Dense Vesicles from Pulse-Labeled

Developing Cotyledons. Five Al of [35S]methionine (70 ACi) wasadministered to the inner surface of the cotyledons on moistenedfilter paper in a Petri dish and incubation was performed at 25°C.After 1 h of [35S]methionine uptake, the tissue was briefly rinsedin water and homogenized in a Petri dish placed in an ice bathby chopping with a stainless razor blade for 5 min using 1 ml ofchilled medium (150 mm Tricine-KOH [pH 7.5], 1 mm EDTA,and 13% [w/vJ sucrose). The homogenates were passed throughtwo layers of cheesecloth and the filtrate was directly layeredonto a stepwise sucrose gradient consisting of a 1.3 ml of 55%(w/w) sucrose, 1.3 ml of 35% (w/w) sucrose, and 1.3 ml of 20%(w/w) sucrose. The gradient solution was then centrifuged at29,000 rpm for 1 h in a Beckman Spinco model L2 ultracentri-fuge using an SW 65 Ti rotor at 4°C. Sucrose densities for thisstepwise gradient (55, 35, and 20%) were determined from thedata reported previously (7). Employing this gradient centrifu-gation, the ER fraction was concentrated between 20 and 35%sucrose interface and the dense vesicle fraction between 35 and55% sucrose interface. The localization of labeled proglobulinwas analyzed using SDS-PAGE and fluorography. The labeledproglobulin in the ER fraction was used as a substrate for theassay of the proteolytic processing in vitro.

Suborganellar Separation of the Isolated Vacuoles. Suborga-nellar fractionation of the isolated vacuoles was performed bythe sucrose density gradient centrifugation essentially as de-scribed previously (8). The isolated vacuoles (0.1 ml) were lysedby adding 0.2 ml of water, and 0.3 ml of the ruptured fractionwas layered on a sucrose density gradient consisting of 0.5 mlcushion of68% (w/w) sucrose, 4 ml ofsucrose solution increasinglinearly from 30 to 68% and 0.5 ml of 15% sucrose. The gradientwas centrifuged at 30,000 rpm for 5.5 h in a Beckman Spincomodel L2-65B ultracentrifuge using an SW 65 Ti rotor at 4C.After centrifugation, fractions (0.3 ml) were collected and theproteolytic processing activity of each fraction was assayed fol-lowing the method described below. The protein content wasanalyzed using the Bio-Rad Protein Assay Kit and the sucrosedensity was measured by a refractometry.Assay for Proteolytic Processing Activity. Either the vacuolar

extract or the whole homogenate was used as the crude enzymeand the labeled proglobulin in the ER fraction was used as asubstrate for assaying the proteolytic processing activity. Reac-tion mixture contained crude enzyme solution (2 Al), labeledproglobulin solution (3 Al), and 0.1 M citrate-phosphate buffer(pH 5.0) (5 Al). To determine the optimum pH of the processing

activity, citrate-phosphate buffer (pH 3, 4, 5, 6, or 7) or Tris-HCIbuffer (pH 8) (final concentration 50 mM) was used. Incubationwas carried out at 30°C for 0 to 4 h. After the termination ofreaction by adding S ,l of 3% SDS solution containing 30 mMTris-HCI (pH 6.8), 7.5% 2-mercaptoethanol, 15% glycerol, and0.003% bromophenol blue, the sample solution was subjected toSDS-PAGE and subsequent fluorography. The fluorogram wasscanned with a Shimadzu dual-wavelength TLC scanner (modelCS 910) to estimate the amount of products formed. The densityof the corresponding band of product, 6 chain of the matureglobulin produced by the processing of proglobulin, was meas-ured from the area of the resulting peak in the densitometrictracing. Following compounds were tested for either inhibitionor activation of the proteolytic processing activities; 25 ,ug/mleach of peptidyl inhibitor, e.g. leupeptin, antipain, chymostatin,pepstatin, or purified pumpkin trypsin inhibitor, 5 mm PMSF,30.01, 0.1, or 1 mm NEM, 0.01, 0.1, or 1 mm pCMB, 0.01, 0.1,or 1 mM IAA, 0.01, 0.1, or 1 mM EDTA, 0.01, 0.1, or 1 mM o-phenanthroline, 1 mM Ca2 , 1 mM Mg2+, 1 mM Zn2, 1 mM g2,1 mM Mn2+, 20 mM DTT, or 0.1 M cysteine. The crude enzymesolution (2 ul) was preincubated with 0.5 ,ul of various inhibitoror activator for 20 min at 25°C, followed by the addition of thelabeled proglobulin solution to start the reaction.To examine the developmental change of the proteolytic proc-

essing activities, the whole homogenates were prepared from thefollowing developmental stages: stage I (2 mm length, 1 mg/cotyledon half), stage II (10.5 mm length, 34 mg/cotyledon half),stage III (17 mm length, 131 mg/cotyledon half), stage IV (17mm length, 244 mg/cotyledon half), and dry half seed. Eachcotyledon half was homogenized using 0.3 ml of 20 mm citrate-phosphate buffer (pH 6.0), and centrifuged at 15,000 rpm for 15min at 4°C in a Hitachi microcentrifuge (HIMAC CR1 5B). Thesupernatant fraction was used as the crude enzyme solution.

Purification of Pumpkin Trypsin Inhibitor. Pumpkin trypsininhibitor was purified exactly following the methods reportedpreviously (4), and the specific rabbit IgG was raised.

RESULTSVacuoles isolated from the developing cotyledons labeled with

[35S]methionine were found to contain both the radiolabeledproglobulin molecules and the llS mature globulin (Fig. 2),indicating that the posttranslational endoproteolytic processingof proglobulin proceeds in the vacuoles. On the other hand, boththe ER and dense vesicle fractions isolated from the pulse-labeleddeveloping cotyledons were shown to contain mainly the labeledprecursor, proglobulin molecule (Fig. 2). The labeled proglobulinin ER fraction could be easily detected in the fluorographywithout immunoprecipitation, and was used as the substrate forassaying the proteolytic processing activities.During incubation of the reaction mixture containing ER and

the vacuolar extracts, the radioactivity in the proglobulin de-creased with a corresponding increase in the radioactivity of thesmaller polypeptide molecules (Fig. 3, right). The latter productsafter cleavage of the proglobulin molecules were shown to co-migrate with the constituent y and 6 polypeptide chains of themature 1 IS globulin molecules localized in the vacuoles isolatedfrom the labeled developing cotyledons (Fig. 3, right). Heatedenzyme was shown to exhibit no processing activity (Fig. 3, left).The results indicate that the vacuolar extracts contained theproteolytic processing activities responsible for the enzymiccleavage of proglobulin.The optimal pH for the proteolytic processing activities in the

isolated vacuoles is at pH 5; no formation of y and 6 chains ofmature 11S globulin being detectable below pH 4. The 20%

3Abbreviations: PMSF, phenylmethylsulfonyl fluoride; pCMB, p-chloromercuribenzoate; NEM, N-ethylmaleimide.

441

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HARA-NISHIMURA AND NISHIMURA

10 Mm pCMB (98mu TAA (77% in

DV v Cui (41% inhibiever, none of PMMn2" was inhibit(mostatin, antipaiinot show any inhiand 0.1 M cysteineproglobulin pretrthe crude enzymprocessed by the

!8 shown); the alkylamolecules resultsthe alkylated pro,be processed in tiulin molecules. 0essing enzyme isThe developmc

zyme activities is

was not detectablbut it increased githe cotyledons ar

FIG. 2. Fluorograms of protein components in the ER, dense vesicles stage of the devel(DV), and vacuoles (V) prepared from the developing pumpkin cotyle-

tindc ating thi

dons. The developing cotyledon pair was pulse-labeled with [35S]methi- , indicating t

onine for 30 min and chased for 0 h (ER and DV) or 2 h (V). After efficiently duringchopping the cotyledon using a solution containing 13% sucrose, 50 mm The suborganeTricine buffer (pH 6.5), and 1 mM EDTA, the homogenate was filtered examined by the

vacuoles isolatedthrough cheesecloth and the filtrate was applied to a sucrose density proles obsegradient centrifugation for 1 h at 4°C using Beckman SW 65Ti rotor. protein peak obs

pG, proglobulin; y and 6, the constituent polypeptide chains of 11S the crystalloid fraglobulin. reported previous

essing activities i

decrease of the maximum activity was observed at pH 6 or 7, fractions of the s

and the 88% decrease was at pH 8. the matrix of theResults presented in Figure 4 show the effects of various types It was found t

of inhibitors and activators on the proteolytic processing activi- isolated from theties. The processing was significantly inhibited in the presence of essing activity to

*ma,

,% inhibition), 10 jsM NEM (90% inhibition), 1ihibition), 1 mM Hg2+ (93% inhibition), 1 mMition), and I mM Zn2+ (22% inhibition). How-[SF, EDTA, o-phenanthroline, Ca2+, Mg2+, or;ory. Peptidyl inhibitors such as leupeptin, chy-Ln, pepstatin, or pumpkin trypsin inhibitor didiibitory action. On the other hand, 20 mm DTTe activated the processing activity. Alternatively,eated with NEM was shown to be processed bye preparation, but proglobulin could not becrude enzyme pretreated with NEM (data notation of thiol group(s) of the processing enzymein the inhibition of the enzyme activity, butiglobulin molecules with NEM were shown tohe same way as that of the nontreated proglob-verall results indicate that the proteolytic proc-a thiol protease.ental change in the proteolytic processing en-;presented in Figure 5. The processing activityile in the cotyledons at developmental stage I,radually during the subsequent development ofnd reaching the maximum activity at the latelopment. Cotyledons at stage IV had about 2ity than that detected in the cotyledons at stageat the processing enzyme is synthesized mostthe developmental stage III to IV.liar distribution of the processing enzyme wassucrose density gradient centrifugation of thefrom the developing cotyledons (Fig. 6). The-erved in the sucrose gradient corresponded toaction, judging from the comparison of resultssly (6). On the other hand, the proteolytic proc-were found to be present in the uppermostucrose gradient, showing that it is localized invacuoles in situ.that the matrix fraction of the protein bodiesdry castor bean endosperm exhibited the proc-convert the proglobulin to globulin exactly in

FIG. 3. Time-course of proteo-lytic processing of proglobulin toglobulin by vacuolar extract withor without heat treatment. ERfractions isolated from the pulse-labeled developing cotyledons bysucrose density gradient centrifu-gation contained the labeled prog-lobulin molecules (Fig. 2) whichwere used as the substrate for theassay. Vacuoles were isolated fromthe developing cotyledons accord-ing to the methods described pre-viously (6). To 2 ,l of the ERfraction was added 3 Al of the vac-uolar fraction and 5 Ml of 0.1 Mcitrate-phosphate buffer (pH 5.0).After incubation for various pe-

_*00 riods up to 4 h, samples were sub-jected to SDS-PAGE and fluorog-raphy. M, markers for proglobulin(pG) and constituent polypeptidechains (-y and 6) of the mature 1 ISglobulin, protrypsin inhibitor(pTI), and trypsin inhibitor (TI) inthe isolated vacuoles.

442 Plant Physiol. Vol. 85, 1987



443POSTTRANSLATIONAL PROCESSING ACTIVITY IN VACUOLES

psu. sueu.ummum urnYuuUU3P

_arn

a e _ -m -_ em emp 0sm .O cUSP -., -I-mn 4* 4.P j]

C

,C~~~>>0 LL < <<

LL (I)~~2 0 aiI- Q~~-c t9~~~- CU U00 coI o cc2t

o N *4 *eczQ.Q.|| w W W 01 c o0 E N

Q)J&Eo X X E EE E E E E BE E EC c ( EB0 0o z : Q . oo00 Z V- r-~0

.~~~~aa - - - S

U S SO pG

_ -. e._ _ _ _

m " -

-I

V U & JY

mOPeof~ ~_41o w

~~~~~~~~~~~~~~~~~++

. . N

C 0 > U U U W U L < C < i C

o 00, UU V Q z z z _ U5

0

pTI

_-- --TI

0 U

a)

-

-

e r~~~~

FIG. 4. Effects of various protease inhibitors or activators on proteolytic processing activity. ER and vacuoles were isolated from the developingcotyledons as described in Figure 3. The crude enzyme fraction (2 pl) was preincubated with 0.5 ul of various inhibitors or activators for 20 min at

room temperature and added to 2 gl of the ER fraction and 5.5 gl of 0.1 M citrate-phosphate buffer (pH 5.0), before incubation for 2 h at 30'C. Thefinal concentration of each inhibitor or activator used was indicated at the bottom of the figure. M, markers for proglobulin (pG) and the constituentpolypeptide chains (y and 6) of I IS globulin, protrypsin inhibitor (pTI) and trypsin inhibitor (TI) in isolated vacuoles.

the same manner as that shown by the matrix fraction of the matrix fraction of the vacuoles isolated from the developingvacuoles isolated from the developing pumpkin cotyledons. cotyledons (Fig. 6). Our previous studies have shown that the

trimeric proglobulin molecules synthesized in the ER are trans-DISCUSSION ported to the vacuoles via the dense vesicles (7). Thus, our overall

results indicate that after the membrane fusion between the denseThe present investigation has demonstrated that the endopro- vesicles and vacuoles, there occurs the transport of trimeric

teolytic processing enzyme engaged in the conversion of the proglobulin molecules into the vacuoles, followed by the cleavageproglobulin to globulin is specifically localized in the soluble catalyzed by the soluble matrix processing enzyme and the final

iAs .ww As 4NO4mw



HARA-NISHIMURA AND NISHIMURA

FIG. 5. Developmental changes in proteolytic processing activity. De-

velopmental stage of the cotyledons was divided into 4 stages, I to IV.

The cotyledon half in stage I was 2 mm length and mg weight; stage

II, 10.5 mm length and 34 mg; stage III, 17 mm length and 131 mg; and

stage IV, 17 mm length and 244 mg. The cotyledons at each stage and

dry seeds were homogenized in 20 mm citrate-phosphate buffer (pH 6.0)

(0.3 ml/half cotyledon). After centrifugation of the homogenates the

supernatant was used as the crude enzyme solution. The reaction mixture

was composed of 2 Mul of ER fraction, 4 Asl of 0.1Im citrate-phosphate

buffer (pH 5.0), and 4 AI of the crude enzyme solution. After incubation

for 0, 1, and 3 h, the reaction mixture was subjected to the SDS-PAGE

and fluorography. M, markers for proglobulin (pG) and the constituent

polypeptide chains (-y and 6) of IlIS globulin, protrypsin inhibitor (pTI)

and trypsin inhibitor (TI) in the isolated vacuoles.

assemblage producing the hexameric mature IlIS globulin mol-

ecules. The mature IS globulin molecules were found to form

the crystalloids which become larger in size during the develop-

ment of the cotyledons and finally budded out of the vacuoles

giving rise to protein bodies (6).

Using proglobulin processing enzyme, we investigated when

$o0 '

40°H

4

and where disulfide bond involved in the linkage of y and 6chains of mature globulin was introduced, resulting that proglob-ulin molecules in ER has already the disulfide bond (5).We found that the enzymic activities ofthe proteolytic cleavage

of proglobulin exhibit the slightly acidic pH optimum around at5.0, coinciding with the pH of the vacuolar sap. It has beenreported that the endoproteolytic enzyme involved in the cleav-age of the precursor molecules of ricin and R. communis agglu-tinin of castor bean endosperm had optimum pH 4.0 (9). How-ever, in our present study the processing enzyme of pumpkinshowed the lower activity at pH 4.0.

It has been reported that the proinsulin- and proglucagon-converting enzymes localized in the secretion vesicles of rat islets(2) or anglerfish islets of Langerhans (3) is a thiol protease andthat the posttranslational processing enzymes present in chloro-plasts (14) or mitochondria (10) are metalloproteases. The proc-essing enzyme associated with the vacuoles presently studied is athiol protease, clearly distinguishable from that in chloroplastsor mitochondria and rather similar to the proinsulin-convertingenzyme. The posttranslational processing in chloroplasts andmitochondria was coupled with the protein transport into theirorganelles, but the processing in vacuoles was not.Although the 11 S globulin molecules are synthesized most

efficiently during developmental stage II, the processing enzymeis shown to accumulate largely during stage III to IV. It isinteresting to note, therefore, that there exists a several-daysdelay between the start of the 11 S globulin biosynthesis and thatof the processing enzyme biosynthesis.Pumpkin trypsin inhibitor was localized in the matrix fraction

of the vacuoles or protein bodies and synthesized as largerprecursor (I Hara-Nishimura, unpublished data). Precursor pro-tein of the pumpkin trypsin inhibitor (protrypsin inhibitor) wasalso converted to the mature trypsin inhibitor by the vacuolarextracts (Fig. 3). The profiles of the effect of inhibitors on theenzyme activities and the developmental change in the activityagainst protrypsin inhibitor were similar to those obtained usingproglobulin as a substrate (Figs. 4 and 5). These results indicatethat the same processing enzyme is likely involved in the con-version of both proglobulin and protrypsin inhibitor. It was alsodemonstrated that the matrix of the protein bodies isolated fromthe dry castor bean is able to process the proglobulin moleculeto the mature globulin. These results suggest that endoproteolyticenzymes capable of processing a wide variety of different precur-

-6003

-i40_.J.

j70 <-n

L~~~~~~~~~~~~~~~~~~~~~~~~~~~~

2001

ok L -J---_ -j -! _ __ r r- 34 5 Eh '8 4

FIG. 6. Suborganellar distribution ofproteolytic proc-i08 essing enzyme in vacuoles. Vacuoles were isolated fromthe cotyledons at the late developmental stage as de-scribed in Figure 3. Isolated vacuoles (0.1 ml) were

- lysed by the addition of 0.2 ml of water and the wholellysate was layered on a sucrose density gradient con-sisting of 0.5 ml cushion of 68% (w/w) sucrose, 4 ml ofa linear sucrose gradient solution (30-68%), and 0.5 ml

X04 of 15% sucrose. The gradient was centrifuged at 29,000rpm for 5.5 h in a Beckman model L2-65B ultracentri-fuge using an SW 64Ti rotor at 4°C. After centrifuga-tion, fractions (0.3 ml) were collected and the proteo-

q} lytic processing activity was assayed by measuring theformation of 6 chain of the mature globulin moleculesfrom fluorogram (]). Sucrose density (... ) and proteinconcentration (-) were also measured.

I I

0 l' 12 13 14 1`:i, i*,j. E.

444 Plant Physiol. Vol. 85, 1987

1.1



POSTTRANSLATIONAL PROCESSING ACTIVITY IN VACUOLES

sor molecules are present in the vacuoles of plant seeds.From comparison of the amino acid sequences of proglobulin

deduced from the nucleotide sequence of pumpkin 11 S globulincDNA clone (M Hayashi, unpublished data), with those of theconstituent y and a chains of the mature 11S globulin (13), itwas found that the processing site in the precursor polypeptidechain is between asparagine and glycine residues. This site exactlycoincides with that of the precursor proteins of several seedproteins such as glycinin (12), concanavalin A (1), and ricin (1 1).However, it is clearly different from that for the proinsulin-converting enzyme which recognizes the site just after pairedbasic amino acids. Overall results indicate therefore that thesubstrate specificity of the processing enzymes in the vacuoles ofvarious plants is unique and the posttranslational processing ofprecursors of various kinds of proteins deposited in the proteinbodies (vacuoles) is possibly catalyzed by the same thiol endo-protease.

Acknowledgments-We deeply thank Prof. H. Beevers, University of Californiaat Santa Cruz, and Prof. T. Akazawa, Nagoya University at Nagoya in Japan, forcritically reading the manuscript and improving the text.

LITERATURE CITED

1. BOWLES DJ, SE MARCUS, DJC PAPPIN, JBC FINDLAY, E ELIOPOULOS, PRMAYCOX, J BURGESS 1986 Posttranslational processing of concanavalin Aprecursors in jackbean cotyledons. J Cell Biol 102: 1284-1297

2. DOCHERTY K, RJ CARROLL, DF STEINER 1982 Conversion of proinsulin toinsulin: Involvement of a 31,500 molecular weight thiol protease. Proc NatlAcad Sci USA 79: 4613-4617

3. FLETCHER DJ, JP QUIGLEY, GE BAUER, BD NOE 1981 Characterization ofproinsulin- and proglucagon-converting activities in isolated islet secretorygranules. J Cell Biol 90: 312-322

4. HARA I, H MATSUBARA 1980 Pumpkin (Cucurbita sp.) seed globulin VI.Proteolytic activities appearing in germinating cotyledons. Plant Cell Physiol21: 233-245

5. HARA-NIsHiMURA I 1987 Introduction of a disulfide bond in proglobulinmolecules during the I 1S globulin biosynthesis in endoplasmic reticulum ofdeveloping pumpkin cotyledons. Agric Biol Chem 51: 2007-2008

6. HARA-NISHIMURA I, M HAYASHI, M NISHIMURA, T AKAZAWA 1986 Biogenesisof protein bodies by budding from vacuoles in developing pumpkin cotyle-dons. Protoplasma 136: 46-55

7. HARA-NISHIMURA I, M NISHIMURA, T AKAZAWA 1985 Biosynthesis and intra-cellular transport of I IS globulin in developing pumpkin cotyledons. PlantPhysiol 77: 747-752

8. HARA-NISHIMURA I, M NISHIMURA, H MATSUBARA, T AKAZAWA 1982 Subor-ganellar localization of proteinase catalyzing the limited hydrolysis ofpump-kin globulin. Plant Physiol 70: 699-703

9. HARLEY SM, JM LORD 1985 In vitro endoproteolytic cleavage of castor beanlectin precursors. Plant Sci 41: 111-116

10. HAY R, P BOHNI, S GASSER 1984 How mitochondria import proteins. BiochimBiophys Acta 779: 65-87

1 1. LAMB FI, LM ROBERTS, JM LORD 1984 Nucleotide sequence of cloned cDNAcoding for preproricin. Eur J Biochem 148: 265-270

12. MOMMA T, T NEGORO, H HIRANO, A MATSUMOTO, K UDAKA, C FUKAZAWA1985 Glycinin A5A4A3 mRNA: cDNA cloning and nucleotide sequencing ofa splitting storage protein subunit of soybean. Eur J Biochem 149: 491-496

13. OHMIYA M, I HARA, H MATSUBARA 1980 Pumpkin (Cucurbita sp.) seedglobulin IV. Terminal sequences of the acidic and basic peptide chains andidentification of a pyrroglutamyl peptide chain. Plant Cell Physiol 21: 157-167

14. ROBINSON C, RJ ELLIS 1984 Transport of proteins into chloroplasts. Theprecursor of small subunit of ribulose bisphosphate carboxylase is processedto the mature size in two steps. Eur J Biochem 142: 343-346

15. STINISSEN HM, WJ PEUMANS, MJ CHRISPEELS 1985 Posttranslational process-ing of proteins in vacuoles and protein bodies is inhibited by monensin.Plant Physiol 77: 495-498

16. TULLY RE, H BEEVERS 1976 Protein bodies of castor bean endosperm. Isola-tion, fractionation, and the characterization of protein components. PlantPhysiol 58: 710-716

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