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Plant Physiology 139:1597-1611 (2005) © 2005 American Society of Plant Biologists Genome Analysis and Functional Characterization of the E2 and RING-Type E3 Ligase Ubiquitination Enzymes of Arabidopsis1,[W]Section of Molecular and Cellular Biology, Division of Biological Sciences (E.K., S.L.S., J.C.) and Plant Biology Graduate Group Program (E.K., J.C.), University of California, Davis, California 95616; and Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520–8104 (L.M., N.S., Y.G., O.-S.L., X.-W.D.)
Attachment of ubiquitin to substrate proteins is catalyzed by the three enzymes E1, E2 (ubiquitin conjugating [UBC]), and E3 (ubiquitin ligase). Forty-one functional proteins with a UBC domain and active-site cysteine are predicted in the Arabidopsis (Arabidopsis thaliana) genome, which includes four that are predicted or shown to function with ubiquitin-like proteins. Only nine were previously characterized biochemically as ubiquitin E2s. We obtained soluble protein for 22 of the 28 uncharacterized UBCs after expression in Escherichia coli and demonstrated that 16 function as ubiquitin E2s. Twelve, plus three previously characterized ubiquitin E2s, were also tested for the ability to catalyze ubiquitination in vitro in the presence of one of 65 really interesting new gene (RING) E3 ligases. UBC22, UBC19-20, and UBC1-6 had variable levels of E3-independent activity. Six UBCs were inactive with all RINGs tested. Closely related UBC8, 10, 11, and 28 were active with the largest number of RING E3s and with all RING types. Expression analysis was performed to determine whether E2s or E3s were expressed in specific organs or under specific environmental conditions. Closely related E2s show unique patterns of expression and most express ubiquitously. Some RING E3s are also ubiquitously expressed; however, others show organ-specific expression. Of all the organs tested, RING mRNAs are most abundant in floral organs. This study demonstrates that E2 diversity includes examples with broad and narrow specificity toward RINGs, and that most ubiquitin E2s are broadly expressed with each having a unique spatial and developmental pattern of expression.
Protein ubiquitination is the covalent attachment of the 76-amino acid eukaryotic molecule, ubiquitin, to substrate proteins. The fate of the ubiquitinated substrate depends upon the type of ubiquitin modification and the choice of ubiquitin lysyl residue used to form the attached polyubiquitin chain (Fang and Weissman, 2004  ; Sun and Chen, 2004) Ligation of a single ubiquitin molecule (monoubiquitination) has been linked to endocytosis and histone modification (Schnell and Hicke, 2003; Umebayashi, 2003). Proteins modified by the attachment of a polyubiquitin chain of four or more ubiquitins linked via ubiquitin lysyl residue 48 are typically targeted for degradation by the 26S proteasome (Thrower et al., 2000). In contrast, modification with a lysyl-63-linked polyubiquitin chain has been implicated in protein activation, stress response, and DNA damage repair (Hoege et al., 2002; Shi and Kehrl, 2003; Zhou et al., 2004).
Ubiquitin conjugation is a multistep reaction, sequentially involving three enzymes referred to as an E1 (ubiquitin-activating enzyme [UBA]), an E2 (ubiquitin-conjugating enzyme [UBC]), and an E3 (ubiquitin ligase; Glickman and Ciechanover, 2002
E2s were originally defined as proteins capable of accepting ubiquitin from an E1 through a thioester linkage via a cysteinyl sulfhydryl group (Glickman and Ciechanover, 2002
Approximately 1,300 genes of the Arabidopsis genome are predicted to encode for E3 components (Smalle and Vierstra, 2004
The ubiquitination pathway modifies a diverse range of proteins, thus placing protein ubiquitination at the center of numerous cellular processes in all eukaryotic species. With over 5% of the Arabidopsis proteome predicted to be involved in the ubiquitination-26S proteasome pathway, it is not surprising that protein ubiquitination is postulated to be involved in many different aspects of plant growth and development (Smalle and Vierstra, 2004
The Arabidopsis Genome Is Predicted to Encode 37 Ubiquitin E2 Proteins
Inspection of the Arabidopsis genome for predicted ubiquitin UBC domain-containing proteins identified a previously unannotated predicted protein (At3g24515) in addition to the 36 compiled either from previous activity assays or from predictions of the annotated genome at the time (Bachmair et al., 2001
Arabidopsis ubiquitin E2 proteins were initially named according to their identity to yeast UBC domain-containing proteins of which 11 of the 13 function with ubiquitin (Bachmair et al., 2001 ). Because there cannot be an association of all UBC numbers between the two species given that Arabidopsis has many more UBC domain-containing proteins than yeast, we classified Arabidopsis UBC proteins into subgroups based on their identity to each other. Phylogenetic analysis was performed, including all yeast UBC proteins that function with ubiquitin, RUB, or SUMO, as well as representative human E2s. Also included in this analysis were Arabidopsis ubiquitin-conjugating E2 enzyme variant (UEV) proteins that contain a UBC domain but lack the active-site cysteinyl residue (Sancho et al., 1998). These include constitutive photomorphogenic AtCOP10 and seven other UEV proteins indicated by Arabidopsis Genome Initiative (AGI) numbers (Fig. 1). Ubiquitin-fold modifier 1-conjugating enzyme (Ufc1) was included to serve as the outgroup. HsUfc1 was recently shown to form a thioester linkage through a conserved cysteinyl residue to the ubiquitin-like protein ubiquitin-fold modifier 1 (Ufm1; Komatsu et al., 2004). Phylogenetic analysis revealed that Arabidopsis UBC domain-containing proteins, excluding the UEV proteins, can be divided into 16 subgroups based on >65% bootstrap support (Fig. 1), with groups I and II functioning in RUB1 and SUMO conjugation pathways, respectively. UEV proteins form several of their own distinct groups, supporting their hypothesized divergence from UBCs with active-site Cys (Fig. 1). Uncharacterized UBC domain-containing proteins, with the exception of the UEV proteins, were given a UBC number based on similarity to previously characterized AtUBCs or to other members of the same subgroup (Table I; Fig. 1).
Most Arabidopsis UBC proteins shared highest similarity with another Arabidopsis protein, suggesting duplications; however, two Arabidopsis E2s displayed higher similarity to yeast and human proteins rather than to other Arabidopsis E2s (Bachmair et al., 2001
Most of the Arabidopsis UBCs do not have additional characterized protein-protein interaction domains. The single exception is UBC27, which has a predicted ubiquitin-binding domain called a ubiquitin-associated domain at its C terminus. Instead, several of the putative E2s contain either an acidic extension, a basic extension, or a predicted transmembrane domain (Bachmair et al., 2001 AtUBC8 clades strongly with seven other Arabidopsis E2s: UBC9 to 12 and 28 to 30 (Fig. 1), which form the largest Arabidopsis E2 subgroup, subgroup VI. AtUBC9 to 11 and 28 are very similar to UBC8, with 92% to 96% amino acid identity. UBC29 and 30 show 87% identity to UBC8, while UBC12 shows 78% identity to UBC8 (Fig. 1). These proteins are more similar to human HsUBC5a to c and yeast ScUBC4 and 5 than to other Arabidopsis UBCs (Fig. 1).
The ability of seven uncharacterized UBC domain-containing proteins to carry thioester-linked ubiquitin was demonstrated (Fig. 2) and is summarized in Table I. For comparison, AtUBC1 (Sullivan and Vierstra, 1993 Another approach to demonstrate ubiquitin E2 activity is to determine whether a UBC domain-containing protein catalyzes E1-dependent protein ubiquitination. Previously uncharacterized Arabidopsis UBCs were expressed in Escherichia coli and tested for their ability to catalyze transfer of ubiquitin to substrate proteins. We were able to test 25 UBC domain-containing proteins for activity; however, we could not produce sufficient soluble protein to assay five others. UBC12, 21, 24, 25, and 31 were insoluble after expression in E. coli and UBC21, 24, and 25 were also insoluble when expressed in cultured insect cells, precluding any activity assays for these five (Table I). Several UBC proteins were sufficiently active in an E3-independent manner under our in vitro assay conditions to prevent their analyses in E3-dependent ubiquitination assays. UBC1 to 6 (data not shown), 20, and 22 (see below) transferred ubiquitin to proteins dependent upon E1, but independent of an E3 ubiquitin ligase. UBC19 exhibited some E3-independent ubiquitination, but it was low enough such that it would not mask E3-dependent activity (Fig. 4; data not shown). In the E3-dependent ubiquitination assays, the previously uncharacterized UBCs, UBC10, 11, 28, 29, 30, 34, 35, and 36, were active in catalyzing polyubiquitination with one or more RING E3 ligases (Fig. 4; Table II; Supplemental Table III), demonstrating that they function as ubiquitin E2s (see below).
Three groups of UBC proteins tested were not active under any assay conditions. All four members of group VII, UBC15 to 18, were expressed and soluble, but exhibited no self-ubiquitination, E3-independent, or E3-dependent ubiquitination (Fig. 4; data not shown; Table II). No similar evidence of any activity was obtained for UBC26 and 37. UBC37 was proteolyzed extensively when expressed in bacteria (data not shown). In summary, at least one representative from nine of the 14 UBC protein subgroups demonstrated E2 activity. Three subgroups had one member that produced sufficient soluble protein to test for activity, but did not exhibit activity: UBC26 in subgroup XI, UBC37 in subgroup XVI, and all members of subgroup VII. Two other subgroups could not be tested because they did not produce sufficient protein for ubiquitination assays (UBC21 and UBC31).
UBC22 consistently showed strong E3-independent activity in in vitro ubiquitination assays when provided with E1 (Fig. 3). The pattern of ubiquitination observed for 6xHis-tagged UBC22 (His-UBC22) does not change with the addition of a number of different types of glutathione S-transferase (GST)-RING E3s (Fig. 3). Conversion of the UBC22 catalytic Cys to Ala results in a loss of protein ubiquitination, thus indicating that the observed activity requires an active UBC22 (data not shown). To determine whether His-UBC22 is self-ubiquitinated, a time-course assay was conducted, followed by western blotting with anti-6xHis antibodies to visualize accumulation of His-UBC22 linked to one or more ubiquitin moieties. Figure 3B shows the accumulation of a higher Mr UBC22 protein over time, indicating the ubiquitination of His-UBC22.
Previous work in our laboratory tested the ability of recombinant RING E3 ligases expressed as GST fusions to catalyze polyubiquitination in vitro with the 6xHis-tagged recombinant AtUBC8 (Stone et al., 2005 The 19 GST-RING proteins inactive with AtUBC8 were tested both with other members of the AtUBC8 subgroup as well as with more diverged UBCs. Two GST-RING proteins, At2g15580 and At2g28840, produced variable results with very low levels of higher Mr ubiquitinated species, so no conclusions regarding the activity of these two were made. The remaining 17 were not active with any of the E2s tested (Table II).
The RING domain is required for E2 interaction and Arabidopsis encodes three canonical RING domain types, as well as five modified RING domain types (Stone et al., 2005 The most generic E2s were members of the UBC8 group. This group was the only group active with both canonical and modified RING types. The relative order of activity was UBC8 = UBC10 = UBC11 > UBC28 > UBC29 > UBC30 (Supplemental Table III), with UBC30 active with the least number of E3s (eight) and UBC8, 10, and 11 active with the most (46, 44, and 46, respectively). UBC8, 10, and 11 were typically active with the same E3s, although there are a few exceptions. Two E3s active with UBC8 were found to be inactive or have questionable activity with UBC10 (At2g28530 and At3g05545; Table II).
To determine in which organs a particular UBC might function, the relative level of expression of 33 E2s on oligoarrays in selected organs and under different developmental conditions was determined as described previously (Ma et al., 2005
These data were compared to that compiled at Genevestigator (Zimmerman et al., 2004 ; https://www.genevestigator.ethz.ch). The expression patterns of UBC28, 29, and 30 were, in general, consistent with the oligoarray data. Similarly, UBC12 expression was very low. In contrast, UBC8, 9, and 11 differed slightly from the data in this study. While UBC8 showed strong expression in leaf tissue, it did not show equivalently strong expression in petals. UBC9 and 11 showed a more general expression pattern. The expression analyses for the other E2 subgroups collectively show expression in most organ and environmental conditions examined (Supplemental Table I). A predicted pseudogene with a UBC domain, At1g35700, showed a relative expression level that was extremely low in all organs (Supplemental Table I). These data give further support to the conclusion that At1g35700 is a pseudogene and was excluded from the UBC nomenclature. Data obtained in our study for representative UBC genes are shown in Supplemental Figure 2A; corresponding data obtained from Genevestigator are included in Supplemental Figure 2B. Interestingly, none of the members within a subgroup showed coordinated expression, suggesting complex and distinct regulation of UBC mRNA levels. Analysis of stress-response data from Genevestigator (https://www.genevestigator.ethz.ch) shows that several E2 mRNAs were up-regulated at least 3-fold in response to various stimuli. Syringolin, a cell death-inducing chemical, induced UBC3, 11, 13, and 27 (data not shown). Several E2 mRNAs were induced by biotic stresses, including UBC17, 20, and 31. The herbicide isoxaben induced UBC20 and 22. UBC6 was induced in response to senescence. UBC16 was induced in response to 6-benzyl adenine and cycloheximide. UBC24 was induced under low nitrogen conditions. UBC31 was induced in high Glc/Suc conditions. UBC32 was induced in abscisic acid treatment and osmotic stress. Once again, distinct regulation of individual members of each UBC subgroup is seen since none showed coordinated changes in mRNA abundance.
The large number of ubiquitin ligases account for the majority of ubiquitination components encoded by the Arabidopsis genome. The translated Arabidopsis genome is predicted to contain over 450 RING-type E3 ligases that can be grouped into eight subgroups based on the type of RING domain (Stone et al., 2005
To get some insight into the relative activity of ubiquitin pathway enzymes, the mRNA levels of the UBCs and RING E3s in different organs and developmental stages were compared. The average expression level of the UBC genes reflects that of the RING genes, with similarly high levels of expression observed in organs such as rosette leaves, petals, and roots (compare Fig. 6, B and C). The majority of RING and UBC genes examined display low levels of expression in a number of organs, including seeds, pistils, siliques, hypocotyls, and cauline leaves. This type of expression pattern is illustrated by UBC1, 6, 27, and 35 (Supplemental Fig. 2A) and RING genes At1g71980 and ARI8 (Fig. 7). With the exception of the RING-D genes, this pattern of expression is observed for the majority of RING genes regardless of RING domain type. The RING genes encoding RING-D domain-containing proteins exhibited the lowest average expression levels in all organs and were expressed predominantly in seeds and petals (data not shown).
To assist in validating the relative expression levels revealed by our analysis, we determined whether our results correlated with previously published data on the expression levels of known RING genes. To do this, we examined the expression levels of the ARI RING gene family whose mRNA abundance was determined previously (Mladek et al., 2003
The ubiquitin E1, E2, and E3 ligase enzymes are the enzymatic core of the ubiquitination pathway. The E2-E3 complex interacts with the substrate, catalyzing ubiquitin addition to the substrate. Thus, the expression, activity, localization, and selectivity of ubiquitin E2s and E3s are important parameters that can serve to regulate ubiquitination. The E2-E3 combination used in the ubiquitination reaction can also influence the fate of the substrate through determining the extent and nature of ubiquitin addition. The majority of substrates observed to date are modified by the attachment of a Lys-48-linked ubiquitin chain, which targets the substrate for degradation by the 26S proteasome. Substrates modified with other types of polyubiquitin chains linked via ubiquitin Lys-6, -11, -29, and -63, or modified with a single ubiquitin, face different or unknown fates. For example, the heterodimeric E2 UBC13/methyl methane sulfonate sensitivity 2 functions with the RING E3 TNF receptor-associated factor 6 to catalyze the formation of Lys-63 polyubiquitin chains involved in protein activation (Deng et al., 2000 ). Monoubiquitination of histone H2B via the UBC2/radiation insensitive 6-Brefeldin A sensitivity 1 E2-E3 pair is required for subsequent methylation of specific Lys on histone H3 (Wood et al., 2003). The function of other ubiquitin linkages in vivo is currently unknown. Specific E2-E3 combinations are also involved in specific functions. In yeast, UBC2/Rad6 along with the RING-type E3s Rad5 and Rad18 are implicated in DNA repair (Jentsch et al., 1987; Bailly et al., 1997). Therefore, identifying and characterizing the components and how they interact in catalyzing ubiquitination is critical for the understanding of E2-E3 combinations required for certain processes.
In vitro ubiquitination assays with RING E3 ligases, while operating without a physiological substrate, appear to be accurate reflections of in vivo activity. The requirements for in vivo and in vitro activity, where compared, appear to be identical. For example, mutations in the mammalian RING proteins Mdm2 (Fang et al., 2000
The E2 enzymes showed little specificity toward different types of RING domain-containing proteins. UBC8, 10, 11, and 28, which show considerable protein sequence similarity to each other, function in vitro with a large number of RING E3 enzymes assayed, including RING E3s with modified RING domains. The high level of expression observed for UBC8, 10, 11, and 28 correlates with the breadth of activity and suggests that these proteins may perform a general ubiquitination function in vivo. Other members of the UBC8 family were not as promiscuous. UBC29 and 30 did not show activity with any of the E3s that contain modified RING domains and only functioned with a fraction of the other RING E3s. UBC29 and 30 are not as similar to UBC8 as other family members and this may account for restricted activity of these E2 enzymes. The changes in the UBC domain of UBC29 and 30 may prevent them from interacting with the modified RING domains (Stone et al., 2005 Greater specificity is observed for UBC35 and 36; both E2s functioned mainly with E3s containing RING-H2 domains. Although UBC35 and 36 are highly similar and functioned with the same set of RING E3s in vitro, their expression patterns are different. Therefore, each E2 may promote protein ubiquitination in different organs or at different times during development. UBC34 functioned with only a few RING-H2 domains. This may be due to the amino acid sequence of the UBC domain that may only allow enhancement of ubiquitin transfer in combination with certain RING domains. In addition, UBC34 shares the greatest identity with E2s known to be endoplasmic reticulum (ER)-associated via their C-terminal transmembrane domain. However, it remains to be determined whether AtUBC34 or the RING proteins it works with in vitro actually localize to the ER and whether it serves a role in ER-associated degradation. The RING-H2 is the most prevalent type of RING protein, so it is not surprising that the majority of E2s function with proteins containing this type of RING domain.
Another interesting observation with E2-E3 activity concerns the types of E2s we found to be active in our study. E2s of group VI and XV showed activity with the greatest number of E3s in this study. Members of these groups consist only of the core catalytic UBC domain. The remainder of the E2s of Arabidopsis, with the exception of UBC21, contains extensions of varying lengths outside the UBC domain or within the catalytic core itself, as in the case of group V members (for review, see Bachmair et al., 2001
Limited studies of E2 activity with other types of E3 ligases have been performed. Mudgil et al. (2004) The E2 enzymes that did not function in our in vitro assays with RING E3s can be divided into two classes, those that showed some E3-independent activity and those that did not. No E2 activity at all was observed for the latter class and the possible reasons for this are multiple. These proteins may not fold properly after expression in E. coli; they may require another type of E3 other than the RING types tested here; they may function with only a specific RING protein not yet tested; or they may be unable to transfer ubiquitin to nonphysiological substrates. Another possibility is that they may require cofactors such as UEVs that are not present in the in vitro assay to promote protein ubiquitination. The fact that these E2s may not conjugate ubiquitin, but instead conjugate a ubiquitin-like protein and require a different E1, should also be considered.
For the former class, the E3-independent conjugation observed for a number of E2s suggests that these enzymes are capable of E1-dependent ubiquitin thioester formation. However, the inability of these E2s to catalyze E3-dependent polyubiquitination could be because they may not be able to function with the RING E3s tested, may not be able to transfer to a nonphysiological substrate, or may require a cofactor or UEV for E3-dependent activity. Self-ubiquitination of one E2, AtUBC22, was easily detected and occurred both in the presence and absence of a RING E3. Whether self-ubiquitination of AtUBC22 or other E2s represents an additional level of regulation in vivo in Arabidopsis is not known. However, there are a few examples from other organisms that suggest self-ubiquitination may serve an in vivo function. Yeast Cdc34p self-ubiquitination is thought to regulate its own levels (Skowyra et al., 1999
To examine whether ubiquitin pathway enzyme expression at the mRNA level was coordinated, we analyzed E2 expression patterns along with that of over 400 RING E3 genes. Overall, specific UBC genes show a generic expression pattern with no UBC gene showing a 3-fold higher level of expression in a single specific organ based on our microarray analysis or data available through Genevestigator (Zimmerman et al., 2004 The ubiquitination system is hierarchical in that few E2 enzymes (dozens) exist in comparison to the large number of E3s (hundreds). In the case of Arabidopsis, two E1s and as many as 34 to 37 ubiquitin E2 enzymes are utilized by hundreds of different E3 enzymes to facilitate ubiquitination of their cognate target proteins. Therefore, it would not be surprising to find a requirement for additional proteins to help guide the specificity of E2-E3 interactions.
Plant Material Seeds from Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col-0) were either sown on soil and grown under photoperiodic cycles of 16 h light and 8 h dark at 16°C with 50% relative humidity or seeds surface sterilized with 30% (v/v) bleach and 0.1% (v/v) Triton X-100 were grown on 1% (w/v) agar with 1x Murashige and Skoog and 1% (w/v) Suc under continuous light.
The UBC domain of Arabidopsis UBC8 was used in BLAST searches against the complete nonredundant Arabidopsis genome (TAIR, April 16, 2003; http://www.arabidopsis.org). The Simple Modular Architecture Research Tool database was used to analyze retrieved sequences (version 4.0, May 28, 2004; http://smart.embl-heidelberg.de) followed by manual inspection to confirm the presence of the complete UBC domain. The previously named UBC12 (Girod et al., 1993
The ClustalX program was used to generate an alignment of the UBC protein sequence. The alignment was generated using a PAM350 protein matrix, with gap opening and gap extension penalty parameters of 35.0 and 0.75, respectively, in pairwise alignment and 15.0 and 0.3, respectively, in the multiple alignments (Thompson et al., 1997
The dataset described in Ma et al. (2005)
Arabidopsis E2 cDNAs were cloned by RT reactions followed by PCR to amplify the predicted ORF for each UBC. RNA isolated from either Arabidopsis ecotype Col-0 10-d-old seedlings or floral tissue from 6- to 7-week-old plants was used. The Qiagen RNeasy plant RNA extraction kit was used to isolate total RNA, according to the manufacturer's instructions. Coding regions for 35 UBCs were first introduced into the Gateway entry vector, pDONR (Invitrogen), and the DNA sequence was determined. Attempts to isolate cDNA for UBC23 failed. Sequences of each UBC cDNA were compared to the predicted ORF available on TAIR (http://www.arabidopsis.org), the Arabidopsis genome annotation database. Sequences obtained for UBC7 were different from TAIR predictions, as previously reported (Bachmair et al., 2001
6xHis UBC fusions were expressed in E. coli strain BL21 AI or BL21-pLysS. Transformed cells were grown at 37°C for 2 to 3 h or to an OD600 of 0.4 to 0.6 before induction with 0.2% Ara or 0.5 to 1.0 mM isopropylthio-
GST-RING fusions were expressed and purified from bacterial extracts as described in Stone et al. (2005)
Thioester assays were performed in a total reaction volume of 30 µL, consisting of 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 mM ATP, 100 ng rabbit E1 (Boston Biochem), 500 ng of recombinant E2s (2 µg for AtUBC32), and 10 µg ubiquitin (Boston Biochem). Reactions were split after incubation for 5 min at 37°C and terminated by SDS sample buffer with dithiothreitol (DTT) or 8 M urea sample buffer without DTT. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DQ027017 to DQ027050 (see Table I).
We would like to thank Christina Tan for assistance with the ubiquitination assays and Mandy Hsia for recombinant E1; Andy Troy and Michael Kerber for their assistance with E2 analysis; and Jemma Jowett and Kate Dreher for comments on the manuscript, as well as other members of the Callis laboratory for helpful discussions. Received July 1, 2005; returned for revision September 30, 2005; accepted October 17, 2005.
1 This work was supported by the National Science Foundation (2010 grant nos. MCB–00115870 and MCB–0519970 to J.C. and X.-W.D.); by the National Institutes of Health (grant no. GM–47850 to X.-W.D. and grant no. GM0007377–27 to E.K.); and by the Natural Sciences and Engineering Research Council of Canada and the International Human Frontier Science Program Organization fellowships (to S.L.S.). Y.J. was the recipient of a Yale University Joseph F. Cullman Jr. fellowship and L.M. was a long-term postdoctoral fellow of the Human Frontier Science Program. 
2 These authors contributed equally to the paper. 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: Judy Callis (jcallis{at}ucdavis.edu).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.105.067983. * Corresponding author; e-mail jcallis{at}ucdavis.edu; fax 530–752–3085.
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ABSTRACT
RESULTS
-galactoside, respectively, for 2 to 3 h at 37°C. Cells were harvested by centrifugation and lysed in lysis buffer containing 25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.01% Triton X-100, and 5 mM imidazole. For purification, nickel-nitrilotriacetic acid agarose (Qiagen) was added to cleared lysates and incubated for 1 h at 4°C. Beads were then washed four times with wash buffer containing 25 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.01% Triton X-100, and 5 mM imidazole. 6xHis fusion proteins were eluted with elution buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.01% Triton X-100 supplemented with 300 mM imidazole. Glycerol was added to the eluted protein to a final concentration of 40%. Proteins were stored at –80°C until needed. 
