INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Living organisms are subjected to numerous biotic or abiotic stresses. Daily, at a cellular level, changing environmental growth conditions trigger the synthesis of new sets of proteins necessary for the acclimation response, and the degradation of regulatory proteins, damaged proteins, and proteins that have become useless. Thus, in plant cells subjected to carbon starvation, the activity of enzymes involved in sugar metabolism and respiration (Journet et al., 1986; Brouquisse et al., 1991; Irving and Hurst, 1993), nitrogen reduction and assimilation (Brouquisse et al., 1992; Peeters and Van Laere, 1992), regulation of cell division and growth (Chevalier et al., 1996), or protein synthesis (Webster and Henry, 1987; Tassi et al., 1992) decreases and, in most cases, the corresponding proteins are likely subjected to proteolysis. In contrast, the activity of enzymes related to the catabolism of proteins (Tassi et al., 1992; James et al., 1993, 1996; Chevalier et al., 1995; Moriyasu and Ohsumi, 1996), amino acids (Brouquisse et al., 1992), or lipids (Dieuaide et al., 1992; Ismail et al., 1997) increases. Genes encoding enzymes involved in protein and lipid catabolism have been shown to be induced by sugar depletion (Koch, 1996), and it is clear that the selective synthesis and degradation of individual proteins are important components of the coordinated response to sugar starvation (Koch, 1996).

In plant cells, protein breakdown is mediated by different proteolytic systems: (a) vacuolar proteolysis, (b) organellar proteolysis, and (c) selective nuclear and cytosolic proteasome-dependent proteolysis (for review, see Vierstra, 1996; Brouquisse et al., 2000). Nonselective vacuolar proteolysis has been reported to increase in plant tissues submitted to sugar starvation (Aubert et al., 1996; Moriyasu and Ohsumi, 1996), and proteolytic enzymes (endopeptidases and carboxypeptidases) are induced during carbon starvation (Tassi et al., 1992; James et al., 1993, 1996; Chevalier et al., 1995). Similarly, plastid aminopeptidases and endopeptidases are induced in sugar beet (Beta vulgaris) cotyledons during prolonged dark growth (El Amrani et al., 1998, and refs. therein). However, the concomitant synthesis and degradation of proteins present in the same compartment (Brouquisse et al., 1992), as well as the tight control of the cell division cycle (Chevalier et al., 1996; Genschik et al., 1998), suggest that, in plant cells, selective proteasome-dependent proteolysis should occur in the response to carbon starvation as already suggested in animal tissues and yeast (Saccharomyces cerevisiae). Thus, in rat (Rattus norvegicus) skeletal muscles submitted to starvation, increased proteolysis has been shown to be related to an increase in proteasome and ubiquitin mRNAs and ubiquitin-protein conjugates (Medina et al., 1995). In yeast, Hilt and Wolf (1992) reported that double mutants defective in two proteasome genes, PRE1 and PRE2, accumulate ubiquitinated proteins upon exposure to starvation, and that ubiquitin mutants are hypersensitive to starvation.

The structure and functions of 20S and 26S proteasomes have been extensively reviewed (Coux et al., 1996; Hershko and Ciechanover, 1998; Voges et al., 1999). Both 20S and 26S forms have been shown to coexist in eukaryotic cells (Yang et al., 1995). The 26S proteasome complex is involved in the rapid ATP-dependent degradation of many rate-limiting enzymes, transcription regulators, regulatory proteins, abnormal proteins, and more generally in the slower degradation of the bulk of proteins in animal cells (for review, see Coux et al., 1996; Voges et al., 1999). It was reported that, besides its function as the proteolytic core of the 26S complex, the 20S proteasome could be involved in the degradation of oxidatively modified proteins (Rivett, 1985; Giulivi et al., 1994; Grune et al., 1995). Oxygen radicals and other activated oxygen species generated as by-products of cellular metabolism or from environmental sources cause modifications to the amino acids of proteins (Dean et al., 1997). Oxidatively modified proteins can undergo chemical fragmentations or form aggregates because of covalent cross-linking reactions and increased surface hydrophobicity. The recognition of hydrophobic amino acid residues of oxidized proteins and their subsequent degradation by the 20S proteasome could be a selective mechanism to remove oxidatively damaged proteins from the cell (Grune et al., 1997). As a consequence of the potential role of the 20S proteasome in the degradation of oxidized proteins, the effects of oxidative treatments on 20S proteasome structure and activities have also been investigated in vitro (Strack et al., 1996; Conconi et al., 1998; Reinheckel et al., 1998). However, whereas the 20S proteasome from chicken (Gallus gallus) erythrocytes has been report to be activated by mild oxidative treatment with either hydrogen peroxide (H₂O₂) or FeSO₄-EDTA-ascorbate (Strack et al., 1996), no change in activity was observed with the 20S proteasome from human erythrocyte (Reinheckel et al., 1998). Moreover, whether the proteasome is modified in vivo by oxidative conditions is not known.

In the context of our protein degradation studies of carbon-starved plant cells, we investigated the fate of the 20S proteasome, and we focused our attention on the occurrence of secondary oxidative effects on its activities. We first tested the hypothesis that the proteasome could be involved in the response of plant cells to carbon starvation. To attain this goal, we purified and characterized the proteasome from maize (Zea mays L. cv DEA 1992) roots, and investigated the changes in 20S proteasome amounts and mRNA levels, in parallel with its proteolytic activities and kinetic properties, in carbon-starved and carbon-fed root tips. Second, we studied the occurrence of potential oxidative modifications of the 20S proteasome in starvation conditions. We report the first biochemical evidence for an in vivo oxidation of the 20S proteasome that activates its proteolytic activity in carbon-starved plant tissues.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Purification of 20S Proteasome from Whole Maize Root

The purification of 20S proteasome (multicatalytic proteinase) was performed through a five-step purification procedure (Table I). The application of the crude extract to Sepharose DEAE Fast Flow (step 2) resulted in the retention of the whole chymotrypsin-like activity and the removal of polyphenols and microsomal fractions that were not retained. The elution of the bound proteins with an NaCl gradient resulted in a single activity peak, with a recovery close to 100%. Active fractions were subjected to gel filtration on Sephacryl S-300 (step 3), which separated two activity peaks. The first peak, which contains the 20S proteasome, eluted in the range of the 600- to 1,000-kD proteins, immediately after the void volume. The second activity peak corresponds to proteins close to 400 to 500 kD, and represents 50% to 60% of the loaded activity that is not because of proteasomeas indicated by a check with anti-20S proteasome antibodies. It should be mentioned that when the 20S proteasome is purified from maize root tips, instead of whole roots, the second activity peak represents less than 20% of the loaded activity (data not shown). The removal of some chymotrypsin-like activity resulted in a low apparent purification factor (Table I), but this step allowed the separation of the proteasome from the bulk of lower molecular mass contaminating proteins. Gradient used for the Mono-Q step was optimized to obtain the proteasome in an individualized sharp peak (around 250 mM NaCl), therefore removing 80% of the unrelated proteins (Table I). The active fractions were brought to 800 mM (NH₄)₂SO₄ and applied to a Phenyl-Superose column. The proteasome did not bind to the column and this step allowed removal of minor contaminants. This last step provided a purified 20S proteasome, showing a single band of protein after native PAGE (Fig. 1A). Under denaturing conditions, 20S proteasome subunits were found to range between 20 and 35 kD (Fig. 1B), and at least 13 different subunits, with pI ranging between 5 and 7, were observed after two-dimensional electrophoresis (data not shown). Native molecular mass of the 20S proteasome was estimated to be 700 ± 30 kD after gel filtration on a Sephacryl S-300 HR column (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 1.   Native-PAGE (A) and SDS-PAGE (B) analysis of purified 20S proteasome preparation obtained from maize roots. The purified 20S proteasome was revealed by Coomassie Brillant Blue coloration after PAGE (6% [w/v] gel; lane 1) or SDS-PAGE (12.5% [w/v] gel; lane 4). Purified antibodies were used to immunodetect 20S proteasome in purified preparation (lanes 2 and 5) or in crude extracts of maize roots (lanes 3 and 6). Pure 20S proteasome (3 µg) was loaded in tracks 1, 2, 4, and 5 and 50 µg of total protein in tracks 3 and 6. The molecular masses of markers are indicated in kilodaltons.

Final purification factor and apparent yield of activity were 27% and 2.6%, respectively (Table I). However, these factors are underestimates because of the presence of the chymotrypsin-like activity removed at the gel filtration step. The five-step purification procedure typically yielded 2 mg of purified 20S proteasome from 60 g of maize roots (Table I). Rabbit polyclonal antibodies were raised against this 20S proteasome. Immunoprecipitation experiments showed that the activity of 20S proteasome was more than 95% inhibited by the addition of 15 µL of crude antiserum (data not shown). Typical western-blot patterns obtained in native and denaturing conditions are shown in Figure 1.

Biochemical Characterization

Substrate Specificity, Optimal pH and Temperature, and Autolysis Test

View larger version (26K): [in this window] [in a new window]   Figure 2.   Determination of 20S proteasome optimal pH (A) and temperature effects (B). Chymotrypsin-like, trypsin-like, PGPH, and caseinolytic activities of purified 20S proteasome (1 µg) were measured in 70 µL of pH 3 to 10 tri-buffer mixture (50 mM acetic acid, 50 mM MES, and 100 mM Tris) for the determination of the pH effects (A) or in Tris-HCl buffer, pH 7.5, and 20 mM NaCl at 25°C, 37°C, 55°C, and 75°C for the determination of the temperature effects (caseinolytic activities were not measured at 75°C because of casein precipitation; B). Activities measured at 37°C were normalized to 100%. Values are means of three independent experiments.

Effects of Inhibitors and Activators

Physiological Characterization

Changes in 20S Proteasome in Sugar-Starved and Non-Starved Excised Root Tips

View larger version (50K): [in this window] [in a new window]   Figure 3.   Total and 20S proteasome activities in starved and non-starved maize root tips. A, Chymotrypsin-like, trypsin-like, PGPH, and caseinolytic total activities were measured in desalted crude extracts of non starved (T0), 24-h Glc-starved (T24 starved), and 24-h Glc-fed (T24 + Glc) root tips. B, The 20S proteasome activities were determined after immunoprecipitation of desalted crude extracts with anti-20S proteasome antibodies. For each activity, the percentage of total activity because of the 20S proteasome is reported in C. n.d., Non-detected, because of the detection limit of the immunoprecipitation method. Values are means of six independent experiments.

1

View larger version (52K): [in this window] [in a new window]   Figure 4.   Western- and northern-blot analysis of the 20S proteasome in starved and non-starved maize root tips. A, Total protein content in T0, T24-starved, and T24 + Glc root tips. Values are means of three independent experiments. B, Protein extracts (1 root tip/track) were separated by PAGE, and the proteasome was immunodetected with anti-20S proteasome antibodies. Relative immunosignal intensities are indicated in brackets. C and E, Northern-blot analysis of two transcripts encoding for 6- and 3-subunits of the maize 20S proteasome (10 µg total RNA/track). D and F, rRNA were used as a loading control. Values are representative of four independent experiments.

View this table: [in this window] [in a new window]   Table IV.   Specific activities and kinetic parameters of 20S proteasomes purified from T0, T24-starved, and T24 + Glc maize root tips Kinetic parameters were determined with the Lineweaver-Burk plot and linear fitting of four plots obtained from four different measurements. Ranges of substrates concentrations are: 10 to 200 µM for Suc-L-L-V-Y-AMC, and 20 to 170 µM for Cbz-G-G-R-NA and Cbz-L-L-E-NA.

Characterization of Oxidized Proteasome in Sugar-Starved Root Tips

View larger version (54K): [in this window] [in a new window]   Figure 5.   Immunodetection of carbonyl residues in 20S proteasomes from starved and non-starved maize root tips. Purified 20S (10 µg) proteasomes from T0, T24-starved, and T24 + Glc-fed root tips were incubated with (A) or without (B) DNPH and separated by 12.5% (w/v) SDS-PAGE. Carbonyl residues were then immunodected with anti-DNP antibodies as described in "Materials and Methods." A, Relative immunosignal intensities are indicated. C, Coomassie Brillant Blue coloration of aliquot fractions of DNPH-treated proteasomes after SDS-PAGE (2 µg/track) was used as loading controls.

View larger version (79K): [in this window] [in a new window]   Figure 6.   Immunodetection of carbonyl residues in 20S proteasomes from starved and non-starved maize root tips after oxidative treatment. Purified 20S proteasomes (2 µg) from T0 and T24-starved root tips were submitted to various oxidative treatments, incubated with DNPH, and separated by 12.5% (w/v) SDS-PAGE. Carbonyl residues were then immunodected with anti-DNP antibodies as described in "Materials and Methods." Lanes 1 and 4, Nonoxidized 20S proteasomes from T0 and T24 starved root tips. Lane 2, 20S proteasome from T0 root tips treated for 2 h at 37°C with 50 mM ascorbate/200 µM FeSO4. Lanes 3 and 5, 20S proteasome from T0 and T24 starved root tips treated for 2 h at 37°C with 50 mM ascorbate/200 µM FeSO4/5 mM H2O2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Proteasome Purification and Characterization

The 20S proteasome from maize roots was purified through a five-step procedure (Table I) close to procedures classically used for animal (Rivett et al., 1994) or plant materials (Ozaki et al., 1992; Skoda and Malek, 1992; Fernandez Murray et al., 1997). After biochemical characterization, maize root proteasome was shown to be similar to most eucaryotic proteasomes with a molecular mass of 700 kD, and containing at least 13 subunits ranging between 20 and 35 kD. When assayed with fluorogenic peptide substrates, it exhibited the three main activities found in all 20S proteasomes: chymotrypsin like, trypsin like, and PGPH, and possessed an endopeptidase activity against casein (Table II). It exhibited optimal pH around 8 to 9, and was not subject to autolysis. Finally, it was sensitive to classical proteasome inhibitors such as PI1, MG132, or chymostatin, and activated by poly-Lys and SDS (Table III), as already reported for other proteasomes (Rivett et al., 1994).

Modifications of 20S Proteasome in Carbon-Starved Tissues

It has been already shown that, in maize roots, carbon starvation stops cell divisions and tissue growth (Chevalier et al., 1996; Brouquisse et al., 1998). In such a situation, proteins are remobilized to supply carbon skeletons to respiration and residual biosynthesis through nonselective autophagic processes involving vacuolar proteolysis (Aubert et al., 1996; James et al., 1996; Moriyasu and Ohsumi, 1996). However, during starvation like in other stress situations, the cessation of cell growth and the remobilization of proteins are accompanied by selective degradation and synthesis of specific proteins (Chevalier, et al., 1996; Brouquisse et al., 1998; and refs. therein). Such selective proteolysis suggests that the 20S proteasome, either as a component of the ubiquitin-dependent proteolysis or by itself, could be involved in the process of acclimation to starvation.

The involvement of proteasome in carbon starvation processes is suggested by the increase in 20S proteasome total activities and amounts in sugar-starved maize root tips (Figs. 3 and 4). These data are slightly different from previous observations showing that 20S proteasome amount levels off, instead of increasing, in roots from whole plants submitted to dark-induced starvation (Brouquisse et al., 1998). This difference may be attributed to different development stages of the roots and to milder starvation conditions in the whole plant. On the other hand, 20S proteasome activities and amounts have also been observed to increase in the nucleus of cancer cells submitted to Glc deprivation (Ogiso et al., 1999). However, contrary to a previous report showing that 20S proteasome-subunit mRNAs increased in starved rat skeletal muscles (Medina et al., 1995), 3- and 6-subunit mRNA levels clearly decreased in 24-h-starved maize root tips (Fig. 4). In Arabidopsis, six -subunits and three -subunits of the 20S proteasome are encoded by at least two genes (Fu et al., 1998). Among them, the 3-subunit is encoded by two genes, PAC1 and PAC2. Thus, in starved maize root tips, the decrease in PAC1-like mRNA level could be balanced by an increase in PAC2-like mRNA level to maintain or increase the amount of 20S proteasome. However, considering (a) the decrease in mRNA level of 6-subunit, which is encoded by only one gene in Arabidopsis (Fu et al., 1998), and (b) the fact that the presence of the 14 subunits is essential for the 20S proteasome structure (Coux et al., 1996; Hochstrasser, 1996), it appears unlikely that the 3-subunit can be expressed from a PAC2-like gene when the 6-subunit is not. Nevertheless, this point, as well as the change in mRNA level of the other 20S proteasome subunits during carbon starvation, will have to be checked when the sequences of the corresponding genes become available. To date, because in most animal, yeast, and plant materials studied so far the expression of the various proteasome subunits seems to occurs via a concerted mechanism (Genschik et al., 1994; Ichihara and Tanaka, 1995; Fu et al., 1998), it may be reasonably assumed that the expression of the other subunits of maize 20S proteasome were similarly decreased. This suggests that, in carbon-starved maize root tips, 20S proteasome expression was modified presumably through: (a) a decrease in proteasome gene transcription, and (b) an increase in proteasome stability and/or in mRNA translation efficiency. In addition to possible changes in translation efficiency, the modification of specific peptidic activities and kinetic parameters in T24-starved root tips (Table IV) suggest that 20S proteasome was subjected to posttranslational modifications during starvation treatment.

In eukaryotic cells, 20S proteasome has been reported to be submitted to various posttranslational modifications such as processing (Schmidtke et al., 1996; Groll et al., 1997), glycosylation (Schliephacke et al., 1991), phosphorylation (Umeda et al., 1997; Bose et al., 1999), or ADP-ribosylation (Ullrich et al., 1999). In the present work, we focused our attention on potential oxidative modifications of the proteasome. As indicated by the increase in carbonyl residues in T24-starved proteasome (Fig. 5), we report, to our knowledge, the first evidence for in vivo oxidation of the 20S proteasome. Sugar deprivation is known to induce metabolic oxidative stress via an increase in oxidized glutathione and intracellular prooxidant levels (Blackburn et al., 1999), and to enhance the sensitivity of the cells to oxidative stress (Zhang et al., 1996). Reactive oxygen species (peroxyl, alkoxyl, and hydroxyl radicals) react with proteins and generate oxidation products such as carbonyl compounds (Chao et al., 1997; Dean et al., 1997). Thus, obviously proteasome may be oxidized, like other intracellular proteins. Furthermore, the changes in specific activities of proteasome during starvation (Table IV) may be related to oxidative modifications because, after a mild oxidative treatment in vitro: (a) the specific activities of proteasomes purified from non-starved tissues (T0 and T24 + Glc) became similar to those of T24-starved proteasome: trypsin-like specific activities decreased, whereas chymotrypsin-like and PGPH ones increased more or less markedly (Table V); and (b) the level of carbonylation is similar between oxidized proteasome from T0 root tips and proteasome from T24 starved once (Fig. 6). Conflicting results have been reported about the chymotrypsin-like activity of 20S proteasome, which was found to be either activated (Strack et al., 1996) or not (Reinheckel et al., 1998) by in vitro oxidative treatment. It has been hypothesized that the activation by H₂O₂ found by Strack et al. (1996) was mediated via an interaction of the proteasome and the PA28 activator (Reinheckel et al., 1998). However, without excluding a possible interaction between the 20S proteasome and the PA28 activator in vivo, our data show that the purified proteasome may be activated by a mild oxidation treatment only, i.e. without H₂O₂ added (Table V). On the other hand, further oxidation of already oxidized proteasome (i.e. T24-starved proteasome) resulted in partial inactivation of the three peptidic activities (Table V), as observed for proteasomes treated with increasing H₂O₂ concentrations (Strack et al., 1996; Conconi et al., 1998; Reinheckel et al., 1998), and in a strong increase in carbonyl residues (Fig. 6). This confirms that 20S proteasome is activated by mild oxidative conditions, but inactivated by strong oxidative treatments. Thus, our data suggest that the in vivo oxidation of the 20S proteasome during starvation is mild enough not only to avoid inactivation of its activities, but to stimulate some of its peptidic and caseinolytic activities.

In mammalian cells, the 20S proteasome has been shown to recognize and selectively degrade oxidatively damaged proteins, such as hemoglobin (Fagan and Waxman, 1991; Giulivi et al., 1994), Gln synthetase (Rivett, 1985; Sahakian et al., 1995), superoxide dismutase (Grune et al., 1995), insulin-B chain (Dick et al., 1991), or Glc-6-phosphate dehydrogenase (Friguet et al., 1994), via ATP-independent degradation processes (for review, see Grune et al., 1997). Moreover, we observed a rise in oxidized protein amounts in Glc-starved maize root tips compared with non-starved ones, and we found that oxidized proteins were preferentially degraded by the proteasome in vitro and in vivo (G. Basset, P. Raymond, and R. Brouquisse, unpublished data). Taken together, the data of the literature and the present study suggest that, in vivo, sugar deprivation conditions induce a mild oxidative stress in the cells that leads to the oxidation of the proteins, including the 20S proteasome. Besides its role in the selective 26S proteasome-dependent proteolysis (Vierstra, 1996), oxidatively activated 20S proteasome could be involved in the degradation of nuclear and cytosolic oxidized proteins, thus contributing, on the one hand, to the detoxification of the cell through the elimination of oxidatively damaged proteins, and on the other hand, to the supply of amino acids for the synthesis of new proteins and as respiratory substrate for energy production.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Materials and Incubation Conditions

Maize (Zea mays L. cv DEA 1992) seeds (Pioneer France Maïs, Toulouse, France) were soaked for 3 h in water and germinated for 3 d on layers of wet filter paper; 3-mm-long primary root tips or 3- to 4-cm-long primary roots were then excised and either immediately used (T0), or incubated for 24 h in the absence (T24-starved) or the presence (T24 + Glc) of 0.2 M Glc. Incubation conditions were essentially as described (Brouquisse et al., 1991) but the incubation medium (medium A) was buffered with 10 mM instead of 100 mM MES (pH 6.0). Incubation medium was renewed every 8 to 12 h.

Preparation of Crude Extracts

Frozen root tissues were crushed at 4°C in a mortar in grinding medium (0.4 mL g fresh weight¹) containing 20 mM HEPES (pH 7.4), 5 mM -mercaptoethanol, and 0.1% to 0.5% (w/v) insoluble polyvinyl polypyrolidone. The brei was transferred into a 1.5-mL microcentrifuge tube and the mortar was rinsed with the same volume of grinding medium, which was then pooled with the brei. The homogenate was centrifuged at 36,000g for 15 min. The supernatant was used for protein and proteolytic activity measurements, and for immunodetection experiments, as described below.

Purification of the 20S Proteasome from Maize Whole Roots or Root Tips

All steps were performed at 4°C. 20S proteasome was followed through its "chymotrypsine-like" activity.

Step 1

Step 2

1

Step 3

1

Step 4

1

Step 5

1

Determination of Molecular Mass

The molecular mass of the purified 20S proteasome was estimated by gel filtration on Sephacryl S-300 HR column (80 × 2.6 cm), equilibrated with buffer A and calibrated with the following proteins as standards: thyroglobulin (669 kD), apoferritin (443 kD), and -amylase (200 kD). Blue Dextran (2,000 kD) was used as an exclusion volume marker.

Enzymic Activity Assays

Chymotrypsin-like, trypsin-like, and PGPH activities of the 20S proteasome were measured respectively with Suc-L-L-V-Y-AMC or Suc-A-A-F-AMC, Cbz-G-G-R-NA, and Cbz-L-L-E-NA. The assay mixture consisted of 10 µL synthetic substrate (stock solutions at 0.1-5 mM in dimethylformamide) and 100 µL proteasome extract plus buffer mixture (50 mM Tris, pH 8.1 at 37°C, and NaCl 20 mM). After a 15-min incubation, at 37°C, the reaction was stopped with the addition of 100 µL of 10% (w/v) SDS and 2 mL of Tris 100 mM, pH 9. The AMC or NA radicals released were measured fluorometrically (excitation 380 nm/emission 460 nm and excitation 335 nm/emission 410 nm, respectively). Activities were calculated using AMC and NA standard curves made in the same conditions. Unless mentioned in the text, Suc-L-L-V-Y-AMC, Suc-A-A-F-AMC, Cbz-G-G-R-NA, and Cbz-L-L-E-NA substrates were routinely used at 100, 100, 170, and 170 µM final concentrations, respectively, in the assays, and dimethylformamide final concentration did not exceed 120 mM (10% [v/v]). 20S proteasome activities against whole protein were measured with iodinated casein. Casein was radiolabeled with ¹²⁵I by the chloramine-T method (Ciechanover et al., 1980). The initial specific radioactivity of [¹²⁵I]-casein was approximately 2 × 10⁴ cpm.µg¹, and the protein concentration was 5 µg µL¹. Twenty microliters of purified proteasome (5 µg of protein), 75 µL of buffer A, and 5 µL of [¹²⁵I]-casein (100,000 cpm µL¹) were incubated for 20 to 30 min at 37°C. The reaction was stopped with 100 µL of 30% (w/v) trichloroacetic acid and the samples were centrifuged for 10 min at 30,000g; 100 µL of supernatant was used for radioactivity counting with a liquid scintillation analyzer (Tri-Carb 2000CA, Packard, Meriden, CT). Linearity of casein degrading activity was checked. Aminopeptidase activities were measured as described by (Sarath et al., 1989) using the -naphtylamine conjugates of L-Ala, L-Leu, L-Arg, L-Phe, or L-Pro as substrates.

Determination of the Optimum pH

The pH dependence of the proteolytic activity against iodinated casein and synthetic substrates was determined using a three-component buffer mixture (50 mM acetic acid, 50 mM MES, and 100 mM Tris).

Effect of Inhibitors and Activators of the 20S Proteasome

Proteasome inhibitors/activators were prepared as the following stock solutions: E-64 (10 mM), iodoacetamide (0.1 M), ATP-Mg²⁺ (0.1 M), Na₂-EDTA (0.5 M), 10% (w/v) SDS, and 10% (w/v) poly-Lys were in water; hemin (50 mM) was in 0.1 mM NaOH; PMSF (0.2 M) was in ethanol; N-acetyl-leucyl-leucyl-norleucinal (MG-132; 5 mM), Z-Ile-Glu(OtBu)- Ala-Leucinal (PI1; 2 mM), and chymostatin (10 mM) were in dimethylformamide. Inhibitors were first preincubated for 15 min with proteasome prior substrate addition, and activities were measured as described above. Control assays were carried out with the corresponding solvent.

MCO of Purified Proteasome

Oxidative treatment of the proteasome in vitro was adapted from Conconi et al. (1998). Ten micrograms of purified 20S proteasome (60-100 µL) was incubated for 2 to 3 h at 37°C with an equal volume of 20 mM sodium phosphate buffer, pH 7.0, with or without (control) 50 mM ascorbate/200 µM FeSO₄. Except when mentioned, no H₂O₂ was added. After treatment, the peptidic activities of oxidized and nonoxidized proteasomes were measured as described above.

Electrophoresis

Native PAGE and SDS/PAGE were performed with 6% (w/v) and 12.5% (w/v) polyacrylamide gels, respectively, by the procedure of (Laemmli, 1970). Gels were fixed, stained with Coomassie Blue or silver stain, and destained using standard methods. Two-dimensional electrophoreses (isoelectric focusing and SDS/PAGE) were as described by O'Farrel (1975).

Preparation of Antibodies and Western-Blot Analysis

Polyclonal antibodies against maize 20S proteasome were produced by subcutaneous injection of the purified enzyme into a Fauve de Bourgogne rabbit as in James et al. (1996). Serum IgG fraction was purified using 1 mL of HiTrap Protein A column (Pharmacia) as recommended by the manufacturer. Western-blot experiments were as described by James et al. (1996).

Immunoprecipitation of the Proteasome

For immunoprecipitation experiments, 500 µL of crude extracts was desalted through an Econo-pac 10DG (Bio-Rad Laboratories, Hercules, CA) column equilibrated with buffer A. Fractions of 160 µL of desalted crude extract or purified proteasome were incubated for 1 h at 4°C with increasing volumes of purified immune or pre-immune serum. Immune complexes were incubated for 1 h at 4°C with a 2-fold (IgG binding) excess of protein A-agarose (Affi-gel, Bio-Rad) and then centrifuged for 5 min at 10,000g. The various proteasome activities were measured in each supernatant fraction as described above.

Immunochemical Detection of Proteasome Carbonyls

Carbonyl content of purified proteasome was measured by reaction with DNPH. Two to 10 µg of purified proteasome were reacted with 200 µL of 10 mM DNPH (in 2 M HCl) or 2 M HCl (control) for 1 h at 25°C. The proteins were precipitated with 20% (w/v) trichloroacetic acid, centrifuged, and washed three times with ethanol:ethyl acetate (1:1 [v/v]). The final protein pellets were dissolved in 2× Laemmli buffer at 100°C for 15 min. The DNPH-derivatized enzymes were separated by 12.5% (w/v) SDS-PAGE. After electrotransfer of the proteins to nitrocellulose membranes, DNPH moieties were detected with rabbit anti-DNP primary antibodies (dilution 1:5,000 [v/v], Sigma, St. Louis) and goat anti-rabbit-IgG-alkaline phosphatase conjugate (dilution 1:30,000 [v/v], Sigma). Immunosignal was quantified by scanning the blots with an Image Master VDS and using the Image Master 1D software (Pharmacia Biotech).

RNA Extraction and Northern-Blot Analysis

Total RNA from maize root tips was extracted using the hot phenol method as described (Chevalier et al., 1995). For northern-blot experiments, total RNA was size fractionated by 6.6% (v/v) formaldehyde and 1.2% (w/v) agarose gel electrophoresis, transferred to Hybond N⁺ (Amersham, Buckinghamshire, UK) membranes by capillarity, and hybridized to random-primed labeled probes. Clones 5c02a05 and 5c01h12 cDNA encoded for partial sequence of 3- and 6-subunits of maize 20S proteasome, respectively (Shen et al., 1994). These are similar to PAC1 and PBF1 Arabidopsis components (Fu et al., 1998), and were kindly provided by the Maize RFLP Laboratory (Columbia, MO). Hybridizations were performed at 65°C as described in (Sambrook et al., 1989).

Protein Determination

Proteins were quantified according to the method of Bradford (1976). Bovine -globulin was used as the protein standard.