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 responses1,2,3,4. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling5, 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.
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We thank A. Fox for providing the HeLaYFP–p68 cell line and other members of the Lamond and the Mann laboratories for help and discussions. Work in the Center for Experimental Bioinformatics (CEBI) is supported by a grant from the Danish National Research Foundation. A.I.L. is a Wellcome Trust Principal Research Fellow and is funded by a Wellcome Trust Programme grant; A.K.L.L. was funded by a Croucher studentship; Y.W.L. was funded by The Human Frontier Science Program, which is also acknowledged for a network grant entitled ‘Functional organization of the cell nucleus investigated through proteomics and molecular dynamics’.
The authors declare that they have no competing financial interests.
This document contains the legends for Supplementary Figures S1 and S2, and legends for Supplementary Tables 1 and 2. (DOC 30 kb)
Electron micrograph details of isolated HeLa nucleoli that are structurally intact and transcriptionally active. (PDF 2216 kb)
Hierarchical clustering of 302 nucleolar proteins arranged by similarity in their response to Actinomycin-D treatment. (PDF 331 kb)
The nucleolar proteome. (XLS 3108 kb)
Nucleolar proteome dynamics after actinomycin D treatment. (XLS 3590 kb)
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Andersen, J., Lam, Y., Leung, A. et al. Nucleolar proteome dynamics. Nature 433, 77–83 (2005). https://doi.org/10.1038/nature03207
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