Subcellular Localization and Possible Functions of �-Glutamyltransferase

in the Radish (Raphanus sativus L.) Plant

Yoshihiro NAKANO,* Satoshi OKAWA, Rafael PRIETO,** and Jiro SEKIYAy;**

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received January 17, 2006; Accepted February 23, 2006; Online Publication, July 23, 2006

[doi:10.1271/bbb.60027]

Previously we reported the purification of soluble �-

glutamyltransferases (GGTs) from radish cotyledon.

Subcellular fractionation of radish cells revealed that

soluble GGT is a vacuolar enzyme. Acivicin, a GGT

inhibitor, mediated the in vivo catabolism inhibition of

the glutathione S-conjugate generated from endogenous

glutathione and exogenously supplied monochlorobi-

mane. Thus soluble GGT is possibly involved in the

catabolism of glutathione S-conjugates.

Key words: �-glutamyltransferase; glutathione; gluta-

thione S-conjugate; radish; vacuole

Xenobiotic detoxification consists of four steps:activation of the xenobiotic, GSH S-conjugate (GSX)formation, GSX sequestration to vacuole, and degrada-tion of GSX.1) In mammals, GSX is sequentiallydegraded by �-glutamyltransferase (GGT) and L-cyste-inylglycine dipeptidase (DPase) reactions.2) The transferof the �-glutamyl moiety of GSH is specificallycatalyzed by GGT.2) In higher plants, GGT is assumedto be involved in the maintenance of GSH homeostasis,and consequently in GSH utilization as a storage andtransport form of L-cysteine.3) In spite of its provenimportance, however, GSH/GSX catabolism in plantshas not yet been successfully characterized. The GSXtransport from cytosol to vacuole has been character-ized,4) but barley vacuolar carboxypeptidase (CPase) isthe only vacuolar enzyme known to be involved in GSXcatabolism in plants.5) On the other hand, even thoughthe presence of vacuolar GGT has been hypothesized,3,5)

sufficient evidence supporting GGT involvement invacuolar GSX catabolism is lacking at present. Variousplants contain GGT isoforms that can be classified intosoluble and bound GGTs.6–10) Plant-bound GGTs appearto be localized to either the cell wall or the plasma

membrane,6,9) but there is no evidence of the subcellularlocalization of soluble GGT.11–13) Here, we focused onthe subcellular localization of radish soluble GGT andits possible involvement in vacuolar GSX catabolism.The subcellular localization of soluble GGT in radish

was examined by protoplast fractionation. Protoplastswere prepared from 10 g of radish cotyledons, asdescribed previously.6) Bound GGT was effectivelyremoved by cell-wall degradation during the protoplastpreparation process. The protoplasts prepared weredivided into two equal parts, used for the preparationof miniplasts and vacuoplasts (experiment 1) and thepreparation of vacuoles (experiment 2) respectively.Experiment 1: Miniplasts and vacuoplasts were pre-

pared from radish protoplasts according to the methodof Sonobe,14) with some modifications. Miniplasts andvacuoplasts were recovered from the bottom of thecentrifuge tube and the surface of the medium respec-tively, as described previously,14) and purified asfollows. The miniplasts were resuspended in 10 volumesof 0.5 M sucrose, pH 5.8, and centrifuged at 200� g for10min. The resulting miniplast pellet was resuspendedin 0.5 M sorbitol, pH 5.8, and used for enzyme assay. Onthe other hand, 5 volumes of 0.4 M sucrose, pH 5.8, wereadded to the vacuoplast suspension, and the mixture wasoverlaid with 0.3 M sorbitol, pH 5.8. After centrifugationat 200� g for 10min, the vacuoplasts were recoveredfrom the interface of the two media, resuspended in0.4 M sucrose, pH 5.8, and used for enzyme assay.Experiment 2: Radish protoplasts were harvested

by centrifugation, and then the protoplast pellet wasresuspended in medium A (20mM Tris–HCl buffer, pH8.0, 5mM EGTA, and 0.3 M sucrose) and the centrifugetube was placed on ice for 15min. One volume of 0.7 M

sucrose was added to the suspension, and a gradient wasformed by overlaying medium B (20mM Tris–HCl

* Present address: Shikoku Research Center, National Agriculture Research Center for the Western Region, NARO, Zentsuji, Kagawa 765-8508,

Japan** Present address: Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Sogabe-cho, Kameoka, Kyoto 621-8555,

Japan

y To whom correspondence should be addressed. Fax: +81-771-29-3429; E-mail: sekiyaj@kyotogakuen.ac.jp

Abbreviations: CPase, carboxypeptidase; Cys-bimane, S-conjugate of L-cysteine with monochlorobimane; CysGly, L-cysteinylglycine; DPase,

L-cysteinylglycine dipeptidase; G6P, glucose-6-phosphate; �-GluCys, �-L-glutamyl-L-cysteine; GGT, �-glutamyltransferase; GS-bimane, S-conjugate

of glutathione with monochlorobimane; GSX, glutathione S-conjugate

Biosci. Biotechnol. Biochem., 70 (7), 1790–1793, 2006

Note

buffer, pH 7.5, 1mM EGTA, 0.2 M sucrose, and 0.2 M

sorbitol) and medium C (20mM Tris–HCl buffer, pH7.5, 1mM EGTA, and 0.3 M sorbitol). The mixture wascentrifuged at 5;000� g for 1 h at 15 �C, and vacuoleswere recovered from the interface of medium B andmedium C. The purified vacuole fraction was resus-pended in medium A and used for enzyme assay.

The formation of protoplasts, miniplasts, vacuoplasts,and vacuoles was confirmed by light microscopy. Thenan aliquot of each fraction was taken for chlorophyllquantification.15) After that, each subcellular fractionwas disrupted by adding of one-tenth volume of 500mM

Tris–HCl buffer, pH 8.0, followed by gentle sonicationon ice, and aliquots of each fraction were used fordetermination of GGT and organelle marker enzymeactivities. G6P dehydrogenase,16) catalase,17) cyto-chrome c oxidase,18) and �-mannosidase19) were meas-ured as organelle markers. GGT activity was measuredas described previously.6) DPase activity was deter-mined according to the production of L-cysteine in areaction mixture (100 ml) containing 40mM Tris–HClbuffer, pH 8.0, 1mM DTT, 5mM L-CysGly, and theenzyme extract. The reaction was terminated by adding25 ml of 20% trichloroacetic acid, and the amount ofL-cysteine was quantified by HPLC.20) Chlorophyllcontent and enzyme activities in each fraction wererepresented as the values originated from 5 g ofcotyledons in Experiment 1 and 2.

Acivicin, a strong GGT inhibitor,2,7) and GSH label-ing reagent monochlorobimane21,22) were used to studythe possible involvement of GGT in GSX catabolism.Radish cotyledons excised from seedlings were infil-trated at �60 kPa with 20mM MES-Tris buffer, pH 5.5,containing 3% sucrose, 50 mM monochlorobimane, and 0or 0.5mM acivicin in Petri dishes. After infiltration for2min, the excess buffer was discarded, and the Petridishes were wrapped with plastic film and incubated at25 �C for 3 and 6 h in the dark. Then the samples wereharvested, frozen in liquid nitrogen and stored at�80 �C. They were homogenized in liquid nitrogenwith mortar and pestle. Thiol compounds were extractedby adding 30 volumes (v/w) of 0.1N HCl to the powderand grinding the samples further. An aliquot of the

mixture was centrifuged at 10;000� g for 10min at 4 �Cand the supernatant was recovered. Total thiol com-pounds in the supernatant were measured by HPLC afterderivatization with monobromobimane.20) To measure invivo monochlorobimane-labeled thiol compounds, thesupernatant was analyzed without monobromobimanederivatization.Table 1 summarizes the distribution of organelle

markers and enzyme activities in protoplasts, miniplasts,vacuoplasts, and vacuoles. Independent experimentsproduced similar results. The recovery of miniplasts ascalculated from G6P dehydrogenase activity was 41%.On the other hand, vacuoplast and vacuole recoveries,which were calculated from �-mannosidase activity,were 18% and 15% respectively. That the miniplastscontained much lower �-mannosidase activity than theprotoplasts is an indication that the vacuoles wereeffectively removed from the protoplasts. Similarly,soluble GGT activity was also greatly reduced in theminiplasts. In contrast, high �-mannosidase and solubleGGT activities were retained in the vacuoplasts. Va-cuole preparations, which were less contaminated byother organelles than the vacuoplasts, had nearly thesame level of soluble GGT activity as the vacuoplasts.Besides the vacuoles, no other organelle markers wererecovered with soluble GGT at a similar ratio. Taking allthe data together, our results indicate that soluble GGTis localized mainly to the vacuoles.To our knowledge, this study is the first to detect

vacuolar soluble GGT activity in plants. Soluble GGThas been reported to be a cytoplasmic enzyme in tobaccocells.11) Similarly, Allium cepa and Allium sativumsoluble GGT activity has been found in the solublefractions of the plant extract,12,13) which suggestscytosolic localization of soluble GGT.23) But in thosereports, the soluble fraction of disrupted cells wasreferred to as the cytoplasmic fraction, without discrim-inating the cytoplasm from the aqueous phases of otherorganelles. In Allium, GGT is thought to be involved inthe biosynthesis of S-alk(en)yl cysteine from S-alk(en)ylGSH.23) We believe that our finding of vacuolar GGTactivity should contribute to the elucidation of thelocation of S-alk(en)yl cysteine biosynthesis in plants.

Table 1. Typical Distribution of Organelle Markers and Enzyme Activities in Protoplast, Miniplast, Vacuoplast, and Vacuole Fractions from Radish

Cotyledon

Marker ProtoplastExperiment 1 Experiment 2

Miniplast Vacuoplast Vacuole

Chlorophyll (mg) 94:4� 0:9 38:0� 0:2ð40Þ 3:1� 0:1ð3Þ 1:4� 0:3ð1ÞG6P dehydrogenase (pkat) 69:3� 5:3 28:5� 0:8ð41Þ 3:2� 0:5ð5Þ <0:1

Catalase (nkat) 1350� 12 486� 4ð36Þ 69� 3ð5Þ 20� 3ð1ÞCytochrome c oxidase (pkat) 156� 11 89� 3ð57Þ 8� 2ð5Þ nd

�-Mannosidase (pkat) 727� 11 12� 1ð2Þ 131� 2ð18Þ 109� 2ð15ÞGGT (pkat) 88:3� 3:1 1:1� 0:2ð1Þ 15:0� 1:2ð17Þ 14:5� 1:2ð16ÞDPase (nkat) 5:81� 0:42 0:64� 0:09ð11Þ 0:66� 0:08ð11Þ 0:59� 0:13ð10Þ

Chlorophyll content and enzyme activities in each fraction are represented as the values originating from 5 g of cotyledons. Recoveries in percentages from protoplasts

are indicated in parentheses. Data are means� SD (n ¼ 3). nd, not detected.

Radish �-Glutamyltransferase 1791

Although DPase activity did not show specificcolocalization with any organelle markers, a significantamount of DPase appeared to be linked to the vacuole(Table 1). The occurrence of soluble GGT and DPase inradish vacuole implies that the GSH/GSX catabolicpathway reported in mammals2) works in plant vacuolesas well.

It has been clearly demonstrated that the S-conjugateformation of GSH with monochlorobimane (GS-bi-mane) is catalyzed by GSH S-transferase and thatthe GS-bimane formed is then transported to thevacuole.21,22) GS-bimane started to accumulate aftermonochlorobimane administration to the radish cotyle-dons (Fig. 1A). The acivicin-treated samples had high

GS-bimane contents as compared with the controlsamples. Total GSH content was also higher in theacivicin-treated samples than in the control samples(Fig. 1C). In vivo labeled cysteine (Cys-bimane) accu-mulated in control cotyledons, whereas only traceamounts of Cys-bimane were observed in the acivicin-treated samples (Fig. 1B). These results indicate that thein vivo labeled Cys-bimane was derived from GS-bimane by the successive reactions catalyzed by GGTand DPase. On the other hand, in vivo labeled �-L-glutamyl-L-cysteine (�-GluCys) and L-cysteinylglycine(CysGly) were below the detection limit in all theanalyzed radish samples. In arabidopsis, it has beenreported that in vivo monochlorobimane-labeled cys-teine, �-GluCys, and CysGly were produced from thedegradation of GS-bimane in the vacuole.22) SolubleGGTs (GGT I and II) purified from radish cotyledonsexhibited about 50% of their maximal activity even atpH 6.0, although their optimal pH was 7.5–8.0.7) Inaddition, soluble GGTs were inhibited more severelythan bound GGTs by acivicin. Hence a possible in vivofunction of radish soluble GGT might be to catalyze thefirst step of GSX catabolism in vacuoles.Taking into account that a GSX specific vacuolar

CPase has been described in barley,5) soluble GGTmight be the second type of enzyme involved in GSXcatabolism in plants. Considering that GGT activity6)

and GSH-specific CPase activity (Okumura and Otoshi,personal communication) vary considerably amongplant species, the predominance of either the GGT-DPase or the CPase-GGT (or �-glutamylcyclotransfer-ase) pathway might differ depending on the plantspecies.

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In v

ivo

lab

eled

GS

H(n

mo

l/g f

resh

wt)

In v

ivo

lab

eled

cys

tein

e(n

mo

l/g f

resh

wt)

0

10

20

B

50

100

150

0

A

0

100

200

300

400

To

tal G

SH

(n

mo

l/g f

resh

wt)

0 6

Incubation time (h)

C

nd nd

nd nd

3

Fig. 1. Contents of in Vivo Labeled GSH (A), in Vivo Labeled

Cysteine (B), and Total GSH (C) in Radish Cotyledons.

The samples were incubated in a medium containing 50mMmonochlorobimane with (white) or without (black) 0.5mM acivicin

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from the samples and determined without further derivatization, as

described in the text. Total GSH means the sum of GSH labeled and

GSH not labeled with monochlorobimane in vivo. Unlabeled GSH

was further derivatized with monobromobimane after extraction.

Data are means� SD (n ¼ 3). nd, not detected.

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Radish �-Glutamyltransferase 1793