Examination of the Contribution of Vacuolar ...

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tease inhibitor, and pepstatin A, an aspartic protease inhibitor, were introduced into the vacuole. With this operation, bovine serum albumin-degrading activity in ...

Plant Physiol. (1 995) 109: i309-1 31 5

Examination of the Contribution of Vacuolar Proteases to lntracellular Protein Degradation in Chara c o r d i n a Yuji Moriyasu*

Department of Biology, Faculty of lnternational Relations, University of Shizuoka, 52-1 Yada, Shizuoka-shi, 422 Japan involvement of the mature vacuole in cellular protein degradation has been done by Canut et al. (1986). Using cultured cells of Acer pseudoplatanus, they performed feeding experiments with [I4C]Phe and 13H]Leu and showed that, when vacuolar proteases are inhibited by increasing the vacuolar pH with benzylamine, more labeled proteins accumulate in the cells. From these results, they concluded that the vacuole is involved in the degradation of intracellular proteins. My associate and I have been investigating the mechanism of intracellular protein degradation using an alga, Chara corallina, as a model system of plant cells. The large size of its internodal cells allows us to perform various surgical operations to control the chemical composition of the vacuole and the cytoplasm (Tazawa et al., 1987). In a previous study, we reported that the central vacuole of Chara internodal cells contains an amount of proteases comparable to those of higher plants and fungi (Moriyasu and Tazawa, 1986). We further showed that these proteases are active in digesting exogenous proteins such as BSA and casein when these proteins are introduced into the vacuole (Moriyasu and Tazawa, 1988). In the study described here, I examined the contribution of proteases in the central vacuole of Chara internodal cells to overall cellular protein degradation using a combination of pulse-chase experiments with [3H]Leu and protease inhibitors.

The contribution of proteases in the central vacuole of Chara coralha internodal cells to overall cellular protein degradation was examined. I measured the decrease in the trichloroacetic acid (TCA)-precipitable radioactivity in the cell for a 6-d chase period after labeling cellular proteins with [3H]leucine. The kinetics of [3H]leucine-labeled protein disappearance showed that the half-life of the cellular soluble proteins was 4 t o 5 d. This value did not change when cells were treated with (2S,3S)-trans-epoxysuccinyl~-leucylamido-3-methyl-butaneethyl ester, a permeant inhibitor of cysteine proteases. This inhibitor mostly inhibited bovine serum albumin-degrading activity in the vacuole. I also measured the release of TCA-soluble radioactivity from the TCA-insoluble fraction i n the cell. This experiment showed that 13% of [3Hlleucinelabeled cellular proteins were degraded in 1 d. This value agreed well with the half-life obtained for soluble proteins i n the above experiment. This value did not change even when both transepoxysuccinyl-~-leucylamido-(4-guanidino)butane,a cysteine protease inhibitor, and pepstatin A, an aspartic protease inhibitor, were introduced into the vacuole. With this operation, bovine serum albumin-degrading activity in the vacuole was almost completely inhibited. These data suggest that the cytoplasmic but not the vacuolar proteases contribute t o cellular protein turnover in Chara internodal cells.

Plant vacuoles have various kinds of hydrolytic enzymes including acid phosphatase, a-mannosidase, proteinase, carboxypeptidase, and RNase (Ryan and Walker-Simmons, 1983; Boller and Wiemken, 1986). In addition, mature plant vacuoles have sometimes, especially in the course of senescence, been observed to include cytoplasm containing organelles such as chloroplasts and mitochondria (Matile, 1975). From these two lines of evidence, Matile (1975) proposed that mature plant vacuoles are a kind of lysosome. However, direct evidence that mature plant vacuoles contribute significantly to intracellular protein degradation in a manner similar to mammalian lysosomes is still lacking. In mammalian cells, the contribution of lysosomes to intracellular protein degradation has been estimated by various investigators using various cells to be as high as 100% to as low as 10% (Dice, 1987). In yeast cells, the contribution of the vacuole (lysosome) to overall protein turnover was estimated to be about 40 and 86% under normal and nutrient-starved conditions, respectively (Teichert et al., 1989). In plant cells, examination of the * E-mail 264-5099.

[email protected] .u-shizuoka-ken.ac.jp; fax

MATERIALS A N D METHODS Plant Material

Chara corallina was used. The alga was grown in a plastic bucket (90 L in volume) at 25 5 1°C in 15-h/9-h light/dark cycles. Two fluorescent lamps (FL20SS; Toshiba, Tokyo, Japan) were set at the top of the bucket for illumination. Mature internodal cells were cut from adjacent internodal cells with scissors and kept in APW (0.1 mM KCI, 0.1 mM NaCI, 0.1 mM CaCl,, and 5 mM Hepes-Na [pH 7.51) at 25 +- 1°C under a dim light for more than 1 d before the experiment. Abbreviations: APW, artificial pond water; AVS, artificial vacuolar

sap; E-64, truns-epoxysuccinyl-~-leucylamido-(4-guanidino)butane; E-64-c, (2S,3S~-tvuns-epoxysuccinyl-~-leucylamido-3-methyl-butane;

81-54-

E-64-d, (2S,3S)-t~u~s-epoxysuccinyl-~-leucylamido-3-methyl-butane ethyl ster; ETC-casein, fluorescein thiocarbamoyl-casein.

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Plant Physiol. Vol. 109, 1995

Moriyasu

Chemicals

~-[4,5-~H]Leu (2.63 TBq/mmol, 37 MBq/mL) was purchased from ICN. E-64, pepstatin A, and leupeptin were from Peptide Institute, Inc. (Minoh-shi, Osaka, Japan). BSA (fraction V) was from Nacalai Tesque, Inc. (Kyoto, Japan). L-Leu was from Wako Chemical Industries, Ltd. (Osaka, Japan). Casein (according to Hammarsten) was from Merk (Darmstadt, Germany). Liquid paraffin was from Kanto Chemical Co., Inc. (Tokyo, Japan). Fluorescein isothiocyanate (isomer I) and PMSF were from Sigma. E-64-c and E-64-d were generous gifts from M. Tamai (Taisho Pharmaceutical Co., Ltd., Oomiya, Japan). FTC-casein was prepared according to the method of Twining (1984). lntroduction of Protein and/or Protease lnhibitors into the Vacuole

To introduce an exogenous protein, BSA, and/or a protease inhibitor(s), I perfused the vacuole with 10 pL of AVS (80 mM KC1,30 mM NaC1,lO mM CaCl,, and 10 mM MgCl,) containing these chemicals (Moriyasu and Tazawa, 1988). The composition of AVS was designed to mimic the concentrations of K+, Na+, Ca2+, Mg2+, and C1- and the osmotic value of the natural vacuolar sap isolated from the internodal cells (Moriyasu et al., 1984). As a result of the perfusion, the original vacuolar sap is diluted with AVS by 20 to 25%, since the volume of the vacuole in an internodal cell is 40 to 50 pL. Electrophoresis and Sample Preparation for Electrophoresis

Each internodal cell was homogenized with 333 p L of the homogenization buffer (0.1 M Hepes-Na [pH 7.51, 100 p~ leupeptin, 1 mM PMSF, and 1 mM EDTA) using a mortar and pestle. The homogenate was centrifuged at 15,OOOg for 10 min. The resulting supernatant was mixed with one-fifth volume of the SDS-PAGE sample-preparing solution, consisting of 6% (w/v) SDS, 0.24 M DTT, 0.012% (w/v) bromphenol blue, and 50% (w/v) SUC.The mixture was boiled at 100°C for 90 s, cooled, and stored at -20°C until electrophoresis. SDS-PAGE was done with Laemmli's discontinuous buffer system (Laemmli, 1970) on 10% gels, which were purchased as ready-to-use gel sandwiches (Multi Gel 10; Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan) or prepared by me. Separation of proteins on gels from these two sources was essentially the same. After electrophoresis, gels were stained with silver. Phosphorylase b (97 kD), BSA (66 kD), egg albumin (45 kD), and carbonic anhydrase (29 kD) were used as molecular mass markers. Measurement of the Decrease in TCA-Precipitable Radioactivity in the Cells

Internodal cells (about 30 cells) were incubated in 6 mL of APW containing 60 pCi of I3H1Leu for 1 d in darkness at 25 ? 1°C. Then cells were washed in 100 mL of APW containing 1 mM Leu for 5 min. After three internodal cells were taken for the samples at time = O, one-half of the remaining cells was transferred into 5 mL of APW contain-

ing 1 mM Leu, 100 p~ E-64-d, and 1%(v/v) methano'l as a carrier, and the other half was transferred into 5 rnL of APW containing 1 mM Leu and 1%(v/v) methanol. The cells were incubated in these solutions in darkness with gentle shaking at 25 2 1°C. After various intervals. cells were taken for the measurement of their radioactivif ies. Each internodal cell was homogenized with 500 pL of the homogenization buffer using a mortar and pestle. The homogenate was centrifuged at 15,0008 for 10 min. Tlie supernatant (50 pL) was spotted onto a glass filter (glass microfiber filter GF/A 2.1 cm, Whatman). The filter was dried and transferred into a scintillation glass vial. It was washed twice with 5 mL of 10% (w/v) TCA for 10 min each and twice with 5 mL of ethanol for 10 min each. Afier the filter was dried again, 5 mL of scintillator (Econofluor, NEN) were added, and the radioactivity of the filter was measured using a liquid scintillation counter (LSC-3100; Aloka, Mitaka, Japan).

Measurement of the Release of TCA-Soluble Radioactivity

This measurement allows us to assay the degradation of a11 proteins, including both soluble and insoluble proteins. Internodal cells (about 16 cells) were incubated in 5 mL of APW containing 50 pCi of [3H]Leu for 1 d in darkness at 25 2 1°C. They were then washed in 100 mL of APW for 5 min and kept in fresh APW until the AVS (10 pL) with and without protease inhibitors was introduced. Subsequently, each cell was incubated in 2.5 mL of APW containing 1 mM Leu in darkness at 25 ? 1°C. Immediately (time = O ) and 1 d after the start of the chase period, the cell was hcimogenized with 500 FL of the homogenization buffer. The homogenate (50 pL) was spotted onto a glass filter. The filter was washed with TCA and ethanol, and its radioactivity, which is regarded as the radioactivity in the total cellular proteins ( P ) was , measured. The remaining homogenate was centrifuged at 15,00Og, and the supernatant (50 pL) was spotted onto each of two glass filters. The radioactivity of one filter was measured directly. The radioactivity of the other filter was measured after it was washed with TCA and ethanol. The difference between these two radioactivities was regarded as TCA-soluble radioactivity in the cell (Siri). The external solution (50 pL) was also spotted onto a filter for the measurement of TCA-soluble radioactivity in it (Sou'). Protein degradation for 1 d (PD) can be defined as in Equation la: (Sy

PD

e

=

- ST') + (S;"

ro'

- SF)

(1a)

where is TCA-insoluble radioactivity in the cell at O d, Sgut is TCA-soluble radioactivity in the external solution at O d, S:ut is TCA-soluble radioactivity in the extern,al solution at 1 d, is TCA-soluble radioactivity in the cell at O d, and Sy is TCA-soluble radioactivity in the cell at 1 d. Thus,

s.b"

lntracellular Protein Degradation in Chara corallina In this experiment, ?", Si", and Sout varied greatly between the cells, and as a result, the PD had large deviations. However, the ratios of various radioactivities in each individual cell were relatively constant. To use the ratios from each individual cell in our calculation, Equation l b was transformed to Equations 4, 5, and 6 as follows: Because the total radioactivity does not change in 1 d for the individual cell,

where is TCA-insoluble radioactivity in the cell at 1 d. Applying Equation 2 to the first term of Equation lb, Equation l b can be transformed to (3) Thus, the PD can be expressed as PD=-

and

A-B 1+A'

B=-.

(4)

srt + sb"

r,"

The individual values of A and B, which represent the ratios of the total TCA-soluble radioactivity to the TCAinsoluble radioactivity at 1 and O d, respectively, can be obtained from each individual cell. Thus, their deviations are minimized. The means (2) and SD values (uA) of A were calculated with the data obtained from five inhibitortreated and five control cells; the mean (E) and SD (gB)of B were from another five cells. Sgut was considered to be O. The means of the PD were calculated by applying à and to Equation 4. The SD of PD (upD)was estimated according to the following equation:

Measurement of Caseinolytic Activity in the Vacuolar Sap lsolated from lnternodal Cells

I

Vacuolar sap was collected from internodal cells as follows (Moriyasu et al., 1984).When the cell lost turgor in the air, both cell ends were cut and liquid paraffin was introduced into the vacuole from one open cell end. The vacuolar sap exuded from the other open end was collected in a glass capillary tube. The collected vacuolar sap was pooled and kept on ice until protease was assayed. The reaction mixture contained 40 p L of 0.1 M acetic-Na (pH 5.0), 10 p L of vacuolar sap, 10 p L of H,O, and 40 p L of a substrate solution (0.5% [w/vl FTC-casein). The reaction was started by the addition of the substrate and the mixture was incubated at 37°C. When the effects of protease inhibitors on the reaction were examined, 10 p L of H,O

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were replaced with the solutions containing inhibitors. When pepstatin A and PMSF were used, the final concentrations of DMSO and methanol were less than 1% (v/v). These solvents did not have any effect on the reaction. The reaction was stopped by the addition of 100 p L of 10% (w/v) TCA. After the mixture was kept on ice for about 1 h, it was centrifuged at 15,0008 for 10 min. The supernatant (150 pL) was mixed with 2 mL of 0.5 M Tris-C1 (pH 8.5) and its fluorescence at 525 nm was measured following excitation at 490 nm. Under this condition, the time-dependent increase in fluorescence was almost linear for at least 2 h.

Treatment of the Cells with Chloroquine, Protein Assay of Vacuolar Sap, and Measurement of Vacuolar pH

Internodal cells were kept in APW containing 0.2 mM chloroquine under a dim light at 25 -C 1°C. After various time intervals, vacuolar sap (10 pL) was isolated as described above and its protein was measured with the Pierce BCA Protein Assay Reagent according to the manufacturer's instructions. BSA was used as a standard protein. The pH of the isolated vacuolar sap was measured with a pH microelectrode (SE1700GC; Fuji Chemical Measurement, Mitaka, Japan). The effect of chloroquine on BSAdegrading activity in the vacuole was examined using the cells that had been kept in APW containing 0.1 or 0.2 miv chloroquine for 1 d. After BSA was introduced, the cells were kept in the same solutions for another 1 d until they were homogenized. RESULTS lnhibition of BSA-Degrading Activity in the Vacuole by a Permeant Cys Protease Inhibitor, E-64-d

After I introduced an exogenous protein, BSA, into the central vacuole of a Chava internodal cell, I analyzed the degradation of BSA with SDS-PAGE (Fig. 1). The BSA disappeared in 1 d (Fig. 1, Control versus t=O). This result is consistent with that reported in a previous paper (Moriyasu and Tazawa, 1988). We have already shown that BSA is degraded within the central vacuole, not outside the vacuole (Moriyasu and Tazawa, 1988). When I treated the cell with a permeant Cys protease inhibitor, E-64-d, 1 d before the introduction of BSA, degradation was mostly inhibited. However, there was still some degradation, since I observed two products of BSA degradation with molecular masses of approximately 50 kD (Fig. 1, E-64-d before loading). One of these bands has been shown to cross-react with an antibody against BSA (Moriyasu and Tazawa, 1988). E-64-d was less effective in inhibiting BSA degradation when the cells were treated with this inhibitor after the BSA introduction (Fig. 1,E-64-d after loading). Effect of E-64-d on the Half-Life of the Cellular Soluble Proteins

Based on the above result that E-64-d can effectively inhibit protease activity in the vacuole, I investigated

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Plant Physiol. Vol. 109, 1995

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Figure 1. The inhibition of protease activity in the vacuole of Chara internodal cells by E-64-d. Ten micrograms of BSA were introduced into the vacuole of a Chara internodal cell. Each cell was homogenized immediately after the introduction (t=0), or kept in APW containing 0.1 mM E-64-d (E-64-d after loading and E-64-d before loading) or 1% methanol (Control) for 1 d and then homogenized. The degradation of BSA (arrowhead) was analyzed by SDS-PACE. The cells, whose proteins were analyzed in the lanes labeled E-64-d before loading, were treated with 0.1 mM E-64-d 1 d before BSA was introduced. The proteins of the cell without the introduction of BSA were also analyzed (Cell). One-twentieth of the water-soluble proteins extracted from an internodal cell was loaded onto each lane. Different lanes represent different cells. Molecular masses (kD) of marker proteins are on the left.

whether or not the turnover of the native soluble proteins is affected by E-64-d. After labeling the overall cellular proteins with [3H]Leu, I measured the radioactivity in the cellular soluble protein fractions at various times during the chase period. The kinetics showed that the half-life of [3H]Leu-labeled soluble proteins was 4 to 5 d (Fig. 2). Although this measurement does not consider the reincorporation of [3H]Leu, this value is consistent with the halflives of Lemna soluble proteins obtained by various methods (Davies, 1982). The kinetics did not change significantly when the cells were kept in 0.1 mM E-64-d and Cys protease activity was inhibited (Fig. 2). I ascertained that BSA-degrading activity in the vacuole was still inhibited after 6 d. These results suggest that a vacuolar Cys protease(s) does not contribute to the turnover of soluble proteins in Chara internodal cells.

Figure 2. The effect of E-64-d on protein turnover in Chara internodal cells. After cellular proteins were labeled with [ 3 H|Leu for 1 d, the cells were kept in APW containing 1 mM Leu and 0.1 mM E-64-d (•) or containing 1 mM Leu and 1% methanol (O). At various intervals, each cell was homogenized and the radioactivity in the TCA-insoluble fraction was measured. The radioactivities per gram of cell fresh weight were standardized with the mean radioactivity at time = 0. Values are the means ± so of six internodal cells from two different experiments.

various protease inhibitors. A Cys protease inhibitor, E-64 (10 yM), and an Asp protease inhibitor, pepstatin A (10 JLIM), inhibited 35 and 86% of the total activity, respectively (Table I). In contrast to these two inhibitors, a Ser protease inhibitor, PMSF (2 mM), had no inhibitory effect (Table I). These results suggest that not only a Cys protease(s) but also an Asp protease(s) is localized in the vacuole and that the Asp protease(s) has a higher activity than the Cys protease(s) in the degradation of FTC-casein. Effects of E-64 and Pepstatin A on BSA-Degrading Activity in the Vacuole

Table I suggests that an Asp protease(s) also contributes to vacuolar protease activity. Thus, I tried to test the hypothesis that the degradation of BSA is also inhibited by pepstatin A. Since pepstatin A does not penetrate the cell, I introduced pepstatin A simultaneously with BSA. This

Table I. The effect of E-64 and pepstatin A on FTC-casein-degrading activity in the isolated vacuolar sap Vacuolar sap was collected from Chara internodal cells. The effects of various protease inhibitors on its protease activity were measured using FTC-casein as a substrate. The remaining activities (percentage) relative to the control are shown. Addition

Effects of Various Inhibitors on Caseinolytic Activity in the Isolated Vacuolar Sap

There remained a possibility that a protease(s) other than a Cys protease(s) in the vacuole contributes to protein turnover. Thus, I tried to characterize proteases in the isolated vacuolar sap using a substrate, FTC-casein, and

Concentration

Remaining Activity

M*

None E-64

10

100 Pepstatin A

1 10

PMSF

2000

100 65 66 13 14 111

Intracellular Protein Degradation in Chara corallina

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Table II. The contribution of vacuolar proteases to cellular protein degradation The release of TCA-soluble radioactivity from [3H] Leu-labeled Chara internodal cells was measured, and protein degradation (percentage) for 1 d was calculated. In the cells with inhibitors (-(-Inhibitors), vacuolar proteases were inhibited by the introduction of E-64 and pepstatin A into the vacuoles. The means ± so of five cells are shown. Cells

Protein Degradation /O

-(-Inhibitors Control

45*-f

Figure 3. The inhibition of protease activity in the vacuole of Chara internodal cells by E-64 and/or pepstatin A. Ten micrograms of BSA were introduced into the vacuole of a Chara internodal cell without protease inhibitors (t=0 and Control), with 0.1 nmol of E-64 (E-64, 10 fj,M), with 1 nmol of E-64 (E-64, 100 /AM), with 0.1 nmol of pepstatin A (Pepstatin, 10 JU.M), and with 1 nmol of E-64 and 0.1 nmol of pepstatin A (E-64 + Pepstatin). The degradation of BSA (arrowhead) was analyzed immediately (t=0) or 1 d after the introduction of BSA (the other lanes) by SDS-PAGE. One-twentieth of the water-soluble proteins extracted from an internodal cell was loaded onto each lane. Different lanes represent different cells. Molecular masses of marker proteins are on the left.

treatment with pepstatin A mostly inhibited the degradation of BSA (Fig. 3; Pepstatin, 10 JAM). Treatment with E-64 in the same way also inhibited the degradation (Fig. 3; E-64, 10 /LIM, and E-64, 100 /K.M). However, in both cases, I could detect significant amounts of degradation products of BSA with molecular masses of approximately 50 kD. When I introduced both E-64 and pepstatin A, the degradation of BSA was almost completely inhibited and I could not detect the degradation products (Fig. 3, E-64 + Pepstatin). Effect of E-64 and Pepstatin A on Intracellular Protein Degradation

Based on the above results showing that both BSA-degrading and caseinolytic activities can be inhibited almost completely by E-64 and pepstatin A, I further examined whether or not the inhibition of both activities affects the cellular protein turnover. I measured the release of TCAsoluble radioactivity from [3H]Leu-labeled cellular proteins and calculated the protein degradation according to Equations 4, 5, and 6 in "Materials and Methods." The protein degradation mainly reflected the release of TCAsoluble radioactivity to the external solution. In the cells, 13.3% of the total cellular proteins were degraded in 1 d (Table II, Control). This value means that

13.9±4.9 13.3±3.7

the half-life of the total cellular proteins is 4.9 d, assuming that the rate of protein degradation is constant. Therefore, the half-life for the total cellular proteins is not much different from that for the soluble proteins shown in Figure 2. The rate of protein degradation did not change significantly even when vacuolar proteases were inhibited by both E-64 and pepstatin A (Table II). This result suggests that vacuolar proteases do not contribute significantly to the cellular protein turnover in Chara internodal cells under the experimental conditions. Effects of Chloroquine on Vacuolar pH, Vacuolar BSADegrading Activity, and Vacuolar Protein Concentration

Lysosomotropic reagents can be expected to have an effect similar to vacuolar protease inhibitors because these reagents may inhibit vacuolar proteolysis by increasing vacuolar pH. I therefore examined the effect of a lysosomotropic reagent, chloroquine, on BSA-degrading activity in the vacuole of Chara internodal cells. With 0.2 mM chloroquine, vacuolar pH increased from 5.0 to 5.6 (Table III) and BSA-degrading activity in the vacuole was indeed inhibited (Fig. 4, 0.2 mM Chloroquine versus Control). With this treatment, however, protein concentration in the vacuole began to increase by about 60 h (Fig. 5). In contrast, I did not observe such an increase with E-64-d for 6 d (data not shown). DISCUSSION

I examined the contribution of vacuolar proteases to cellular protein turnover in Chara internodal cells by inhibiting vacuolar proteases with specific protease inhibitors. I first checked the effect of several protease inhibitors, given from outside the cell, on vacuolar proteases. Leupeptin, Table III. The effect of chloroquine on the vacuolar pH of Chara internodal cells Chara internodal cells were kept in APW containing 0.2 mM chloroquine or in APW (Control) for 1 d. Vacuolar sap was isolated from the cell, and its pH was measured with a glass microelectrode. Values are shown as the means ± so (the numbers of the cells used). Cells

Vacuolar pH

0.2 mM Chloroquine Control

5.59±0.03(4) 4.95±0.08(5)

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Figure 4. The inhibition of protease activity in the vacuole of Chara internodal cells by chloroquine. After Chara internodal cells were kept in APW containing 0.1 or 0.2 mM chloroquine or in APW (Control) for 1 d, 10 jig of BSA were introduced into the vacuole of each internodal cell. After the introduction, cells were returned to the original solutions. The degradation of BSA (arrowhead) was analyzed 1 d after the introduction by SDS-PAGE. One-twentieth of the watersoluble proteins extracted from an internodal cell was loaded onto each lane. Different lanes represent different cells. Molecular masses of marker proteins are on the left.

antipain, E-64-c, E-64, or pepstatin A had no effect; they may not penetrate the cell. I found that a Cys protease inhibitor, E-64-d, could reduce the vacuolar protease activity. It effectively inhibited the degradation of BSA introduced into the vacuole (Fig. 1), but it did not affect the half-life of [3H]Leu-labeled cellular soluble proteins (Fig. 2). The central vacuole of a Chara internodal cell contains both a Cys protease(s) and an Asp protease(s) (Table I). The introduction of both Cys and Asp protease inhibitors, E-64 and pepstatin A, almost completely inhibited the degradation of BSA in the vacuole (Fig. 3). This operation, however, did not have any effect on the degradation rate of [3H]Leulabeled cellular proteins (Table II). These results suggest that cytoplasmic but not vacuolar proteases contribute to protein turnover in Chnra internodal cells. In contrast, Canut et al. (1986) reported that the vacuoles in Acer cultured cells do degrade cellular proteins. There are some explanations for the difference between the experiments of Acer pseudoplatunus cultured cells and those of Chara corallina internodal cells. First, in the experiments in which Acer cells were used, the cells were dividing and growing and vacuole genesis was occurring in the cells, whereas the vacuole in the Chara internodal cells used in the present study were mature. Because autophagy is known to occur during the formation of plant vacuoles (Marty, 1978), the data from Canut et al. (1986) may include the contribution of vacuolar proteases to protein degradation during vacuole formation. Second, it is possible that the contribution of plant vacuoles to intracellular protein degradation was affected by some environmental factors, and these factors may explain the difference between the data. In mammalian and yeast cells, the contribution of the lysosomes to intracellular protein degradation increases

Plant Physiol. Vol. 109, 1995

following nutrient starvation (Schworer and Mortimore, 1979; Teichert et al., 1989; Takeshige et al., 1992). Indeed, Canut et al. (1986) further reported that the contribution of the vacuole to cellular proteolysis increased when they fed the cells with an amino acid analog. Third, there remains a possibility that lysosomotropic reagents such as benzylamine, which Canut et al. (1986) used to inhibit proteolysis in the vacuole, may have had some effects other than inhibiting vacuolar proteases. In this study, I confirmed that protease activity in the vacuole of Chara cells is indeed inhibited by chloroquine, a lysosomotropic reagent like benzylamine (Fig. 4). However, with chloroquine, protein concentration in the vacuole increased after 2 to 3 d (Fig. 5), whereas such accumulation of cellular proteins in the vacuole did not occur during 6 d of treatment with E-64-d. A Cys protease inhibitor, E-64, and an Asp protease inhibitor, pepstatin A, but not a Ser protease inhibitor, PMSF, inhibited FTC-casein-degrading activity in the isolated vacuolar sap. E-64 and pepstatin A also inhibited the degradation of BSA introduced into the vacuole. These results suggest that the vacuole of Chara cells has at least one Cys protease and one Asp protease. Mammalian lysosomes have two major Cys proteases, cathepsins B and L, and a major Asp protease, cathepsin D (Bohley and Seglen, 1992), whereas yeast vacuoles contain a Ser protease, proteinase B, an Asp protease, proteinase A, and no Cys proteases (Jones, 1991). Thus, the composition of proteases in the vacuole of Chara internodal cells is more akin to that in the lysosome of mammalian cells rather than to that in the

vacuole of yeast cells. There still remain two important questions. One question is what proteases in the vacuole of Chara cells do. It is difficult to suppose that vacuolar proteases are merely left over from the early developmental phase of vacuoles, in which autophagy is known to occur, because vacuolar proteins may turn over like other cellular proteins (Canut et al., 1985). There may be some situations in which vacuolar proteases work in the cells. As mentioned above, a lysosomal/vacuolar contribution to cellular proteolysis is

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80

Time (h) Figure 5. The effects of chloroquine on protein concentration in the vacuoles of Chara internodal cells. Chara internodal cells were kept in APW containing 0.2 mM chloroquine (•) or in APW (A). At various intervals, vacuolar sap was isolated and its protein content was measured. The protein concentrations (mg protein/mL vacuolar sap) from five internodal cells are shown as the means ± so.

lntracellular Protein Degradation in Cbara corallina

known t o be activated upon nutrient starvation in m a m malian and yeast cells (Schworer and Mortimore, 1979; Teichert e t al., 1989; Takeshige e t al., 1992). A similar situation c a n be expected i n plant cells and i n Chara cells a s well. Journet e t al. (1986) reported that n e t protein degradation occurs upon Suc starvation i n Acer cultured cells, which grow heterotrophically. Such proteolysis is likely t o occur i n vacuoles. Furthermore, a possibility that vacuolar proteases act as defensive enzymes against pathogens m u s t be considered (Boller, 1986). The other question is what kind of proteases contribute t o protein turnover in Chara cells. In this study, I showed that vacuolar proteases do not contribute significantly t o t h e cellular protein turnover a n d suggested that extravacuolar proteases a r e involved. I n mammalian and yeast cells, proteasomes are known t o w o r k in t h e degradation of several proteins w i t h short half-lives (Hershko and Ciechanover, 1992). In addition, calpains, cytosolic proteases found i n various animal cells (Pontremoli and Melloni, 19861, a r e thought t o contribute t o some specific proteolysis (Wang e t al., 1989). Also, in higher plant cells, a proteasome is shown t o be involved i n t h e degradation of proteins such a s phytochrome (Vierstra, 1993). Furthermore, chloroplasts a n d mitochondria a r e thought t o have their own proteolytic systems. In Chara internodal cells, we c a n easily w a s h vacuolar proteases, which m a y interfere t h e assay of extravacuolar proteases, o u t of the cell by vacuolar perfusion. Taking advantage of this material, we are searching for extravacuolar proteases. So far, we h a v e found a proteasome a n d a protease activated b y Ca2+ (Moriyasu and Tazawa, 1987). Such an approach m a y help us t o unders t a n d t h e mechanism of protein degradation in plant cells. ACKNOWLEDCMENTS

I thank Dr. Tetsuro Mimura (Hitotsubashi University) and Dr. Randy Wayne and Dr. Mark Staves (Cornell University) for critica1 reading of the manuscript and Dr. Masaharu Tamai (Taisho Pharmaceutical Co., Ltd.) for providing E-64-c and E-64-d. I also thank Prof. Yasuhiro Miyoshi for constant encouragement. Received July 10, 1995; accepted September 12, 1995. Copyright Clearance Center: 0032-0889/95/l09/1309/07. LITERATURE ClTED

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Boller T, Wiemken A (1986) Dynamics of vacuolar compartmentation. Annu Rev Plant Physiol 37: 137-164 Canut H, Alibert G, Boudet AM (1985) Hydrolysis of intracellular proteins in vacuoles isolated from Acer pseudoplatnnus L. cells. Plant Physiol 79: 1090-1093 Canut H, Alibert G, Carrasco A, Boudet AM (1986) Rapid degradation of abnormal proteins in vacuoles from Acer pseudoplatanus L. cells. Plant Physiol 81: 460-463 Davies DD (1982) Physiological aspects of protein turnover. In D Boulter, B Parthier, eds, Encyclopedia of Plant Physiology, Vol 14A. Springer-Verlag, Berlin, pp 189-228 Dice JF (1987) Molecular determinants of protein half-lives in eukaryotic cells. FASEB J 1:349-357 Hershko A, Ciechanover A (1992) The ubiquitin system for protein degradation. Annu Rev Biochem 61: 761-807 Jones EW (1991) Three proteolytic systems in the yeast Saccharomyces cerevisiae. J Biol Chem 266 7963-7966 Journet E, Bligny R, Douce R (1986) Biochemical changes during sucrose deprivation in higher plant cells. J Biol Chem 261: 3193-3199 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680-685 Marty F (1978) Cytochemical studies on GERL, provacuoles, and vacuoles in root meristematic cells of Euphorbia. Proc Natl Acad Sci USA 7 5 852-856 Matile P (1975) The Lytic Compartment of Plant Cells. SpringerVerlag, Vienna, Austria Moriyasu Y, Shimmen T, Tazawa M (1984) Vacuolar pH regulation in Chara australis. Cell Struct Funct 9: 225-234 Moriyasu Y, Tazawa M (1986) Distribution of severa1 proteases inside and outside the central vacuole of Chara austualis. Cell Struct Funct 11: 81-86 Moriyasu Y, Tazawa M (1987) Calcium-activated protease in the giant alga Chara australis. Protoplasma 140: 72-74 Moriyasu Y, Tazawa M (1988) Degradation of proteins artificially introduced into vacuoles of Chara australis. Plant Physiol 88: 1092-1096 Pontremoli S, Melloni E (1986) Extralysosomal protein degradation. Annu Rev Biochem 55: 455481 Ryan CA, Walker-Simmons M (1983) Plant vacuoles. Methods Enzymol96: 580-589 Schworer CM, Mortimore GE (1979) Glucagon-induced autophagy and proteolysis in rat liver: mediation by selective deprivation of intracellular amimo acids. Proc Natl Acad Sci USA 76: 3169-31 73 Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y (1992) Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol 119: 301-311 Tazawa M, Shimmen T, Mimura T (1987) Membrane control in the Characeae. Annu Rev Plant Physiol 38: 95-117 Teichert U, Mechler B, Miiller H, Wolf DH (1989) Lysosomal (vacuolar) proteinases of yeast are essential catalysts for protein degradation, defferentiation, and cell survival. J Biol Chem 264: 16037-16045 Twining SS (1984)Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Ana1 Biochem 143: 30-34 Vierstra RD (1993) Protein degradation in plants. Annu Rev Plant Physiol Plant Mo1 Biol 44: 385-410 Wang KKW, Villalobo A, Roufogalis BD (1989) Calmodulinbinding proteins as calpain substrates. Biochem J 262: 693-706

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