Use of RNA interference and complementation to study the function of the Drosophila and human 26S proteasome subunit S13.
The S13 subunit (also called Pad1, Rpn11, and MPR1) is a component of the 19S complex, a regulatory complex essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome. To address the functional role of S13, we combined double-stranded RNA interference (RNAi) against the Drosophila proteasome subunit DmS13 with expression of wild-type and mutant forms of the homologous human gene, HS13. These studies show that DmS13 is essential for 26S function. Loss of the S13 subunit in metazoan cells leads to increased levels of ubiquitin conjugates, cell cycle defects, DNA overreplication, and apoptosis. In vivo assays using short-lived proteasome substrates confirmed that the 26S ubiquitin-dependent degradation pathway is compromised in S13-depleted cells. In complementation experiments using Drosophila cell lines expressing HS13, wild-type HS13 was found to fully rescue the knockdown phenotype after DmS13 RNAi treatment, while an HS13 containing mutations (H113A-H115A) in the proposed isopeptidase active site was unable to rescue. A mutation within the conserved MPN/JAMM domain (C120A) abolished the ability of HS13 to rescue the Drosophila cells from apoptosis or DNA overreplication. However, the C120A mutant was found to partially restore normal levels of ubiquitin conjugates. The S13 subunit may possess multiple functions, including a deubiquitinylating activity and distinct activities essential for cell cycle progression that require the conserved C120 residue.
Lundgren
Josefin
J
Department of Molecular Biology and Functional Genetics, Stockholm University, S-10691 Stockholm, Sweden.
Masson
Patrick
P
Realini
Claudio A
CA
Young
Patrick
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D015153:Blotting, Western; D002453:Cell Cycle; D002460:Cell Line; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D017353:Gene Deletion; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism*; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics*; D025801:Ubiquitin / Q000378:metabolism
D008164:Luminescent Proteins; D012330:RNA, Double-Stranded; C047013:RPS13 protein, human; D012269:Ribosomal Proteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D008666:Metalloendopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease