A Kunitz-type cysteine protease inhibitor from cauliflower and Arabidopsis

Plant Science 170 (2006) 1102–1110 www.elsevier.com/locate/plantsci

A Kunitz-type cysteine protease inhibitor from cauliflower and Arabidopsis Coralie E. Halls a,1, Sally W. Rogers a,1, Mohammed Oufattole a, Ole Østergard b,2, Birte Svensson b,2, John C. Rogers a,* a

Institute of Biological Chemistry, Washington State University, Pullman, WA 99064-6340, USA b Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Received 20 January 2006; accepted 20 January 2006 Available online 28 February 2006

Abstract A Kunitz-type protease inhibitor co-purified from cauliflower florets with a granulin domain cysteine protease that cleaved barley proaleurain to yield a molecular form the same size as that for mature aleurain. The purified cauliflower protease required treatment with SDS detergent to become active. This observation raised the question of whether the protease inhibitor might have the ability to interact with the granulin domain protease. Here we express an Arabidopsis homolog of the protease inhibitor as a recombinant protein and demonstrate that it is a potent inhibitor of the recombinant proaleurain maturation protease and of papain when assayed at pH 4.5 but not at pH 6.3. In a pull-down assay, the inhibitor bound tightly to papain, but only weakly to the aspartate protease pepsin. When the cauliflower protease inhibitor was transiently expressed in tobacco suspension culture protoplasts, it colocalized with BP-80, a vacuolar sorting receptor that interacts with proaleurain and traffics to prevacuolar compartments for lytic vacuoles. Our results indicate that the cauliflower and Arabidopsis protease inhibitors would traffic through cellular compartments where proaleurain also traffics. Their ability to inhibit a cysteine protease implicated in maturation of proaleurain to active form at the acidic pH found in vacuoles raises the possibility that they could participate in regulating activation of aleurain. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Granulin domain; Vacuole; Prevacuolar compartment; BP80; Proaleurain

1. Introduction Kunitz-type protease inhibitors, comprising the soybean trypsin inhibitor family [1–3], are abundant plant proteins found in legume and cereal seeds and in tubers. They are thought to protect the seeds or tubers from insect or animal predators. The best studied members of the family have activity against serine proteases, but others inhibit thiol or aspartic proteases [3], and bifunctional activity against both serine proteases and plant a-amylases [4], against both serine and

* Corresponding author at: National Science Foundation, 4201 Wilson Boulevard, Arlington, VA 22230, USA. Tel.: +1 703 292 7139; fax: +1 703 527 3278. E-mail address: [email protected] (J.C. Rogers). 1 These authors contributed equally. 2 Current address: Biochemistry and Nutrition Group, BioCentrum-DTU, The Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark. 0168-9452/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2006.01.018

aspartic proteases [5], or against both serine and cysteine proteases [6] have been reported. From the perspective of plant cell biology, there is a distinction between seed storage proteins, which are largely only expressed in developing seeds, and proteins stored in vegetative tissues, such as tubers [7,8]. Protease inhibitors of the latter type are frequently expressed in other plant tissues, and their accumulation and storage in vacuoles may be induced by wounding [7]. This observation raises the question of whether plant protease inhibitors could act against endogenous proteases that also are targeted to vacuoles as a mechanism to regulate their activity in response to developmental or environmental cues. Here we characterize a Kunitz-type protease inhibitor from cauliflower and its homolog from Arabidopsis. The cauliflower inhibitor co-purified from cauliflower florets with a granulin domain-containing cysteine protease that could proteolytically process the proenzyme, proaleurain, to a mature form [9]. The purified protease required treatment with 2% SDS for optimal activation; possible explanations for this

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observation included displacement of an auto-inhibitory domain from the active site, or displacement of the Kunitz inhibitor [9]. Our results now indicate that the Arabidopsis Kunitz inhibitor protein is a potent inhibitor of two cysteine proteases, the proaleurain maturation protease and papain. The cauliflower Kunitz inhibitor traffics within cells in a pathway followed by proaleurain. Thus, the possibility exists that this type of Kunitz inhibitor has a regulatory function within plant cells. 2. Materials and methods 2.1. Cloning of the cauliflower and Arabidopsis Kunitz inhibitor genes We identified an expressed sequence tag cDNA clone, GenBank #AV537756, for the Arabidopsis Kunitz inhibitor gene, At1g72290, and obtained the plasmid from Dr. Nobumi Kusuhara, Kazusa DNA Research Institute, Chiba Japan (http://www.kazusa.or.jp). The coding sequence for the protein lacking a signal peptide (beginning with residue His 22) was amplified by polymerase chain reaction (PCR) using oligonucleotide primers At-5 = 50 -GGGGATCCCACGGAAATGAACCCG-30 , and At-4 = 50 -GGGTCGACACCCGGGAAGTATAA-30 . This yielded a fragment flanked by a BamHI site at the 50 end and a SalI site at the 30 end that was inserted into the BglII–SalI interval of plasmid pBAD/gIII A (Invitrogen, Carlsbad, CA) to yield an in frame fusion with the gene III signal sequence at the 50 end and a (His)6 sequence at the 30 end. The Arabidopsis At1g72290 gene lacks introns. We therefore hypothesized that the cauliflower homolog would also lack introns and amplified its gene from cauliflower genomic DNA using oligonucleotide primers derived from the drought-induced cDNA sequence GenBank #Z25770, where numbers indicate nucleotide positions within that sequence: C1 = 17-GATGTCATCATTCCCATTGGTC-38; C2 = 771AATCCAGGTACATGCATGAGG-751. The resulting amplification fragment was re-amplified with primers: C3.3 = 50 GGGGATCCGAAAATCGCGTGAATGAC-30 and 50 -GGGAGCTCTCACTTACTGTCGTCATCCTTGTAACCCGGGAAGTATAA-30 . This resulted in a DNA fragment containing the full coding sequence of the cauliflower gene flanked at the 50 end with a BamHI restriction site and fusing a FLAG epitope in frame to the 30 end [10] followed by a stop codon and a SacI restriction site. This fragment was inserted into the BamHI–SacI interval of LJ526 [10] to yield an expression construct with the cauliflower mosaic virus 35S promoter controlling expression of the cauliflower Kunitz inhibitorFLAG fusion protein. 2.2. Cell culture and protein expression Cloning of the proaleurain maturation protease lacking its granulin domain into the pBAD/gIII expression vector (Invitrogen, Carlsbad, CA), expression of the recombinant protein into the E. coli periplasmic space, and purification of the recombinant enzyme has been described previously [9].

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Expression of recombinant At1g72290p was performed in a similar manner, in accordance with instructions provided by Invitrogen. An overnight culture of TOP 10 E. coli cells (Invitrogen) transformed with the appropriate plasmid was used to inoculate a 500 ml culture of Luria broth containing 50 mg/ ml carbenicillin and growth was continued until the A600 exceeded 0.5. Arabinose was added to a final concentration of 0.002% for induction of protein expression and growth was allowed to continue for another 4 h. To release the proteins secreted into the periplasmic space, pelleted bacteria were resuspended in an osmotic shock buffer of 20 mM Tris–HCl pH 8, 20% sucrose and 200 mg/ml lysozyme, with the addition of phenylmethylsulfonylfluoride to 100 mM at A600 = 5 and incubated on ice for 15 min. Following centrifugation, the supernatant was dialyzed overnight at 4 8C against 0.1 M Na phosphate–0.01 M Tris pH 8 containing 100 mM phenylmethylsulfonylfluoride for three times, 1 l per dialysis, and then passed over a Ni2+–nitriloacetic acid–agarose (Qiagen, Chatsworth, CA) column equilibrated with the same buffer. Proteins were eluted from the washed column with 1 ml aliquots of 0.1 M imidazole, pH 7.7 and assayed by SDS-PAGE and immunoblot using a monoclonal (His)6 antibody (Serotec, Oxford, UK) at 1 mg/ml. Samples containing the appropriate protein were pooled and concentrated using an Amicon Ultra-4 Centrifugal Filter Device (Millipore, Billerica, MA) and the protein concentration determined by BCA assay (Pierce, Rockford, IL). Aliquots were stored at
20 8C. 2.3. Enzyme assays Activity of the proaleurain maturation protease was assayed as described [11]. The assay mix contained appropriate fluorescent peptide at a concentration of 0.1 mM in 0.1 M Na acetate pH 4.5, 1 mM dithiothreitol and 0.2% NP40 detergent (Sigma, St. Louis, MO). Fifty microliters of concentrated enzyme was used to start the reaction, and a zero time sample was removed before incubating tubes at 35 8C. At various time points, a 2 ml aliquot was removed and diluted into 2 ml of 0.1 M Na acetate pH 4.5 for fluorescence determination in a fluorimeter using filters with excitation at 380 nm and emission at 460 nm. The average background from an assay mix containing no enzyme was subtracted from each reading and the results were plotted using CricketGraph III software (Computer Associates International, Islandia, NY). Assays were repeated independently three times; data presented represent one of the three consistent obtained results. 2.4. Binding of At1g72290 to papain-agarose and pepstatin-agarose gels Papain-agarose (P4406, Sigma) and pepsin-agarose (P0609, Sigma) were rehydrated in water to give a 50% slurry representing 3000–5000 and 90–150 units/ml, respectively. 50 ml of gel was distributed into 1.5 ml ultracentrifuge tubes, which were centrifuged briefly at 200  g and the supernate was removed. To each tube was added 45 ml of reaction buffer (0.1 M Na acetate pH 4.5, 1 mM dithiothreitol, 0.1% NP40) and

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5 ml of concentrated At1g72290p recombinant protein preparation (3 mg), and tubes were incubated at room temperature with gentle mixing for 30 min. After brief centrifugation, the supernates were removed and designated as Drain (D) fractions. An additional 50 ml of reaction buffer was added to each, the gels were resuspended by mixing and incubated for 5 min. The beads again were pelleted and the supernate was removed and designated as Wash (W) fraction. To each pellet was added 50 ml of SDS-PAGE sample buffer containing 2% SDS, and the gels were resuspended and centrifuged to obtain the eluat (E) fraction. Similarly, the D and W fractions were diluted with 5 sample buffer. All were heated at 100 8C for 5 min and then electrophoresed on 4–20% acrylamide gradient gels and At1g72290p was detected by immunoblot analysis with anti(His)6 antibodies. 2.5. Transient expression in tobacco suspension culture protoplasts and immunolocalization Methods for culture of TxD suspension culture cells, protoplast preparation, electroporation to transfect plasmid DNA, and immunolocalization by confocal immunofluorescence have been previously described [10]. 3. Results 3.1. Identification of a putative Kunitz-type protease inhibitor We previously partially purified proaleurain maturation protease activity from cauliflower florets, separated proteins in that preparation by SDS-PAGE, and identified the peptide, NSGGGLLPVPVK, by mass spectrometry (MS) and tandem MS analyses of a tryptic digest from an 30 kDa protein band [9]. From a second, separate preparation of the maturation protease analyzed in a similar manner, we identified two peptides, TTAQYLILPLSPR and LQPLCPLGISQSSV. All three peptide sequences were present in the GenBank accession #S36621 for a probable drought-induced protein from Brassica rapa (turnip). When tested in GenBank searches, S36621 was homologous to known Soybean Trypsin Inhibitor/Kunitz-type protease inhibitors (MEROPS inhibitor family I3, clan IC http://merops.sanger.ac.uk/ [12]); both the homologous inhibitor sequences and S36621 were predicted to contain an Nterminal signal peptide (data not shown). We were concerned, however, because S36621, the sequence of which was predicted from GenBank #Z25770, a drought-induced mRNA, contained an unpaired Cys residue. This would be highly unusual for a protein that was translocated into the endoplasmic reticulum and then trafficked through the secretory pathway. We therefore utilized the nucleotide sequence of Z25770 to design polymerase chain reaction (PCR) primers, and obtained a clone of the homologous cauliflower sequence by PCR amplification of cauliflower genomic DNA. The nucleotide sequence of the cauliflower gene lacked introns; it is deposited as GenBank #DQ177284, and the predicted encoded protein sequence is presented in Fig. 1(a). The mature protein, after

cleavage of the signal peptide, would contain two Cys residues, consistent with having a single intramolecular disulfide bond. The signal peptide cleavage site is predicted to be between residues 24/25 (SignalP 3.0; http://www.cbs.dtu.dk/services/ SignalP), and the mature protein would contain 226 amino acids with a molecular mass of 22,392 Da. The mRNA for a closely related protein from B. napus, BnD22, was reported to be induced by water stress [13]. The gene for a closely related sequence in Arabidopsis, At1g72290, similarly lacks introns and was also obtained by PCR amplification; alignment of its predicted protein sequence with the cauliflower inhibitor is presented in Fig. 1(b). The two proteins, that predicted from DQ177284 and from At1g72290, respectively, maintain a high degree of sequence identity over their lengths, including conservation of the two Cys residues. For simplicity, the text will refer to the two proteins as the cauliflower inhibitor and At1g72290p, respectively. 3.2. Expression and purification of recombinant proaleurain maturation protease and At1g72290p For both proteins, we desired a recombinant form that would be properly folded and would contain appropriately formed intramolecular disulfide bonds. To achieve that goal, as previously [9], the proaleurain maturation protease lacking its granulin domain was expressed in E. coli such that it was secreted into the periplasmic space, an oxidizing environment where disulfide bond formation is achieved [14]. At1g72290p was similarly expressed and partially purified. As a control, we also similarly expressed and purified the lumenal domain of the receptor-like RMR protein JR702 [15]. This was chosen as a control because it is of a size approximately similar to the Kunitz inhibitor, contains internal disulfide bonds, and was efficiently expressed and secreted to the periplasmic space in the E. coli system. Immunoblot and SDS-PAGE analyses of the various preparations are presented in Fig. 2. The level of expression of recombinant At1g72290p was very low. The concentrated preparation obtained after elution from Ni2+-agarose contained two molecular species of 25 and 27 kDa as identified on an immunoblot with anti-(His)6 antibodies (Fig. 2(a)); it is likely that the larger form represents molecules that did not undergo cleavage by the bacterial signal peptidase [14]. When the immunoblot signal was aligned with the other half of the Coomassie blue-stained gel carrying lanes identical to those transferred for immunoblot analysis, the lower band from the immunoblot corresponded to a minor protein band on the gel (Fig. 2(a), indicated by arrows). The levels of expression obtained for the proaleurain maturation protease and for the RMR protein were substantially higher, and the protein bands representing each are indicated by the closed arrow and open arrow, respectively, in Fig. 2(b). 3.3. Fluorescent peptide assay for the proaleurain maturation protease The original assay used in purification of the protease measured conversion of 42 kDa proaleurain to 32 kDa aleurain

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Fig. 1. (a) Sequence of the cauliflower Kunitz inhibitor. Arrowhead, predicted signal peptide cleavage site; bold type, putative vacuolar targeting signal; single and double underlines, peptide sequences identified by mass spectrometry in two separate proaleurain maturation enzyme preparations. Note that predicted peptide sequence SNGGGLLPVPVK here differs by inversion of two residues from the peptide NSGGGLLPVPVK previously determined by mass spectroscopy [9]. (b) Alignment of cauliflower (top) and At1g72290 (bottom) sequences. Identical residues are indicated with dots, while conserved residues are indicated by vertical lines.

as followed by SDS-PAGE and immunoblot. We wished to develop an assay using a synthetic peptide, where proteolytic cleavage resulted in release of a fluorescent methylcoumarin tag [16]. Using the bacterial recombinant protease under conditions found optimal for the proaleurain maturase assay [9], we tested two peptides, N-t-Boc-Leu-Arg-Arg-7-amido-4methylcoumarin, and N-t-Boc-Val-Leu-Lys-7-amido-4methylcoumarin [11] (abbreviated Val-Leu-Lys-Mec). Activity was detected only with the latter, and it therefore was used preferentially in the subsequent experiments. We used the unusual requirement for activation by 2% SDS [9] as a criterion to define proteolytic activity from the recombinant proaleurain maturation protease versus activity that might result from a contaminating E. coli protease present in the partially purified preparation. Freshly prepared recombinant maturation protease was either not treated, or treated with 2% SDS; then each sample was diluted with ten volumes of 1% NP40 in assay buffer, equal aliquots were incubated with Val-Leu-Lys-Mec substrate at 35 8C, and substrate hydrolysis was monitored by the increase of fluorescence over time. As can be seen in Fig. 3(a), the SDS-treated enzyme (open squares) released 4.7 pmol amino-4-methylcoumarin (AMC) per minute with its activity linear over the 120 min assay, while the enzyme not treated with SDS (closed squares) showed little activity for 90 min but then slowly became activated such that its rate of substrate hydrolysis approached that measured for the SDS-

activated enzyme after 150 min of incubation. Thus, SDSactivation was necessary for optimal enzyme activity, but the enzyme could slowly become activated without SDS treatment over time. Incubation in the presence of 0.1% SDS [11] could not substitute for 2% SDS treatment (data not shown). This unusual requirement for activation by SDS strongly supports the concept that hydrolysis of Val-Leu-Lys-Mec was due to the activity of the proaleurain maturation protease. 3.4. Kunitz inhibitor activity against proaleurain maturation protease Copurification of the Kunitz inhibitor with the proaleurain maturation protease raised the possibility that the two proteins might interact. We therefore asked if the recombinant Kunitz inhibitor protein could block activity of the maturation protease. We tested its possible effect at two different pH values, 6.3 and 4.5 (Fig. 3(b)). The protease activity towards Val-Leu-Lys-Mec was greater at pH 6.3 than 4.5: 2.5 pmol/min (open squares) versus 0.94 pmol/min (open circles). No effect of the inhibitor was observed at pH 6.3 (compare open versus closed squares). In contrast, the inhibitor had a substantial effect at pH 4.5: 0.94 pmol/min (open circles) versus 0.25 pmol/min (closed circles). To test the specificity of this effect, we compared the proaleurain maturase activity in the presence of the Kunitz inhibitor to that in the presence of an

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Fig. 2. Expression and purification of recombinant proteins. (a) Purification of recombinant At1g72290p. The recombinant Kunitz inhibitor was purified from E. coli periplasmic space as described in Section 2. The purity of the protein was estimated from an immunoblot, detected with anti-(His)6 antibodies (Ab, lane 1), and from a replicate loading on the same 4–20% acrylamide SDS-PAGE gel as used for the immunoblot, stained with Coomassie blue (P, lane 2). Left, positions of molecular mass markers (M) for immunoblot; lane 3, positions of molecular mass markers (M) for Coomassie-stained gel. Arrows, positions of protein corresponding to Kunitz inhibitor with properly cleaved bacterial signal peptide. (b) Purification of recombinant proaleurain maturation protease and RMR protein. The protease was purified from E. coli periplasmic space by chromatography on a Ni2+-agarose gel and eluted with imidazole in three fractions (1–3, lanes 2–4), where the protein was detected with anti-(His)6 antibodies. Fractions containing the protein (arrows) were concentrated and electrophoresed on a 10% acrylamide SDS-PAGE gel (G, lane 5); solid arrow indicates position of the protease. RMR protein was similarly purified and analyzed (R, lane 6); the recombinant protein is indicated by the open arrow. Lanes 1 & 7 (M), molecular markers; masses indicated to sides.

equal amount of recombinant RMR protein as a control (Fig. 3(c)). The RMR protein had no effect on maturation protease activity: 2.3 pmol/min alone (open circles) versus 2.2 pmol/min in the presence of RMR (squares), while the presence of the Kunitz inhibitor reduced the rate of substrate hydrolysis by half (1.2 pmol/min; closed circles). The proaleurain maturation protease used in Fig. 3(b) and (c) was from two different preparations. 3.5. Kunitz inhibitor activity against papain The ability of the Kunitz inhibitor to act against other cysteine proteases was tested with papain. As shown in Fig. 4(a), addition of 11 mg inhibitor preparation to 10 ng of papain resulted in inhibition of papain activity at either pH 6.3 or 4.5. (It should be noted that the small amount of inhibitor

protein in this semipurified preparation makes an estimate of inhibitor:protease stoichiometry impossible.) The rate of substrate hydrolysis was not linear in the absence of inhibitor at either pH. Papain’s activity at pH 6.3 was 3 times greater than at pH 4.5, yet in both incubations the reaction rate decreased in a similar manner after 20 min. We interpreted this decrease in protease activity to reflect instability of the protease itself, perhaps from autoproteolysis, rather than from substrate depletion, because in the latter instance we would have expected the rate of reaction to remain linear for 3 times longer at pH 4.5. Therefore, to calculate reaction rates, as indicated by the corresponding lines on Fig. 4(a), we used only the data points up to 20 min for those two reactions. The effect of the Kunitz inhibitor was greater at pH 4.5 than at pH 6.3: 400 pmol/min versus 193 pmol/min with inhibitor at pH 6.3; 124 pmol/min versus 20 pmol/min with inhibitor at pH 4.5.

Fig. 3. Proaleurain maturation protease assays. (a) SDS effect. Recombinant protease was assayed without treatment with SDS (solid squares) or after treatment with SDS (open squares). Abscissa, time in minutes; ordinate, pmol of amino-methylcoumarin released. (b) Effect of At1g72290p at pH 4.5 and 6.3. Open squares, pH 6.3 minus inhibitor; closed squares, pH 6.3 plus inhibitor; open circles, pH 4.5 minus inhibitor; closed circles, pH 4.5 plus inhibitor. (c) Comparison of the effects of equal amounts of recombinant RMR protein and of At1g72290p on protease activity. Open circles, no addition; open squares, plus RMR protein; closed circles, plus At1g72290p.

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Fig. 4. Papain assays. (a) Effect of At1g72290p at pH 4.5 and 6.3. Open squares, pH 6.3 minus inhibitor; closed squares, pH 6.3 plus inhibitor; open circles, pH 4.5 minus inhibitor; closed circles, pH 4.5 plus inhibitor. (b) Comparison of the effects of equal amounts of recombinant RMR protein and of At1g72290p on protease activity. Open circles, no addition; open squares, plus RMR protein; closed circles, plus At1g72290p. Ordinate and abscissa as in Fig. 3.

As a control, we again compared the effect of the Kunitz preparation with that of the RMR protein preparation at pH 4.5. As shown in Fig. 4(b), in the presence of 3.5 mg of RMR protein, the rate of substrate hydrolysis by 2 ng of papain decreased from 3.8 pmol/min to 2.3 pmol/min, while the presence of 3 mg of Kunitz inhibitor preparation decreased the rate to 0.8 pmol/min. These rate estimates were derived from a single linear fit to all of the data points for each reaction and do not address the two components that comprise each reaction. Similar to the experiments in Fig. 4(a), the reactions for papain alone and for papain plus RMR demonstrated more rapid substrate hydrolysis during the first 20 min, 6.2 and 4.1 pmol/ min, respectively, and then the rates slowed (separate fits to the data for two components are not shown in Fig. 4(b)). In contrast, in the presence of the Kunitz inhibitor, complete inhibition of papain activity occurred at the onset of the experiment because substrate hydrolysis was first detected only at 30 min, but then the rate gradually increased in a nonlinear manner thereafter. A similar delay was observed in the experiment in Fig. 4(a). We interpreted this phenomenon to indicate that the Kunitz inhibitor preparation blocked papain activity in the beginning of the experiments, but then with time was degraded, thereby releasing previously inactive papain molecules that could act on substrate.

represented about a quarter of that applied (L). Thus it is possible that papain degraded a substantial portion of the Kunitz inhibitor mixed with the papain-agarose (or degraded the C-terminal (His)6 recognized by the antibody). Nevertheless, the experiment demonstrates clearly that the smaller band of the inhibitor doublet preferentially interacted with the papain-agarose. As a control, to exclude non-specific interaction of the inhibitor protein with agarose, we tested its ability to be retained on pepsin-agarose (Fig. 5(b)). The presence of 10 mM

3.6. The Kunitz inhibitor physically interacts with papain We tested the ability of the Kunitz inhibitor to interact with papain by incubating the inhibitor preparation with papainagarose beads. As shown in Fig. 5(a), the presence of 200 mg/ ml E-64 was necessary to prevent degradation of the Kunitz inhibitor protein during the incubation. In the presence of E64, the larger band of the inhibitor protein doublet bound poorly to the papain-agarose and most was eluted in the initial drain fraction (Fig. 5(a), lane 2). In contrast, essentially all of the smaller doublet band bound to the gel because it was not present in either the drain or wash fractions, but instead was eluted in the presence of SDS (E fraction, indicated by arrow). As judged from the signals obtained in each lane, the amount of inhibitor protein recovered in the D, W, and E fractions

Fig. 5. Binding of At1g72290p to papain-agarose and pepsin-agarose. (a) Papain-agarose. At1g72290p was incubated at pH 4.5 with papain-agarose in the presence (lanes 2–4) or absence (lanes 5–7) of 200 mg/ml E-64. Protein in various fractions was detected on an immunoblot with anti-(His)6 antibodies. L, amount initially mixed with the gel; D, fraction not binding; W, fraction washed from gel; E, fraction eluted from gel with SDS buffer; arrow, smaller molecular form specifically retained on gel. (b) Pepsin-agarose. At1g72290p was incubated at pH 4.5 with pepsin-agarose in the presence (lanes 2–4) or absence (lanes 5–7) of 10 mM pepstatin A. L, D, W, E as in (a); arrow indicates smaller band not retained on gel.

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pepstatin A was necessary to prevent degradation of the inhibitor by the immobilized pepsin. Again, the larger band of the inhibitor protein doublet did not bind to the gel and was recovered exclusively in the drain fraction. However, in contrast to the results with papain-agarose, most of the amount of the smaller band of the inhibitor protein doublet that was recovered was also present in the drain fraction (arrow, lane 2), with a smaller amount present in the E fraction (arrow, lane 4). The total amount of the smaller band recovered from the gel was less than the amount applied, again indicating that a substantial proportion of the protein was probably degraded by the pepsin. However, in comparison to the results with papain-agarose, little was retained on the gel. We conclude that the smaller Kunitz inhibitor band preferentially interacted with papain-agarose as compared to pepsin-agarose. This result is consistent with the prior enzyme assays, and supports the concept that inhibition of the proaleurain maturase and of papain resulted from physical interactions between the Kunitz inhibitor and the proteases. 3.7. The cauliflower Kunitz inhibitor colocalizes with BP80 when transiently expressed in tobacco suspension culture protoplasts Co-purification of the cauliflower Kunitz inhibitor and the proaleurain maturation protease raised the possibility that the two proteins might interact within the secretory pathway. While we could not test this possibility directly, we could determine if the Kunitz inhibitor was present in organelles where proaleurain, the maturation protease substrate, was known to localize. Pea BP-80 is a vacuolar sorting receptor that binds the vacuolar sorting determinant in the proaleurain propeptide with high affinity in vitro [17,18], and BP-80 traffics within cells to prevacuolar compartments [19] where proaleurain is proteolytically processed to mature aleurain [10,20]. We therefore co-expressed pea BP-80 and the cauliflower Kunitz inhibitor carrying a C-terminal FLAG epitope in tobacco suspension culture protoplasts, and localized the two proteins by confocal immunofluorescence. As would be expected because the antibodies recognize endogenous BP-80 [19], essentially all cells demonstrated punctate green labeling. We searched for cells expressing the recombinant Kunitz inhibitor as judged by the presence of red dots, indicating labeling by anti-FLAG antibodies. In 35 cells so identified, all red dots colocalized with green dots, while in three cells some red dots were separate from green dots. Examples of complete co-localization are shown in Fig. 6(a), where BP-80 localized to small punctate organelles typical for prevacuolar compartments [19] (examples indicated by arrow). The Kunitz inhibitor, detected by an anti-FLAG monoclonal antibody, localized to organelles with a similar appearance (Fig. 6(b), arrow). When the two images are superimposed, in every instance the BP-80 and Kunitz signals colocalize as indicated by the yellow color (Fig. 6(c)). Thus the Kunitz inhibitor traffics to organelles where proaleurain also would be present.

Fig. 6. Co-expression of the cauliflower Kunitz inhibitor and BP-80 in tobacco suspension culture protoplasts. Labeling of two protoplasts is shown. (a) BP-80; (b) cauliflower Kunitz inhibitor; (c) superimposition of (a) and (b) where yellow indicates colocalization of the two proteins; (d) transmitted light image of the fixed, permeabilized protoplasts labeled with antibodies. V, central vacuole; n, position of nucleus; arrow, cluster of three organelles labeled with both antibodies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

4. Discussion Cystatins are well-characterized inhibitors of plant cysteine proteases [11,21,22]. Some members of the cystatin family, such as soybean cystatin [22] lack signal peptides and presumably act within the cell cytoplasm, while others, such as maize cystatin [GenBank #P3172611] carry typical signal peptides and would traffic through the endomembrane system. Soybean cystatin has been implicated in regulating programmed cell death [22]. In contrast, Kunitz-type protease inhibitors all carry signal peptides and would perform their functions within organelles of the secretory pathway or after being secreted from cells. The cauliflower and Arabidopsis Kunitz inhibitors described here are part of a conserved gene family that is expressed in response to environmental stresses. The GenBank annotation for the inhibitor from B. rapa, S36621, indicates that its mRNA is induced by drought stress. A closely related member of the family, BnD22 from Brassica napus, is not expressed in seeds [13]. Rather, it was induced by drought stress or abscisic acid treatment in leaves, where it accumulated to more than 1% of the total protein. The authors noted that drought stress accelerates leaf senescence, a process that is associated with increased protease activities, and speculated that BnD22 might serve to regulate senescence-related processes by its protease inhibitory activities [13]. Our purification of the cauliflower Kunitz inhibitor from florets is consistent with expression of these family members in vegetative tissues.

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Cysteine protease mRNAs are induced during senescence in Arabidopsis [23,24] and other plants [25,26]. Among senescence-induced Arabidopsis mRNAs are those for SAG2, equivalent to Arabidopsis aleurain [23,27], and for SAG12, a papain-like cysteine protease [24]. A SAG12-green fluorescent protein fusion protein localized to senescence-associated vacuoles in Arabidopsis leaf cells that were separate from the central vacuole [28]. These senescence-associated vacuoles concentrated the dye lysotracker red, a marker for autophagyderived vacuoles [29]; this observation suggests that targeting of SAG12 to that destination probably would involve mechanisms different from those used for targeting proaleurain [10,30]. Thus our finding that the Arabidopsis Kunitz protein is an inhibitor of the cauliflower proaleurain maturation protease and of papain raises the possibility that it, and by extension the cauliflower Kunitz protein and BnD22, may have regulatory functions. Their function would depend upon the compartment within the secretory pathway to which they were targeted. The cauliflower protein has the amino acid sequence NPLRT positioned near the N-terminus of the mature protein, in a region that is known to be exposed on the surface of the soybean trypsin inhibitor [31]. The corresponding sequence in the Arabidopsis Kunitz inhibitor is NPLNT. In sporamin, a Kunitztype serine protease inhibitor from sweet potato tubers [32,33], the sequence NPIRL is similarly positioned in a propeptide. This sequence contains the information necessary and sufficient to target prosporamin to vacuoles [34], and Leu can substitute for Ile to maintain function [35]. A similarly positioned NPINL is present in a number of potato protease inhibitors [36]. The NPIR-containing sequences in proaleurain and prosporamin also are the ligands for binding by BP-80, a vacuolar targeting receptor [36]. We therefore thought it likely that the cauliflower Kunitz protein would traffic in the pathway followed by BP-80. Indeed, when co-expressed in tobacco protoplasts, the two proteins colocalized in organelles whose morphology would be consistent with prevacuolar compartments or Golgi. These considerations indicate that the cauliflower protein DG177284, Arabidopsis At1g7220p, and BnD22 likely comprise a Kunitz inhibitor family defined by their cysteine protease inhibitor activity and by their targeting through the BP-80 pathway to vacuoles. As such, they could regulate activity of the proaleurain maturation protease, as well as other vacuolar cysteine proteases. This hypothesis might be tested in the future by overexpressing one of the proteins in plant cells and measuring its affect on conversion of proaleurain to mature aleurain. Overexpression might also allow a further test of the hypothesis that inhibition of vacuolar cysteine proteases can limit or prevent changes of senescence [25]. Acknowledgements This research was supported by grants DE-FG0397ER20277 from DOE and MCB-9974429 from NSF. A portion of the mass spectrometry analyses were performed at Pacific Northwest National Laboratory under EMSL User Proposal #2503. OØ was supported by the Danish Academy of Technical Sciences, Grant #EF803.

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