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Plant Physiol. Feb 2006; 140(2): 726–733.
PMCID: PMC1361338

Evidence for Functional Conservation, Sufficiency, and Proteolytic Processing of the CLAVATA3 CLE Domain1,[W][OA]

Abstract

Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) is hypothesized to act as a ligand for the CLV1 receptor kinase in the regulation of stem cell specification at shoot and flower meristems. CLV3 is a secreted protein, with an amino-terminal signal sequence and a conserved C-terminal domain of 15 amino acids, termed the CLE (CLV3/ESR-related) domain, based on its similarity to a largely unstudied protein family broadly present in land plants. We have tested the function of 13 Arabidopsis CLEs in vivo and found a significant variability in the ability of CLEs to replace CLV3, ranging from complete to no complementation. The best rescuing CLE depends on CLV1 for function, while other CLEs act independently of CLV1. Domain-swap experiments indicate that differences in function can be traced to the CLE domain within these proteins. Indeed, when the CLE domain of CLV3 is placed downstream of an unrelated signal sequence, it is capable of fully replacing CLV3 function. Finally, we have detected proteolytic activity in extracts from cauliflower (Brassica oleracea) that process both CLV3 and CLE1 at their C termini. For CLV3, processing appears to occur at the absolutely conserved arginine-70 found at the beginning of the CLE domain. We propose that CLV3 and other CLEs are C-terminally processed to generate an active CLE peptide.

The CLAVATA loci (CLV1, CLV2, and CLV3) encode putative signaling components critical for stem cell specification and organogenesis in Arabidopsis (Arabidopsis thaliana). CLV1 encodes a Leu-rich repeat-containing receptor kinase (Clark et al., 1997) and CLV2 encodes a Leu-rich repeat-containing receptor-like protein lacking a cytoplasmic signaling domain (Jeong et al., 1999). CLV3 encodes a small secreted peptide (Fletcher et al., 1999; Rojo et al., 2002). CLV loci promote differentiation of stem cell daughters at shoot and flower meristems by limiting the expression of the stem cell-promoting WUS transcription factor (Laux et al., 1996; Brand et al., 2000; Schoof et al., 2000). In the absence of CLV function, shoot and flower meristems accumulate massive pools of stem cells, leading to severe disruption of organogenesis and development (Clark et al., 1993, 1995; Kayes and Clark, 1998).

Little is known about the mechanism of CLV signaling. CLV1 and CLV2 are hypothesized to form a heterodimer that acts as the receptor for a CLV3-based ligand (Trotochaud et al., 1999). CLV3 is expressed in cells adjacent to those expressing CLV1 (Clark et al., 1997; Fletcher et al., 1999). CLV3 has been shown to be secreted in several assays, while CLV3 overexpression leads to meristem differentiation, dependent on CLV1 function (Brand et al., 2000; Rojo et al., 2002). Genetic evidence also suggests CLV3 diffusion can be sequestered by CLV1 (Lenhard and Laux, 2003). Finally, a recent report demonstrated that a 14-amino acid peptide corresponding to the C-terminal CLE domain of CLV3 could dramatically alter root development when added exogenously (Fiers et al., 2005). These lines of evidence suggest that CLV3 is a secreted ligand for CLV1 that may be processed to an active ligand in vivo, but definitive data for these hypotheses are lacking.

Resolving these issues is critical for several reasons. First, stem cell maintenance and differentiation are essential for plant development. Second, each member of the CLV pathway is a member of a large gene family: more than 400 receptor-like kinases, 60 receptor-like proteins, and 25 CLV3-related (CLE) proteins are found encoded within the Arabidopsis genome (Cock and McCormick, 2001; Shiu and Bleecker, 2001). Finally, CLV1-related receptor-like kinases, especially the closely related BAM1, BAM2, and BAM3 receptors, are critical for myriad aspects of plant growth and development, indicating that understanding receptor signaling is essential for understanding a large number of developmental events (DeYoung et al., 2006).

Here we report analysis of CLV3 and related CLE proteins in Arabidopsis. We show that many CLEs can replace CLV3 function in vivo, suggesting the importance of the conserved CLE domain common to this family of proteins. Using domain swaps, we provide evidence that the CLE domain of CLV3 alone is sufficient to replace CLV3 function in vivo. We show variability in whether the ability of CLEs in replacing CLV3 function is dependent on CLV1 or other receptor kinases. Finally, we characterize a proteolytic activity from plant extracts that is capable of C-terminal processing of both CLV3 and CLE1 in in vitro reactions. We propose that CLV3 is processed in vivo, releasing the active CLE peptide from a mature CLV3 precursor protein.

RESULTS

Cross-Complementation between CLE Family Members and CLV3

The Arabidopsis genome contains a large number of putative genes that encode proteins with similarity to CLV3 within the CLE domain (Cock and McCormick, 2001; DeYoung and Clark, 2001; Sharma et al., 2003; Supplemental Fig. 1). Given recent evidence suggesting the functional significance of this domain, we sought to determine whether any CLE proteins could replace CLV3 function in vivo. We designed a transformation vector in which CLV3 upstream and downstream cis-regulatory elements could be used to drive CLE expression (Fig. 1A).

Figure 1.
A vector to express CLEs under the control of CLV3 cis-regulatory elements. A, Modified pCB302 vector with CLV3 upstream and downstream cis-elements (see “Materials and Methods”). RB, Right border; LB, left border. B, CLV3 was placed in ...

To test the function of this vector, we first placed the CLV3 cDNA into the vector and transformed clv3-1 mutants. clv mutants lead to stem cell accumulation at the flower meristem, eventually giving rise to supernumary floral organs, especially carpels (Clark et al., 1993, 1995). Whereas wild-type flowers develop an invariant two carpels per flower, clv3-1 mutants initiate around five carpels per flower (Fig. 1B). We have shown previously that carpel number is a sensitive measurement for meristem size (Clark et al., 1993, 1995; Yu et al., 2000). For multiple PCLV3:CLV3 clv3-1 transgenic lines, we observed complete to nearly complete rescue (Fig. 1B).

Thirteen intronless CLE genes were amplified from Landsberg erecta (Ler) genomic DNA and cloned into the same binary vector containing CLV3 cis-regulating elements (Fig. 1A). Each PCLV3:CLE was transformed into clv3-1, and multiple independent transgenic lines were isolated and analyzed. The majority of CLEs tested were able to rescue the clv3-1 meristem defects to some degree (Fig. 2; Table I). CLE1 and CLE6 were nearly equivalent to CLV3 in terms of ability to rescue clv3-1 (Fig. 2). In descending order, CLE13, CLE19, CLE9, CLE22, CLE12, CLE21, and CLE11 provided only partial rescue of clv3-1, with the mean number of carpels per flower between 3.0 and 4.0 averaged over multiple independent lines. CLE8, CLE14, CLE25, and CLE26 provided little to no rescue of clv3-1. None of the CLEs showed clear evidence of dominant-negative effects.

Figure 2.
CLEs provide variable complementation of clv3-1. A, Gynoecia phenotypes for wild-type Ler, clv3-1, and clv3-1 transformed with PCLV3:CLEs (left to right: Ler, PCLV3:CLE1, PCLV3:CLE6, PCLV3:CLE12, PCLV3:CLE19, PCLV3:CLE22, PCLV3:CLE26, clv3-1). B, Mean ...
Table I.
CLE complementation of clv3-1

The CLE Domain Alone Is Sufficient for CLV Signaling

In comparing CLV3 and the CLEs, the sequences between the putative signal peptide and the CLE domain, termed the variable domain, are largely unrelated (Supplemental Fig. 1). The fact that a number of the CLEs were able to replace CLV3 function leads us to hypothesize that the CLE domain is the critical domain allowing for the function of these CLEs. To test this idea, we designed a domain-swap experiment to determine whether we could restore function to a nonfunctional CLE.

The C-terminal domain from the nonfunctional CLE8 was replaced by the CLV3 C-terminal domain, and the chimeric reading frame was placed into the pCB302-CLE expression vector to generate PCLV3:E8C3. When transformed into clv3-1, this transgene provided nearly complete rescue in multiple independent lines (Fig. 3A), indicating that addition of the CLV3 CLE domain was capable of restoring function.

Figure 3.
Chimeric proteins reveal importance of the CLV3 CLE domain. A, Chimera of CLE8 and CLV3 was generated in the CLV3 expression vector (PCLV3:E8C3), transformed into clv3-1, and multiple independent homozygous lines were isolated and analyzed for the mean ...

These results would suggest that the CLE domain is the function region of the protein; however, we could not rule out a role for the variable domain in providing the correct context for the CLE domain. To test whether the CLE variable domain was necessary for CLV3/CLE function, we replaced the signal sequence and variable domain of CLV3 with the first 66 amino acids, including the signal sequence, from the unrelated receptor kinase ERECTA (Torii et al., 1996). Thus, in this ERC3 chimera, the only CLV3 sequences were from the CLE domain. The PCLV3:ERC3 transgene transformed into clv3-1 provided nearly complete rescue, indicating that the CLV3 variable domain is dispensable for function (Fig. 3B).

Some CLEs Require CLV1, Others Do Not

CLV1 is the proposed receptor for CLV3. All clv1 missense mutants are dominant-negative alleles, whereas clv1 null alleles, like clv1-11, display weak phenotypes (Diévart et al., 2003). These facts suggest the existence of redundant receptors in CLV signaling and also suggest that the proposed partnership between CLV3 and CLV1 is not exclusive. Thus, it was unclear from the results above whether the CLE proteins expressed in the meristem were acting through CLV1 or through other receptor kinases.

To resolve this issue, we chose three CLEs with differing abilities to replace CLV3 function and tested their function in the absence of CLV1. When CLE1, which provides nearly complete rescue of clv3-1, was assessed in a clv3-1 clv1-11 background, we observed a major, but not complete, reversal of the rescue (Fig. 4). This suggests that CLE1 acts largely, although not exclusively, through CLV1. CLE11-driven rescue was significantly affected by the removal of CLV1 (t test, P < 0.01), but was affected to a lesser degree than CLE1. CLE22, which also provided only partial rescue of clv3-1, was not significantly affected by the removal of CLV1 (t test, P = 0.04), suggesting CLE22 primarily acted through other receptors (Fig. 4).

Figure 4.
CLEs differ in reliance on CLV1 for activity in the meristem. clv3-1 plants homozygous for PCLV3:CLE1, PCLV3:CLE11, and PCLV3:CLE22 were crossed to clv1-11. Lines homozygous for clv3-1 and clv1-11, and carrying at least one copy of the transgene, were ...

Proteolytic Processing of CLV3 and CLE1

Given the results presented here that the CLE domain of CLV3 is sufficient for function and recently published data on the ability of a peptide corresponding to the CLV3 CLE domain to alter development in roots (Fiers et al., 2005), we hypothesized that the CLE domain might be proteolytically released from the CLV3 mature protein.

We expressed mature CLV3 without the signal peptide as a glutathione S-transferase (GST)-tagged fusion protein in Escherichia coli (GST-mCLV3; Fig. 5A). We then incubated this with cauliflower (Brassica oleracea) protein extracts. We observed proteolytic processing of CLV3, revealing two smaller mass fragments, while GST alone was not processed (Fig. 5A). The smaller GST-mCLV3 forms were detected with anti-GST antibodies, suggesting C-terminal processing. His-tagged mature CLV3 was also processed at the carboxyl end upon incubation with cauliflower extracts (data not shown). Processing occurred in extracts generated in both the presence and absence of triton, suggesting that the processing activity is not membrane associated (Supplemental Fig. 2). No cross-reacting bands were detected in cauliflower extracts alone (Fig. 5A; Supplemental Fig. 2).

Figure 5.
CLE processing activity in cauliflower extracts. A, GST-tagged mature CLV3 (GST-mCLV3), GST, or no proteins (none) were untreated, incubated for 2 h in buffer, incubated for 2 h with cauliflower extracts, or incubated for 2 h with cauliflower extracts ...

To test more rigorously whether processing was carboxy terminal, we repurified, using GST affinity, a large-scale reaction of GST-mCLV3 with cauliflower extracts. As shown in Figure 5B, the repurified proteins contained intact GST-mCLV3 (i), two processed forms (p1 and p2), and the background band observed as a contaminant in the GST-mCLV3 synthesis (b). Individual bands were subjected to in-gel trypsin digestion followed by time-of-flight (TOF)-mass spectrometry (MS) analysis. The amino-terminal MSPILGYWK fragment from GST was definitively identified among tryptic fragments of p1 and p2 (Supplemental Fig. 3). This indicates an intact GST N terminus in p1 and p2 and reveals that cleavage was C terminal.

To determine the sites of proteolytic cleavage, our repurified processing mixture (Fig. 5B) was subjected to intact MS analysis (Supplemental Fig. 4). GST-mCLV3 and the two processed forms (but not the nonspecific band) were each represented among singly and doubly charged species as five peaks, presumably as a result of either modification in E. coli or by MS preparation (Supplemental Figs. 3 and 4). By averaging mass differences between corresponding peaks for each protein species, we estimate a mass difference of approximately 3,067 D between full-length GST-mCLV3 and the larger processed form (Fig. 5B, f1). This would place the cleavage for the f1-processed form at Arg-70, which is at the beginning of the CLE domain and is absolutely conserved among predicted CLE proteins (Fig. 6). Our peptide mass fingerprinting of the f1-processed form revealed that the tryptic fragment GLHEELR is present within f1, suggesting that the cleavage occurs to the carboxyl side of this residue (Supplemental Fig. 3).

Figure 6.
Alignment of CLE domains. Alignment of C-terminal domains from CLV3 and CLEs utilized in this study. The top cluster is best-functioning CLEs, the middle cluster is partially functioning CLEs, and the bottom cluster is nonfunctioning CLEs. The estimated ...

For the smaller f2-processed form, average peak mass difference from full-length GST-mCLV3 was 6,296 D (Supplemental Fig. 4). This would place the cleavage at Met-41, among a stretch of four Met residues in the variable domain (Supplemental Fig. 1).

To determine whether the cauliflower processing activity would use other CLEs as substrates, we generated GST-tagged mature CLE1 (GST-mCLE1) and repeated the assays. As for CLV3, we observed processing of GST-mCLE1 to two smaller mass forms (Fig. 5C). A time course indicated that the two processed forms arose simultaneously and they did not appear to be the result of sequential processing events (Fig. 5C).

The Arg-70 residue that appears to be a site of proteolysis for CLV3 incubated with cauliflower extracts is absolutely conserved among predicted CLE proteins. To test the importance of this residue for in vitro proteolysis, we generated both Lys (R63K) and Asn (R63N) substitutions of the corresponding Arg-63 residue in CLE1. When incubated with extracts, we observed no obvious alteration in processing for these substituted forms, indicating that this residue is not absolutely essential for processing in vitro (Fig. 5D).

To test the specificity of the processing reaction, we first used a control GST protein. No processing of GST could be detected after a 2-h incubation (Fig. 5A). We also examined the stability of the proteins from the cauliflower extracts in a mock processing reaction. The extracts generated in both the presence and absence of triton showed no apparent general proteolysis or loss of specific proteins, indicating that the extracts do not contain significant amounts of general protease activity (Fig. 5E).

DISCUSSION

The Role of the CLE Domain

We have characterized the ability of many CLEs to replace CLV3 function in vivo and observed extensive, but variable, functional overlap. This suggested that the CLE domain, which is the only region with similarity among these proteins, is the functional domain. This was supported by the ability to turn nonfunctional CLE8 (nonfunctional in terms of replacing CLV3 in vivo) into a functional protein simply by replacing the CLE domain with that from CLV3. Definitive evidence on the sufficiency of the CLE domain came from the full function of a chimeric protein in which the CLV3 signal sequence and variable domain were replaced with sequences from the unrelated ERECTA receptor kinase, leaving only the C-terminal domain from CLV3. Taken together with recent evidence on the ability of the CLE domain peptide to drive developmental changes when added exogenously to Arabidopsis roots (Fiers et al., 2005), this would suggest that the key to CLV3 and CLE function is indeed the CLE domain alone.

What is the role of the rest of the CLV3 protein? The signal peptide is obviously critical for secretion by targeting CLV3 for translation into the endoplasmic reticulum. The variable domain may simply act to allow for translation/translocation of the CLE domain into the lumen of the endoplasmic reticulum.

Despite the sufficiency of the CLE domain, an alignment of the CLE domains of those we tested in clv3-1 cross-complementation reveals that higher sequence similarity to CLV3 does not correlate with being functionally equivalent to CLV3 (Fig. 6). CLE1 and CLE6, which are highly functional (in terms of replacing CLV3), are not particularly similar to CLV3 among CLEs, with five nonconservative differences between CLV3 and CLE1/CLE6 over the 15-amino acid CLE domain. By comparison, the weakly functional CLE11 has only two nonconservative differences with CLV3. Examining the CLE domains as aligned by CLV3-equivalent function does little to suggest which residues might be critical for CLV3 function. One candidate is the Asp at position 11. This is common between CLV3, CLE1, and CLE6 and is an Asn in all partially and nonfunctional CLEs. The only exceptions are the nonfunctional CLEs, CLE25 and CLE26, which both contain an Asp; however, CLE25 and CLE26 contain predicted, but not verified, hydrophobic extensions that might independently disrupt function. Another potential critical site is the acidic residues at positions 1 and/or 2 found in all of the functional and partially functional CLEs and within none of the nonfunctional CLEs.

Receptor Specificity

Using the clv1-11 null allele to test the requirement of the CLEs for CLV1 indicated differences in receptor specificity, at least on a genetic level. CLE1 function was largely dependent on CLV1, CLE11 less so, and CLE22 not at all. This suggests that these CLEs differ in the receptor targets, with CLE22 utilizing receptors other than CLV1. Interestingly, the PCLV3:CLE22 clv3-1 phenotype was very similar to clv1-11 mutants, suggesting that CLE22 function represents the activity of the as-yet-unidentified CLV1-redundant receptors.

We have recently shown that three CLV1-related receptors, BAM1, BAM2, and BAM3, have similar biochemical functions as CLV1, but very different developmental roles (DeYoung et al., 2006). BAM receptors are expressed broadly throughout plant development and regulate a wide range of developmental events, from vascular patterning to anther development to meristem development. As CLV1 can replace BAM1 throughout the plant in cross-complementation studies, this suggests that CLV3-equivalent CLEs make excellent candidates for BAM ligands in various tissues. It will be interesting to compare the precise expression profiles at the cellular level for BAM and CLE genes, and to test relationships between gene pairs expressed in adjacent cell populations. With data on the broad expression of many members of the CLE protein family (Sharma et al., 2003), it is interesting to speculate that processed CLE peptides with the ability to activate a broad receptor pool may be present in nearly all plant tissues.

CLE Processing

Several lines of evidence are consistent with processing of CLV3 and other CLEs at the C terminus to release an active peptide. First, the CLE domain is sufficient to carry out CLV3 activity. Second, addition of a peptide corresponding to the CLV3 CLE domain can dramatically alter development of Arabidopsis roots when added exogenously (Fiers et al., 2005). Third, CLE19 overexpression in the roots can drive similar changes, and these can be ameliorated by mutations in a putative carboxy peptidase, SOL1 (Casamitjana-Martinez et al., 2003). With this in mind, we tested the ability of cauliflower extracts to process CLV3 and found activities that can process CLV3 at its carboxy terminus. The processing activities are not membrane associated, can process both CLV3 and CLE1, and are resistant to protease inhibitors used during production of the cauliflower extracts (see “Materials and Methods”).

Interestingly, one of the sites utilized by the in vitro processing activity appears to fall at Arg-70 in CLV3, an absolutely conserved residue among CLEs found at the beginning of the CLE domain. This raises the possibility that this activity may represent a key enzyme in the production of an active CLE peptide. The observation that altering this residue does not abolish processing suggests that the protease may recognize a motif upstream or downstream of this site.

The significance of the second processing site around Met-41 is unclear. Whether this is carried out by the same enzyme is unknown. CLE1 is also processed at a second site and also contains paired Met residues at approximately the same position as Met-41 in CLV3 (Supplemental Fig. 1).

At this point, we cannot rule out that these processing activities are nonspecific proteases present in cauliflower extracts. The fact that both CLV3 and CLE1 are processed in a similar fashion and that neither control proteins nor general cauliflower proteins are processed would suggest some level of specificity, but this is not definitive. A key test would be to determine whether related processing occurs in vivo; however, the likelihood that any processed fragments would be rapidly turned over might make this very difficult to detect. Combined with the effect of the CLV3 CLE peptide on roots, our results would suggest that following CLV3 localization and diffusion may be experimentally quite difficult.

MATERIALS AND METHODS

Plant Growth

Seeds were sowed on a combination of Metromix 360 (Scott), medium vermiculite, and coarse perlite mixed in a 1:1:1 ratio, supplemented with approximately 1 g Osmocote 14-14-14 fertilizer (Scott) per 3.5-inch pot, and topped with a thin layer of Metromix 360. After 7 d of cold treatment at 4°C, plants were grown at 22°C under constant cool-white fluorescent light.

Construct Generation

For CLV3 cis-elements, an approximately 0.6-kb fragment downstream of the CLV3 stop codon was amplified from Columbia plants with primers CLV3T-5′ Bam (CGGATCCTAATCTCTTGTTGCTTTAAAT) and CLV3T-3′ (TAAGGATAATAATTAGCTCTAGG). This fragment was regarded as a CLV3 terminator and cloned into a binary vector pCB302 (Xiang et al., 1999) through restriction sites BamHI and EcoRI (pCB302-C3T).

An approximately 3.5-kb fragment upstream of the CLV3 start codon from primer CLV3P-5′-Xba (CTCTAGAACCGAAAGAATCGGAACC) to primer CLV3P-3′-MluSmaBam (AGGATCCCGGGACGCGTGAGAGAGATAAAGAGAGAAAT) was amplified from Columbia plants as the CLV3 promoter region. This fragment was inserted into pCB302-C3T to generate pCB302-C3PT.

All CLE sequences were amplified from Landsberg erecta (Ler) genomic DNA based on current annotation with attachment of the MluI site to the 5′ end and the BamHI site to the 3′ end, with the exception of CLE22, where the 3′ end was BglII. The CLE open reading frames were then ligated into pCB302-C3PT by the attached restriction sites (pCB302-CLE), integrated between the CLV3 promoter and terminator sequences.

The portion of the CLV3 coding sequence corresponding from His-66 to the stop codon was used to replace the portion of CLE8 corresponding from Phe-57 to the stop codon by fusion PCR. The PCR fragment was ligated into pCB302-C3PT through sites MluI and BamHI to generate pCB302-E8C3.

ERECTA sequences corresponding to the signal sequence and the following 46 amino acids were amplified, fused to CLV3 coding sequences corresponding from His-66 to the stop codon, and cloned into pCB302-C3PT to generate pCB302-ERC3.

Cauliflower Processing and Detection

Cauliflower (Brassica oleracea) meristem protein extracts were prepared as described before (Trotochaud et al., 1999) with or without 0.1% Triton X-100. Note that 1 mL of protease inhibitor cocktail for use with plant cell extracts (P9599; Sigma) was added in the extraction buffer per 300 g of tissues. Before use, the extracts were centrifuged at 40,000g for 30 min at 4°C.

Escherichia coli-purified proteins (His-CLV3, GST-CLV3, or GST-CLE1) were incubated with cauliflower protein extracts or mock (distilled, deionized water or elution buffers for individual tagged proteins) for 2 h at 4°C on a rotor. SDS loading buffers were added to end the reactions, and samples were boiled at 100°C for 5 min before analysis.

Western protein gel blots were performed as described previously (DeYoung et al., 2006). Chicken anti-GST antibodies were kindly provided by Ken Cadigan and detected with horseradish peroxidase-conjugated rabbit anti-chicken secondary antibodies.

Proteolytic Fragment Isolation and Analysis

Approximately 100 μg of purified GST-mCLV3 protein was incubated with cauliflower protein extracts for 2 h at room temperature with rotation. After incubation, GST-tagged proteins were repurified with glutathione Sepharose 4B (Amersham) and eluted in 10 mm glutathione, 50 mm Tris-HCl, pH 8.0. Repurified, processed GST-mCLV3 fragments were submitted to the Michigan Proteome Consortium (http://www.proteomeconsortium.org) in liquid for intact MS analysis and in SDS-PAGE gel for peptide mass fingerprinting analysis on each protein band.

MS analysis was carried out as follows. Protein solutions were prepared for matrix-assisted laser-desorption (MALDI)-TOF MS analysis by purification on a C4 ZipTip (Millipore). Protein was eluted with three 10-mL aliquots of 60% acetonitrile/0.1% trifluoroacetic acid and spotted in two 1-mL aliquots onto a MALDI target with 1-mL sinapinic acid as matrix (10 mg/mL in 60% acetonitrile/0.1% trifluoroacetic acid). MS spectra of intact proteins were acquired on a DE-STR (Applied Biosystems) operated in linear positive ion mode with an accelerating voltage of 25 kV and a grid voltage of 23.25 kV. The guide wire was 0.1 kV and a delay time was 750 ns. MS spectra were acquired for the mass range 2,000–70,000 D. Spectra were summed from a variable number of laser shots using a nitrogen laser operating at 337 nm and 10 Hz. External calibration was performed using carbonic anhydrase I (CAI; m/z 28,740). A three-point calibration was utilized from the +1 and +2 molecular ions of CAI and the CAI dimer. Protein masses are reported as average masses with a mass accuracy better than 200 ppm.

Peptide mass fingerprinting was conducted as follows. Each band on the SDS-PAGE gel was subjected to in-gel trypsin digestion followed by MALDI-TOF MS analysis. Tandem MS spectra were acquired on a 4700 proteomics analyzer (Applied Biosystems) operated in positive ion mode with a source voltage of 8.0 kV and a grid voltage of 6.8 kV. Atmosphere was used as the collision gas with a pressure of 6 × 10−7 Torr and collision energy of 1 kV. Spectra were acquired using 8,000 to 10,000 laser shots from a Nd-YAG laser operating at 354 nm and 200 Hz. For calibration, fragmentation of angiotensin II was used; mass accuracy was better than 50 ppm based on peptide fragment assignments.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank Ken Cadigan for providing anti-GST antibodies and Kathleen Noon for assistance in interpreting MS data.

Notes

1This work was supported by the National Institutes of Health (grant no. 1R01GM62962–01A1 to S.E.C.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Steven E. Clark (ude.hcimu@skralc).

[W]The online version of this article contains Web-only data.

[OA]Open Access articles can be viewed online without a subscription.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.072678.

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