The RAD23 family provides an essential connection between the 26S proteasome and ubiquitylated proteins in Arabidopsis

Abstract

The ubiquitin (Ub)/26S proteasome system (UPS) directs the turnover of numerous regulatory proteins, thereby exerting control over many aspects of plant growth, development, and survival. The UPS is directed in part by a group of Ub-like/Ub-associated (UBL/UBA) proteins that help shuttle ubiquitylated proteins to the 26S proteasome for breakdown. Here, we describe the collection of UBL/UBA proteins in Arabidopsis thaliana, including four isoforms that comprise the RADIATION SENSITIVE23 (RAD23) family. The nuclear-enriched RAD23 proteins bind Ub conjugates, especially those linked internally through Lys-48, via their UBA domains, and associate with the 26S proteasome Ub receptor RPN10 via their N-terminal UBL domains. Whereas homozygous mutants individually affecting the four RAD23 genes are without phenotypic consequences (rad23a, rad23c, and rad23d) or induce mild phyllotaxy and sterility defects (rad23b), higher-order mutant combinations generate severely dwarfed plants, with the quadruple mutant displaying reproductive lethality. Both the synergistic effects of a rad23b-1 rpn10-1 combination and the response of rad23b plants to mitomycin C suggest that RAD23b regulates cell division. Taken together, RAD23 proteins appear to play an essential role in the cell cycle, morphology, and fertility of plants through their delivery of UPS substrates to the 26S proteasome.

Figure 1.

Domain Architectures of the Arabidopsis UBL/UBA Proteins. (A) Protein domain organization of RAD23a, DSK2a, DDI1, NUB1, and UBL1. Numbers on the right indicate the amino acid length of each protein. STI, stress-inducible-1 domain; RVP, retroviral aspartyl-protease domain. (B) Amino acid sequence alignment of the UBL domains compared with Ub. Arrowheads indicate the conserved Lys residues in Ub that are used in poly-Ub chain assembly. Asterisks identify residues that form the hydrophobic patch in Ub (Leu-8, Ile-44, and Val-70) needed for RPN10 binding. (C) Amino acid sequence alignment of the UBA domains. For RAD23a-d and NUB1, both UBA sequences are shown. Diamonds identify residues of the hydrophobic MGF loop (Met-Gly-Phe) that promotes UBA-Ub association. For (B) and (C), black and gray boxes denote conserved and similar residues, respectively. Dots indicate gaps. Numbers on the left and right indicate the amino acid positions of each sequence.

Figure 2.

Gene Expression Patterns and Protein Localization for Arabidopsis RAD23 Isoforms. (A) Relative transcript abundance of RAD23a-d in various tissues determined from the GENEVESTIGATOR DNA microarray data set (https://www.genevestigator.ethz.ch). Number of representatives in the EST database (www.Arabidopsis.org) is listed for each locus. Juv, juvenile; Mat, mature; Sen, senescing. Error bars represent the se from different arrays. (B) Detection of RAD23, DSK2, and DDI1 proteins in nuclear (N)- and cytosol (Cy)-enriched fractions. The fractions were prepared from 1-week-old wild-type seedlings by Percoll gradient centrifugation and subjected to SDS-PAGE and immunoblot analyses with the indicated antibodies. PBA1, β 1 subunit of the CP; RPN5, lid subunit of the RP; RPN10 and RPN1, base subunits of the RP. Antibodies against WIP-1, histone H3 (H3), PUX1, and SUMO1 were used to verify enrichment of the nuclear and cytosolic fractions, respectively. An equal amount of total protein was analyzed in each lane. Cr, crude extract. (C) to (F) Subcellular localization of RAD23b fusions to GFP in intact seedlings. (C) Immunoblot analyses of transgenic rad23b-1 plants stably expressing 35S-GFP-RAD23b. Crude seedling extracts were subjected to SDS-PAGE and immunoblot analyses with anti-RAD23b and anti-GFP antibodies. Filled arrowheads indicate GFP-RAD23b, open arrowheads indicate RAD23b, and asterisks indicate free GFP. GFP represents wild-type plants expressing GFP alone. Equal protein loads were verified by immunoblot analysis with antibodies against PBA1. (D) Rescue of the rad23b-1 phyllotaxy phenotype by the 35S-GFP-RAD23b transgene. Fourteen-day-old plants of the indicated genotypes are shown. (E) Confocal fluorescence microscopy of root tip cells from 2-week-old 35S-GFP-RAD23b rad23b-1 and GFP plants. Arrowheads identify nuclei. Bars = 5 μ m. (F) Subcellular localization of GFP-RAD23a-d transiently expressed by the 35S promoter in protoplasts. Protoplasts were prepared from 2-week-old leaves and imaged by confocal fluorescence microscopy 24 h after transfection with plasmids encoding GFP alone or fused to each RAD23 isoform. Green, GFP; red, chloroplasts. Arrowheads identify nuclei. Bars = 5 μ m.

Figure 3.

Arabidopsis RAD23s Preferentially Bind Lys-48–Linked Poly-Ub Chains and Ub-Conjugates. (A) Poly-Ub chain binding in vitro. A mixture of Ub and poly-Ub chains linked via Lys-48 (K48) or Lys-63 (K63) was incubated with recombinant GST or GST fused to RAD23b, RAD23c, and RAD23d and precipitated with glutathione beads. The Ub moieties were resolved by SDS-PAGE and detected by immunoblot analysis with anti-Ub antibodies. Left panel, Ub mixtures added to the reactions. Right top panel, poly-Ub chains recovered with the RAD23 proteins. Right bottom panel, amount of GST or GST-RAD23 used in each pull-down assay as shown by staining the gel with Coomassie blue. Arrowheads show the migration of Ub and Ub polymers of various lengths and the RAD23-GST and GST proteins. (B) Co-IP of RAD23 binding proteins from wild-type Arabidopsis seedlings using anti-RAD23b antibodies. Crude extracts (Cr) from 1-week-old seedlings were incubated with Protein A beads alone or decorated with anti-RAD23b immunoglobulins. The precipitated fractions were analyzed by SDS-PAGE and immunoblot analyses with antibodies against Ub, RAD23b, RPN10, DSK2a, DDI1, and PBA1. Anti-UBC1 antibodies were used as a control for nonspecific binding. The positions of the Ub monomer, free poly-Ub chains, and Ub-protein conjugates are indicated.

Figure 4.

Arabidopsis RAD23 Proteins Interact with the 26S Proteasome (A) Copurification of RAD23 with the 26S proteasome. The PAG1 subunit of the Arabidopsis CP was replaced by a Flag epitope-tagged version and used to enrich for the 26S proteasome from crude seedling extracts with anti-Flag antibodies. Where indicated, ATP was included to preserve the association of the CP and RP subcomplexes. The immunoprecipitates from PAG1-Flag pag1-1 and wild-type plants were subjected to SDS-PAGE and immunoblot analyses with antibodies against RAD23b, DSK2a, and DDI1 and the RPN10, RPN1a, and PBA1 subunits of the 26S proteasome. (B) Co-IP of Ub conjugates and RAD23 from wild-type seedlings using the UIM-containing region from Arabidopsis RPN10. Crude extracts (Cr) were incubated with glutathione beads decorated with GST or GST fused to the UIMs. The precipitated fractions were analyzed by SDS-PAGE and immunoblot analyses with antibodies against Ub, RAD23b, DSK2a, DDI1, and PBA1. The migration positions of Ub, free poly-Ub chains, and Ub conjugates are indicated.

Figure 5.

Organization of the Arabidopsis RAD23 Genes and Descriptions of the rad23 Mutations. (A) Diagrams of the RAD23a, RAD23b, RAD23c, and RAD23d genes. Boxes and lines denote protein coding regions and introns, respectively. Black boxes, UBA domains; cross-hatched boxes, UBL domains; gray boxes, stress-inducible 1 (STI1) domains. The locations of the various T-DNA insertions are shown. The arrows locate the positions of the primers used for the RT-PCR analyses in Figure 6A. (B) Effect of the rad23a-1 T-DNA insertion on the RAD23a open reading frame. The nucleotide sequence of rad23a-1 downstream of the ATG initiator codon is aligned with the wild-type RAD23a sequence. The boxes locate the predicted N-terminal Met of each protein. The brackets demarcate the aberrant mRNA sequence generated by the rad23a-1 insertion. The introduced nonsense codon (asterisk) is underlined. (C) Ball-and-stick three-dimensional structure of Ub highlighting the region predicted to be missing from the rad23a-1 protein. β -Strands are in light gray, random coils are in dark gray, and the α helix is in black. The arrowhead indicates the position of the T-DNA insertion within the coding sequence for rad23a-1 gene. The thicker lines denote the deleted sequence predicted for the rad23a-1 UBL with the box identifying the expected N-terminal Met (M), Met-24. The positions of the Lys residues (K) conserved between Ub and RAD23a-d and the N-terminal Met (M1) and the C-terminal Gly (G76) in Ub are indicated.

Figure 6.

Molecular and Biochemical Descriptions of the Arabidopsis rad23 Mutants. (A) RT-PCR analyses of the rad23a-1, rad23b-1, rad23b-2, rad23c-1, and rad23d-1 mutants. Total RNA isolated from wild-type and mutant seedlings was subjected to RT-PCR using the primers shown in Figure 5A. A primer pair specific to PAE2 was used as an internal control. (B) RNA gel blot analyses of total RNA from 1-week-old wild-type, rad23 mutant, and 35S-RAD23b seedlings using probes for RAD23a, RAD23b, RAD23c, and RAD23d. Equal loading of the blot was confirmed by probing with β -tubulin4 (TUB4). (C) Immunoblot analysis of crude extracts from wild-type, rad23 mutant, and 35S-RAD23b seedlings with anti-RAD23b antibodies. The SDS-PAGE migration positions of the four RAD23 isoforms are indicated. Equal protein loads were confirmed by probing with anti-PBA1 antibodies.

Figure 7.

Phenotypic Analysis of Arabidopsis rad23b Mutants. (A) rad23b mutants have altered leaf phyllotaxy. Wild-type and single homozygous rad23 mutant seedlings were grown in LD for 7 (top) or 14 d (bottom). (B) Homozygous rad23b-1 roots grow slower and have fewer lateral roots. Seedlings were grown for 1 week on vertical hard-agar plates under LD conditions. (C) rad23b plants are semisterile. Pictured are siliques from self-fertilized wild-type and homozygous rad23b-1 plants. Aborted ovules are indicated by white arrowheads. (D) to (F) Rescue of the rad23b phenotype with a 35S-RAD23b transgene. (D) Genotyping of a rad23b-1 complementation line. DNA was isolated from wild-type, rad23b-1, and 35S-RAD23b rad23b-1 seedlings and PCR amplified with primers specific for RAD23b, the T-DNA in rad23b-1, or the 35S-RAD23b transgene. (E) Immunoblot analysis with anti-RAD23b antibodies showing the reintroduction of the RAD23b protein in 35S-RAD23b rad23b-1 seedlings. Equal protein loads were confirmed by probing with anti-PBA1 antibodies. (F) Phenotypic rescue of the rad23b-1 mutant. Pictured are 14-d-old plants showing the restoration of normal seedling growth and leaf phyllotaxy for a 35S-RAD23b rad23b-1 line.

Figure 8.

Exposure to MMC Induces the rad23b Phyllotaxy Defect in Wild-Type and rad23a, c, and d Seedlings. (A) Two-week-old seedlings grown under LD in the absence or presence of 5 μ g/mL MMC. (B) Fresh weight ( ±sd) of seedlings grown on increasing concentrations of MMC and normalized to their respective growth without MMC. Number of seedlings analyzed per line was 25 (wild type), 21 (rad23a-1), 17 (rad23b-1), 20 (rad23b-2), 20 (rad23c-1), and 20 (rad23d-1).

Figure 9.

The rad23b-1 Mutant Acts Synergistically with the rpn10-1 Mutant. (A) to (E) Phenotypes of Arabidopsis plants homozygous for rpn10-1 and heterozygous for rad23b-1 when grown under a short-day photoperiod (8 h light/16 h dark). Bars = 2 mm. (A) Ten-week-old plant showing a disorganized rosette, reduced inflorescence branches, and secondary rosettes (arrowheads) on the inflorescence stem. (B) Six-week-old rosette with unusual pin-like organs emerging at or near the axillary meristems. Inset: 2-week old seedling displaying the rpn10-1 phenotype (Smalle et al., 2003). (C) An inflorescence terminating in a pin-like structure. (D) Pin-like structures emerging at or near the axillary meristems on the inflorescence stem. (E) A pin-like structure (arrowhead) covered in trichomes emerging from the midvein on the abaxial surface. (F) and (G) Two-week-old seedling phenotype of double homozygous rpn10-1 rad23b-1 plants. (F) Homozygous rpn10-1 seedling. (G) Homozygous rad23b-1 seedling. (H) Double homozygous rpn10-1 rad23b-1 seedling.

Figure 10.

Phenotypic and Biochemical Descriptions of the Combinatorial rad23 Mutants. (A) Two-week-old seedlings grown under LD. (B) Flowering plants grown for 8 weeks under LD. (C) and (D) Immunoblot analyses of crude extracts prepared from 2-week-old seedlings with anti-RAD23b antibodies. The migration positions of the RAD23a-d isoforms are shown on the right. The arrowhead in (D) marks the position of the presumed rad23a-1 truncation. Immunoblot analysis with anti-PBA1 antibodies was included to verify equal loading.