Proteomic analysis of yeast mutant RNA exosome complexes.
The yeast exosome is a conserved multiprotein complex essential for RNA processing and degradation. The complex is formed by a nine-subunit core that associates with two hydrolytic 3'-5' exoribonucleases. Although catalytically inert, the assembly of this nine-subunit core seems to be essential for the exosome activity, as mutations in regions that do not directly bind RNA or are not in the active sites of the exonucleases impair the function of the complex. Previously isolated mutations in the exosome core subunit Rrp43p have been shown to negatively affect the function of the complex. With the aim of investigating the effect of these mutations on the complex stability and activity, Rrp43p and its mutant forms were purified by means of the TAP method. Mass spectrometry analyses showed that lower amounts of the exosome subunits are copurified with the mutant Rrp43p proteins. Additionally, by decreasing the stability of the exosome, other nonspecific protein interactions are favored (the data have been deposited to the ProteomeXchange with identifier PXD000580). Exosome copurified with mutant Rrp43p exhibited increased exonuclease activity, suggesting higher dissociation constants for these mutant complexes. Therefore, data reported here indicate that complexes containing a mutant Rrp43p exhibit decreased stability and provide information on additional protein interactions.
Department of Biochemistry, Chemistry Institute, University of São Paulo , Av. Prof. Lineu Prestes 748, 05508-000 São Paulo, Brazil.
Rogério F
RF
Lourenço
Adriana F P
AF
Leme
Carla C
CC
Oliveira
D004591:Electrophoresis, Polyacrylamide Gel; D005092:Exonucleases / Q000235:genetics* / Q000378:metabolism; D063326:Exosome Multienzyme Ribonuclease Complex / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D008958:Models, Molecular; D058977:Molecular Sequence Annotation; D009154:Mutation; D055550:Protein Stability; D040901:Proteomics; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013194:Staining and Labeling
D012331:RNA, Fungal; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005092:Exonucleases; D063326:Exosome Multienzyme Ribonuclease Complex