The Ubiquitin-Related Protein RUB1 and Auxin Response in Arabidopsis

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Science  12 Jun 1998:
Vol. 280, Issue 5370, pp. 1760-1763
DOI: 10.1126/science.280.5370.1760


The AXR1 (auxin-resistant) protein, which has features of the ubiquitin-activating enzyme E1, is required for normal response to the plant hormone auxin in Arabidopsis thaliana. ECR1 functions together with AXR1 to activate members of the RUB/NEDD8 family of ubiquitin-related proteins. Extracts from mutant seedlings lacking AXR1 did not promote formation of the RUB-ECR1 thiolester, indicating that AXR1 is the major activity in this tissue. AXR1 was localized primarily to the nucleus of dividing and elongating cells, suggesting that the targets of RUB modification are nuclear. These results indicate that auxin response depends on RUB modification of one or more nuclear proteins.

Plant growth and development depends on the coordinated action of several phytohormones. One of these hormones, indole-3-acetic acid (IAA or auxin), has been implicated in the control of diverse developmental processes (1). Auxin regulates these processes by promoting changes in cell division and cell elongation (2). Recessive mutations in the AXR1 gene result in decreased auxin response, including reduced auxin-induced gene transcription (3). A screen for suppressors of the axr1 phenotype resulted in identification of the SAR1 gene. By genetic criteria,SAR1 acts downstream of AXR1 (4). Mutations in another gene, TIR1, also result in a defect in auxin response (5). Genetic studies indicate thatAXR1 and TIR1 function in the same or overlapping pathways.

The molecular characterization of AXR1 and TIR1implicates the ubiquitin pathway in auxin response. AXR1encodes a protein related to the NH2-terminal half of the ubiquitin-activating enzyme, E1 (6), and proteins related to AXR1 can be found in yeast and mammals (7). Despite extensive similarity with E1, all lack the active-site cysteine required for thiolester bond formation with ubiquitin. TIR1encodes an F-box protein related to yeast Grr1p and human SKP2 (5). F-box proteins have been shown to function in a ubiquitin-ligase complex (E3) called the SCF (for Skp1 Cdc53 F-box). These results suggest that auxin response is mediated by ubiquitin or a ubiquitin-like protein modification.

Ubiquitin (UBQ) is one of the most conserved proteins among eukaryotes. Covalent attachment of UBQ to other proteins targets them for degradation by the 26S proteosome (8). UBQ is activated by E1 in an adenosine 5′-triphosphate (ATP)–dependent reaction in which a thiolester bond is formed between the COOH-terminus of UBQ and a cysteine within the E1 enzyme. The UBQ moiety is transferred to a cysteine residue on a UBQ-conjugating enzyme (E2). Finally, UBQ is covalently attached to a target protein by an isopeptide linkage directly from the E2 or by a UBQ-protein ligase such as the SCF (9).

Two conserved families of UBQ-related proteins (Smt3p/SUMO and RUB/NEDD8) have also been identified (10). InSaccharomyces cerevisiae, activation of Smt3p is performed by the E1-like heterodimer Aos1p/Uba2p rather than E1 (11). Aos1p is a member of the AXR1 family, while Uba2p is similar to the COOH-terminal half of E1 and contains the active-site cysteine. Smt3p is conjugated in vivo to other proteins, but these targets are yet to be identified (11). A protein related to Smt3p in humans, called SUMO-1, is covalently attached to the RanGAP1 (12) and PML proteins (13). This posttranslational modification affects the subcellular localization of these proteins rather than their metabolic stability.

Another member of the AXR1 family, yeast Enr2p, is required for Rub1p (for “related to ubiquitin”) conjugation to the cell-cycle protein Cdc53p (14). Enr2p functions as a second bipartite activating enzyme, together with Uba3p (14)

Because several RUB genes have been identified in Arabidopsis thaliana (15), we investigated whether AXR1 was required for RUB activation. Through homology with Uba2p, we identified an expressed sequence tag (EST) from Arabidopsis that encodes a 55-kD protein (ECR1) related to the COOH-terminus of the E1 enzyme. Based on similarity to Uba2p and Uba3p, ECR1 contains a possible active-site cysteine at position 215 (Fig.1A). The ECR1 gene was mapped, using the recombinant inbred (RI) lines (16), to position 7.4 on chromosome 5, between CHS and g4560 (Fig. 1B). When recombinant AXR1 and ECR1 were mixed and incubated with radiolabeled RUB1 and ATP (17), a species of 65 kD size was observed (Fig.2). This product was destroyed upon incubation with the reducing agent dithiothreitol (DTT) indicating that formation of the labeled complex involves a thiolester bond (Fig. 2). No thiolester products were generated when either AXR1 or ECR1 were omitted from the reaction. To investigate the specificity of the AXR1-ECR1 heterodimer, we determined if UBQ and NEDD8, a mouse protein with 84% identity to RUB1, could form a thiolester bond with ECR1. We found that AXR-ECR1 was capable of activating NEDD8 (Fig. 2), but not UBQ (18). To determine whether the thiolester bond is formed with AXR1 or ECR1, we used a glutathione S-transferase (GST)–ECR1 fusion protein in the thiolester assay. In this reaction, the DTT-sensitive product was detected at a size of ∼85 kD (Fig. 2), indicating that the thiolester bond is formed between the COOH-terminus of RUB1 and ECR1. Furthermore, ECR1 mutants in which Cys215was replaced with Ala were unable to activate RUB1 or NEDD8 (Fig. 2). This suggests that Cys215 forms the thiolester bond with the COOH-terminus of RUB1. On the other hand, the axr1-3mutation, which replaces the Cys154 with Ala, also prevents thiolester formation between ECR1 and the RUB/NEDD8 proteins (Fig. 2). This missense mutation in the AXR1 gene results in reduced auxin responses in the axr1-3 plants, confirming that the auxin defect is related to a decrease in RUB activation (6).

Figure 1

ECR1 (GenBank AF051135) is related to yeast Uba2p (GenBank U32274) and Uba3p (EMBL Y16891) (24). (A) Alignment of ECR1 with Uba2p and YPR066W. Boxed residues are identical. The conserved active-site cysteine is indicated with an asterisk. (B) Map position of ECR1 on chromosome 5.

Figure 2

Conjugation of RUB1 and NEDD8 with ECR1. Radiolabeled RUB1 or NEDD8 was incubated with recombinant AXR1 and ECR1 in a thiolester reaction (17). The reaction was terminated with SDS loading buffer in the absence or presence of DTT. The position of molecular size markers is indicated beside the gel. The arrow indicates the ECR1-RUB1 or ECR1-NEDD8 complex at ∼65 kD. Radiolabeled RUB1 was incubated with the recombinant GST-ECR1 protein in a thiolester assay. The asterisk indicates the DTT-sensitive product GST-ECR1-RUB at 85 kD. Replacement of Cys215 in ECR1 or Cys154 in AXR1 with Ala were performed with the Stratagene direct mutagenesis kit. Neither AXR1-ECR1-C215 nor AXR1-C154-ECR1 heterodimers were able to form any DTT-sensitive product.

To investigate RUB activation in various auxin response mutants, we developed an in vitro RUB1 activation assay (19). When radiolabeled RUB1 was incubated with total protein extract from wild-type, axr1-12 (4), tir1-9(6), sar1-1 (5), or axr1-12 sar1-1 double mutant plants, two DTT-sensitive products at ∼50 and 65 kD were detected in wild-type, tir1-9, andsar1-1 extracts, but not in axr1-12 andaxr1-12 sar1-1 extracts (Fig.3A). The 65-kD product is likely the ECR1 protein conjugated to one molecule of RUB1, and the 50-kD protein could be either an E2-RUB1 or E3-RUB1 thiolester. To confirm that these products were AXR1-dependent, recombinant AXR1 protein was added to axr1-12 protein extract. This resulted in the reappearance of the DTT-sensitive 65- and 50-kD products (Fig. 3B). These results suggest that AXR1 is responsible for the majority of RUB1 activation, at least in seedlings. Further, our results show that thesar1-1 suppressor does not act by restoring RUB activation in axr1 plants. This indicates that sar1 acts to bypass the effects of the axr1 mutation and is consistent with downstream function for SAR1 (4).

Figure 3

RUB1-thiolester formation in protein extracts is dependent on AXR1. (A) Radiolabeled RUB1 was incubated with total plant protein from mutants as described (19). Arrows indicate two RUB1 thiolester adducts that were destroyed with DTT incubation (18). Free RUB1 protein ran off the gel. (B) Total protein extract fromaxr1-12 mutant plants was incubated with RUB1. The biochemical phenotype (thiolester adducts at 50 and 65 kD) was restored when increasing amounts of recombinant AXR1 were supplied to the thiolester reaction.

In addition to the DTT-sensitive products described above, we also observed a broad DTT-resistant band of labeled protein migrating above the free RUB1 (Fig. 2 and Fig. 3, A and B). When the reaction was performed in the presence of [α-32P]ATP and unlabeled RUB1, a labeled protein of this size was evident on the gel (18). This labeled protein was not present when the reaction was done with [γ-32P]ATP. This result argues strongly that the smaller product is RUB1–adenosine 5′-monophosphate, an intermediate in RUB1 activation. Several other high molecular weight DTT-resistant products were also observed upon prolonged exposure. These proteins are likely to be RUB1 conjugates, indicating that the plant extracts are also capable of isopeptide bond formation between RUB1 and target proteins (18).

Analysis of AXR1 genes expression by in situ hybridization indicate that AXR1 expression is restricted to dividing and elongating cells (18). To determine the cellular location of AXR1, tissue sections of Arabidopsis seedlings were treated with AXR1 antiserum (20). Staining was associated primarily with the nuclei of dividing and expanding cells (Fig.4, A and B). This staining was not detected in wild-type sections treated with preimmune serum oraxr1-12 mutant sections treated with AXR1 antiserum, which indicates that the AXR1 antiserum is specific (Fig. 4, C and D).

Figure 4

AXR1 is located in the nucleus. Sections of 5-day-old Arabidopsis seedlings were used for immunolocalization of AXR1 protein by the method described (20). (A) Wild-type sections were labeled with AXR1 antiserum, which detected AXR1 protein in the nuclei of shoot meristem (sm) and expanding cotyledon (c) cells. (B) Higher magnification of expanding cotyledon cells showing the nuclear localization of AXR1. The identity of the nuclei in these sections was confirmed by staining with 4′,6′-diamidino-2-phenylindole (18). (C) No stain was observed with preimmune serum on wild-type sections. (D) Sections from anaxr1-12 plant were labeled with AXR1 antiserum. Theaxr1-12 mutation introduces a stop codon in the exon 11 of the AXR1 gene (6). AXR1 antiserum did not detect this truncated protein. Bars represent 100 μm in (A), (C), and (D), and 20 μm in (B).

On the basis of these results, we propose that auxin response is mediated, at least in part, through modification of one or more proteins by RUB1 or a related protein. The nuclear localization of AXR1 suggests that these targets are probably nuclear proteins, which is consistent with studies in mammalian cells which show that most NEDD8-modified proteins are nuclear (21). Because the RUB/NEDD8 protein is conserved among the three eukaryotic kingdoms, it seems likely that RUB/NEDD8 modification will have an important regulatory function in all eukaryotes. In support of this, a human homolog of AXR1 called APP-BP1 interacts with the cytoplasmic domain of the amyloid precursor protein (APP), suggesting a role in APP function (7). In addition, a temperature-sensitive hamster cell line called ts41 carries a mutation in a hamster homolog of AXR1called SMC1 (22). Mutations in SMC1result in a complex cell-cycle defect at the nonpermissive temperature (22). Further studies will be required to determine the biochemical function of RUB/NEDD8 modification in both plant and animal systems.

In yeast, the most abundant Rub1p-modified protein is Cdc53p (14). Genetic evidence suggests that Rub1p modification regulates the activity of SCFCdc4, the E3 responsible for conjugation of UBQ to the CDK inhibitor Sic1p at the G1-to-S phase transition. It is possible that RUB1 has a similar function in plant cells. For example, theArabidopsis F-box protein TIR1 may be part of an SCF complex that is required for the degradation of negative regulators of auxin response. RUB1 may modify the activity of this SCF, perhaps in response to auxin (23). A homolog of CDC53 exists inArabidopsis, and it will be interesting to see if CDC53 is a target of RUB1 conjugation in plants.

  • * Present address: Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148, USA.

  • To whom correspondence should be addressed. E-mail: mestelle{at}


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