title: Nucleolar proteome dynamics.
abstract: The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.
authors.affiliation: Department of Biochemistry and Molecular Biology, Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Jens S
authors.initials: JS
authors.lastname: Andersen
authors.forename: Yun W
authors.initials: YW
authors.lastname: Lam
authors.forename: Anthony K L
authors.initials: AK
authors.lastname: Leung
authors.forename: Shao-En
authors.initials: SE
authors.lastname: Ong
authors.forename: Carol E
authors.initials: CE
authors.lastname: Lyon
authors.forename: Angus I
authors.initials: AI
authors.lastname: Lamond
authors.forename: Matthias
authors.initials: M
authors.lastname: Mann
mesh_terms: D000595:Amino Acid Sequence; D002466:Cell Nucleolus / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival; D003609:Dactinomycin / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D009687:Nuclear Proteins / Q000032:analysis / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D040901:Proteomics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics
chemical_list: D009687:Nuclear Proteins; D020543:Proteome; D012333:RNA, Messenger; D003609:Dactinomycin