title: The defective proteasome but not substrate recognition function is responsible for the null phenotypes of the Arabidopsis proteasome subunit RPN10.
abstract: 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) domain-containing 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.
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Republic of China.
authors.forename: Ya-Ling
authors.initials: YL
authors.lastname: Lin
authors.forename: Shu-Chiun
authors.initials: SC
authors.lastname: Sung
authors.forename: Hwang-Long
authors.initials: HL
authors.lastname: Tsai
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Yu
authors.forename: Ramalingam
authors.initials: R
authors.lastname: Radjacommare
authors.forename: Raju
authors.initials: R
authors.lastname: Usharani
authors.forename: Antony S
authors.initials: AS
authors.lastname: Fatimababy
authors.forename: Hsia-Yin
authors.initials: HY
authors.lastname: Lin
authors.forename: Ya-Ying
authors.initials: YY
authors.lastname: Wang
authors.forename: Hongyong
authors.initials: H
authors.lastname: Fu
mesh_terms: D017360:Arabidopsis / Q000033:anatomy & histology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D035264:Flowers / Q000033:anatomy & histology / Q000502:physiology; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D018515:Plant Leaves / Q000033:anatomy & histology / Q000502:physiology; D030821:Plants, Genetically Modified; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; D021122:Protein Subunits; C475163:RPN10 protein, Arabidopsis; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D045643:DNA Repair Enzymes