Characterization of Arabidopsis and rice DWD proteins and their roles as substrate receptors for CUL4-RING E3 ubiquitin ligases

Abstract

A subset of WD40 proteins that contain a DWD motif (for DDB1 binding WD40) is reported to act as substrate receptors for DDB1-CUL4-ROC1 (for Damaged DNA Binding 1-Cullin 4-Regulator of Cullins 1) based E3 ubiquitin ligases in humans. Here, we report 85 Arabidopsis thaliana and 78 rice (Oryza sativa) proteins containing the conserved 16-amino acid DWD motif. We show by yeast two-hybrid and in vivo coimmunoprecipitation that 11 Arabidopsis DWD proteins directly interact with DDB1 and thus may serve as substrate receptors for the DDB1-CUL4 machinery. We further examine whether the DWD protein PRL1 (for Pleiotropic Regulatory Locus 1) may act as part of a CUL4-based E3 ligase. PRL1 directly interacts with DDB1, and prl1 and cul4cs mutants exhibited similar phenotypes, including altered responses to a variety of stimuli. Moreover, AKIN10 (for Arabidopsis SNF1 Kinase Homolog 10) was degraded more slowly in cell extracts of prl1 and cul4cs than in cell extracts of the wild type. Thus, both genetic and biochemical analyses support the conclusion that PRL1 is the substrate receptor of a CUL4-ROC1-DDB1-PRL1 E3 ligase involved in the degradation of AKIN10. This work adds a large new family to the current portfolio of plant E3 ubiquitin ligases.

Figure 1.

Core Sequence of the DWD Motif and the Structure of Arabidopsis DWDs. (A) Conserved amino acids of DWD motifs in various DWD proteins. Hy, hydrophobic amino acids; Sm, small amino acids. (B) Structure of DWD proteins in Arabidopsis and rice. Each type is labeled A to N corresponding to groups in Supplemental Figures 1 and 2 online. S_T Kc, catalytic domain of Ser/Thr protein kinase. (C) Numbers of DWD proteins and DWD motifs in Arabidopsis and rice.

Figure 2.

Phylogenetic Trees Based on DWD Motifs in Arabidopsis and Rice. (A) A phylogenetic tree constructed from the 101 Arabidopsis DWD motifs and divided into subgroups A1, A2, B, C, and D. Numbers indicate percentage values after 1000 replications; values below 50% are not shown. The bar represents the amino acid substitutions per site for a unit branch length. (B) Phylogenetic tree generated with the 96 rice DWD motifs divided into subgroups E1, E2, E3, E4, F, and G. Gene names are from The Institute for Genomic Research (TIGR) Rice Genome Annotation.

Figure 3.

Interactions between DDB1a and Arabidopsis DWD Proteins Detected by Yeast Two-Hybrid Assays. Growth of yeast strains harboring the indicated DWD protein as prey and DDB1a as bait on X-gal and plates lacking leucine is shown, together with their β-galactosidase activity. CUL4 was used as a positive control since it is known to bind DDB1. Empty vector and GFP proteins were used as negative controls. Values are means ± sd (n = 3). At4g15900m is an At4g15900 mutant whose Arg residues at positions 290 and 332 were changed into His residues.

Figure 4.

In Vivo Interaction between DDB1b and DWD Proteins and between CUL4 and PRL1. (A) In vivo interaction of DDB1b with DWD proteins. Total protein (left two lanes) or extracts immunoprecipitated for FLAG (right two lanes) from transgenic plants overexpressing FLAG-DDB1b detected with antibodies to the indicated DWD proteins (right labels). Total, 5% of the crude extract used for coimmunoprecipitation assays; F, FLAG-DDB1b/ddb1a transgenic plants; F-TRIP-1, FLAG-TRIP-1 trangenic plants. The arrowhead on the α-CUL4 panel represents the position of the CUL4 protein. The asterisk and arrowhead on the anti-TRIP-1 panel indicate the positions of FLAG-TRIP-1 and endogenous TRIP-1 protein, respectively. The immunoblot using anti-RPN6 was used as a negative control. (B) In vivo interaction of CUL4 with PRL1. As above, total protein (left two lanes) or extracts immunoprecipitated for FLAG (right two lanes) from transgenic plants overexpressing FLAG-CUL4 detected with antibodies to the indicated DWD proteins (right labels). Total, 5% of the crude extract used for coimmunoprecipitation assays; F-CUL4, FLAG-CUL4 trangenic plants. The immunoblot using anti-RPN6 was used as a negative control.

Figure 5.

Coimmunoprecipitation of DDB1b and Arabidopsis DWDs in FLAG-DDB1b/ddb1a/MYC-DWD Transgenic Plants. (A) Coimmunoprecipitation of DDB1b and CUL4 in FLAG-DDB1b/ddb1a transgenic plants. Arrowhead indicates CUL4 protein. Total, 5% of the crude extract used for coimmunoprecipitation assays; F, FLAG-DDB1b/ddb1a transgenic plants. (B) Coimmunoprecipitation of DDB1b and Arabidopsis DWDs in FLAG-DDB1b/ddb1a/MYC-DWD transgenic plants. Arrowhead on At1g04140-MYC panel indicates the position of At1g04140-MYC. Total, 5% of the crude extract used for coimmunoprecipitation assays.

Figure 6.

Phenotypic Comparison of cul4cs and prl1 at the Seedling and Adult Stages. (A) Three-day-old light-grown wild-type (Columbia [col]), cul4cs, and prl1 seedlings. (B) Five-week-old wild-type, cul4cs, and prl1. (C) Wild-type, prl1, and cul4cs siliques. (D) Rosette leaves from wild-type, prl1, and cul4cs plants.

Figure 7.

Phenotypes of Wild-Type, cul4cs, and prl1 Seedlings Treated with Sucrose, Glucose, and Various Hormones. (A) Effects of 6% sucrose on wild-type, cul4cs, and prl1 light-grown seedlings. (B) Effects of 4% glucose on light-grown seedlings. (C) Effects of 0.5 μM kinetin on light-grown seedlings. (D) Effects of 0.1 μM ABA on light-grown seedlings. (E) Effects of 1 μM ACC on dark-grown seedlings. Bars = 2 mm in (A) to (E). (F) Root lengths of wild-type, cul4cs, and prl1 seedlings after treatment with sucrose, glucose, cytokinin, or ABA. Values are means ± sd (n = 20). (G) Hypocotyl lengths of wild-type, cul4cs, and prl1 seedlings after treatment with ACC. Values are means ± sd (n = 20).

Figure 8.

Transcriptional Derepression of Various Sugar-, Cytokinin-, and ABA-Responsive Genes in prl1 and cul4cs. (A) Expression of various sugar-, cytokinin-, and ABA-responsive genes in the wild type, prl1, and cul4cs. (B) Relative expression of various sugar-, cytokinin-, and ABA-responsive genes in the wild type, prl1, and cul4cs.

Figure 9.

Comparison of PRL1 Protein and mRNA Levels in Wild-Type, cul4cs, and prl1 Seedlings. Total proteins ([A] and [C]) and RNA (B) were extracted from wild-type, cul4cs, and prl1 seedlings. In (B), total RNA was reverse-transcribed, and then PCR was performed with specific forward and reverse primers for PRL1. Anti-RPN6 and RT-PCR with RPN6a-specific primers were used as controls for equivalent loading for the protein and RT-PCR gels, respectively. (A) PRL1 and CUL4 protein levels in 3-d-old wild-type, cul4cs, and prl1 seedlings. (B) PRL1 mRNA levels in 3-d-old wild-type, cul4cs, and prl1 seedlings. (C) PRL1 and CUL4 protein levels in 7-d-old wild-type, cul4cs, and prl1 seedlings.

Figure 10.

Degradation of His₆-Tagged AKIN10 in Cell Extracts of Wild-Type, cul4cs, and prl1 Seedlings and the Expression Level of AKIN10 Protein in Wild-Type, cul4cs, and prl1 Seedlings. (A) Effect of MG132 on cell-free degradation of AKIN10. His₆-tagged AKIN10 protein was incubated in extracts (20 μg) of 3-d-old wild-type seedlings in the presence or absence of 10 μM MG132 for the indicated times at 30°C. His₆-tagged AKIN10 protein levels were determined by protein gel blotting with anti-His antibody. (B) AKIN10 protein degradation in extracts of prl1, cul4cs, and wild-type seedlings. His₆-tagged AKIN10 proteins were incubated in extracts (20 μg) of 3-d-old prl1, cul4cs, and wild-type seedlings. His₆-tagged AKIN10 protein levels were determined by protein gel blotting with anti-His antibody. Equivalence of loaded extracts was determined by tubulin protein levels. (C) The expression level of AKIN10 protein in 3-d-old wild-type, cul4cs, and prl1 light-grown seedlings. Equivalence of loaded extracts was determined by RPN6 protein levels.