The Defective Proteasome but Not Substrate Recognition Function Is Responsible for the Null Phenotypes of the Arabidopsis Proteasome Subunit RPN10

• Published in: The Plant cell Vol. 23; no. 7; pp. 2754 - 2773
• Main Authors: Lin, Ya-Ling, Sung, Shu-Chiun, Tsai, Hwang-Long, Yu, Ting-Ting, Radjacommare, Ramalingam, Usharani, Raju, Fatimababy, Antony S., Lin, Hsia-Yin, Wang, Ya-Ying, Fu, Hongyong
• Format: Journal Article
• Language: English
• Published: United States American Society of Plant Biologists 01.07.2011
• Subjects: Arabidopsis - anatomy & histology; Arabidopsis - genetics; Arabidopsis - growth & development; Arabidopsis - metabolism; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Binding Sites; Complementation; DNA Repair Enzymes - genetics; DNA Repair Enzymes - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Flowers; Flowers - anatomy & histology; Flowers - physiology; Genetic Complementation Test; Humans; Leaves; Molecular Sequence Data; Ovules; Phenotype; Phenotypes; Plant cells; Plant Leaves - anatomy & histology; Plant Leaves - physiology; Plants; Plants, Genetically Modified; Pollen; Proteasome Endopeptidase Complex - chemistry; Proteasome Endopeptidase Complex - metabolism; Protein Binding; Protein Subunits - genetics; Protein Subunits - metabolism; Proteolysis; Receptors; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Substrate Specificity; Substrates; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitins; Ubiquitins - metabolism
• ISSN: 1040-4651; 1532-298X
• DOI: 10.1105/tpc.111.086702

Ubiquitylated substrate recognition during ubiquitin/proteasome-mediated proteolysis (UPP) is mediated directly by the proteasome subunits RPN10 and RPN13 and indirectly by ubiquitin-like (UBL) and ubiquitin-associated (UBA) domaincontaining factors. To dissect the complexity and functional roles of UPP substrate recognition in Arabidopsis thaliana, potential UPP substrate receptors were characterized. RPN10 and members of the UBL-UBA-containing RAD23 and DSK2 families displayed strong affinities for Lys-48-linked ubiquitin chains (the major UPP signals), indicating that they are involved in ubiquitylated substrate recognition. Additionally, RPN10 uses distinct interfaces as primary proteasomal docking sites for RAD23s and DSK2s. Analyses of T-DNA insertion knockout or RNA interference knockdown mutants of potential UPP ubiquitin receptors, including RPN10, RPN13, RAD23a-d, DSK2a-b, DDI1, and NUB1, demonstrated that only the RPN10 mutant gave clear phenotypes. The null rpn10-2 showed decreased double-capped proteasomes, increased 20S core complexes, and pleiotropic vegetative and reproductive growth phenotypes. Surprisingly, the observed rpn10-2 phenotypes were rescued by a RPN10 variant defective in substrate recognition, indicating that the defectiveness of RPN10 in proteasome but not substrate recognition function is responsible for the null phenotypes. Our results suggest that redundant recognition pathways likely are used in Arabidopsis to target ubiquitylated substrates for proteasomal degradation and that their specific roles in vivo require further examination.