Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function

Abstract

The ubiquitin-related protein RUB/Nedd8 is conjugated to members of the cullin family of proteins in plants, animals, and fungi. In Arabidopsis, the RUB conjugation pathway consists of a heterodimeric E1 (AXR1-ECR1) and a RUB-E2 called RCE1. The cullin CUL1 is a subunit in SCF-type ubiquitin protein ligases (E3s), including the SCF(TIR1) complex, which is required for response to the plant hormone auxin. Our previous studies showed that conjugation of RUB to CUL1 is required for normal SCF(TIR1) function. The RING-H2 finger protein RBX1 is a subunit of SCF complexes in fungi and animals. The function of RBX1 is to bind the ubiquitin-conjugating enzyme E2 and bring it into close proximity with the E3 substrate. We have identified two Arabidopsis genes encoding RING-H2 proteins related to human RBX1. Studies of one of these proteins indicate that, as in animals and fungi, Arabidopsis RBX1 is an SCF subunit. Reduced RBX1 levels result in severe defects in growth and development. Overexpression of RBX1 increases RUB modification of CUL1. This effect is associated with reduced auxin response and severe growth defects similar to those observed in axr1 mutants. As in the axr1 mutants, RBX1 overexpression stabilizes the SCF(TIR1) substrate AXR2/IAA7. The RBX1 protein is a component of SCF complexes in Arabidopsis. In addition to its direct role in SCF E3 ligase activity, RBX1 promotes the RUB modification of CUL1 and probably functions as an E3 ligase in the RUB pathway. Hypermodification of CUL1 disrupts SCF(TIR1) function, suggesting that cycles of RUB conjugation and removal are important for SCF activity.

Figure 1.

RBX Genes in Arabidopsis. (A) Alignment of RBX proteins in Arabidopsis (At) with human (Hs) and budding yeast (Sc) orthologs. Arabidopsis RBX1a is encoded by At5g20570 and RBX1b is encoded by At3g42830. Identical residues in all five proteins are indicated with asterisks. The predicted zinc binding Cys residues are boxed. (B) RNA gel blot showing RBX1a expression in all tissues examined. Fifteen micrograms of total RNA from 30-day-old soil-grown plants was loaded in each lane.

Figure 2.

RBX1a Is a Component of the SCF Complex in Arabidopsis. (A) Recombinant GST-RBX1a or GST was added to extracts prepared from Arabidopsis seedlings. The GST pulldowns were examined for the presence of CUL1 by protein gel blot analysis using α-CUL1 antibody. The two CUL1 species are unmodified and RUB-modified protein. The three lanes are from the same autoradiogram. (B) As in (A) except that extract was prepared from a tir1 line expressing TIR1-myc. CUL1, TIR1-myc, and GST-RBX1a were detected in the pulldown by protein gel blot analysis. The left lane is a control pulldown using GST alone.

Figure 3.

Reduced Expression of RBX1 Results in Severe Growth Defects. (A) Nine-day-old Col(α-RBX1) seedling. (B) Nine-day-old Col(pROKII) seedling. (C) Thirty-five-day-old Col(α-RBX1). Bar = 1 cm. (D) Higher magnification of the inflorescence of a Col(α-RBX1) plant showing a floral cluster.

Figure 4.

Effects of RBX1a Overexpression on Plant Growth and Development. (A) RNA gel blot showing increased RBX1a expression in transgenic lines. RNA was extracted from 7-day-old Columbia (lane 1), Col(35S::RBX1)2d (lane 2), and Col(35S::RBX1)1a (lane 3) seedlings. (B) to (D) Five-day-old dark-grown seedlings of Columbia (B), Col(35S::RBX1)1a (C), and Col(35S::RBX1)2d (D). (E) to (H) Twenty-three-day-old plants of Columbia (E), axr1-12 (F), Col(35S::RBX1)1a (G), and Col(35S::RBX1)2b (H). (I) to (K) Thirty-five-day-old plants of Columbia (I), Col(35S::RBX1)1a (J), and Col(35S::RBX1)2d (K). (L) Morphology of (from left to right) Columbia, axr1-12, Col (35S::RBX1)1a, and Col(35S::RBX1)2b flowers. (M) Morphology of (from left to right) Columbia, Col(35S::RBX1)1a, and Col(35S::RBX1)2b siliques. (N) Forty-day-old plants of axr1-12 (left) and axr1-12(35S::RBX1)1a (right). Bars = 1 cm.

Figure 5.

RBX1a Overexpression Results in Reduced Auxin Response and Stabilization of AXR2/IAA7. (A) Response of seedling roots to auxin. Columbia (open squares), Col(35S::RBX1)1a (open circles), and Col(35S::RBX1)2e (closed circles). sd was <20% for each data point. (B) RNA gel blot showing DEX-regulated accumulation of RBX1a RNA. RNA was isolated from 7-day-old seedlings treated with DEX for 12 h. Lane 1, Col(BA3::GUS); lane 2, Col(BA3::GUS,GVG::RBX1)a; lane 3, Col(BA3::GUS,GVG::RBX1)b. (C) Seven-day-old Col(BA3::GUS,GVG::RBX1)a seedlings were pretreated with either Arabidopsis thaliana medium + sucrose buffer (left) or DEX (right) for 12 h followed by treatment with 0.2 μM 2,4-D for 8 h and staining for GUS activity. (D) Pulse-chase analysis of AXR2/IAA7. Seedlings were treated as described in Methods. Labeled AXR2/IAA7 was immunoprecipitated as described by Gray et al. (2001). The half-life of AXR2/IAA7, based on four independent experiments, was 11.3 ± 1.4 min in wild-type and 14.7 ± 2.0 min in Col(35S::RBX1)1a seedlings.

Figure 6.

Increased RUB-CUL1 Formation in 35S::RBX1 Lines Is AXR1 Dependent. Extracts were prepared from 7-day-old seedlings and examined by protein blot analysis using α-CUL1 antiserum. Lane 1, Columbia; lanes 2 and 3, Col(35S-RBX1)1a; lane 4, axr1-12; lanes 5 and 6, axr1-12 (35S-RBX1)1a. The lines indicate CUL1 and RUB-CUL1. The control panels (bottom) show levels of an unknown cross-reacting protein.