Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins.
BACKGROUND
The nucleolus is a subnuclear organelle in which rRNAs are transcribed, processed, and assembled with ribosomal proteins into ribosome subunits. Mass spectrometry combined with pulsed incorporation of stable isotopes of arginine and lysine was used to perform a quantitative and unbiased global analysis of the rates at which newly synthesized, endogenous proteins appear within mammalian nucleoli.


RESULTS
Newly synthesized ribosomal proteins accumulated in nucleoli more quickly than other nucleolar components. Studies involving time-lapse fluorescence microscopy of stable HeLa cell lines expressing fluorescent-protein-tagged nucleolar factors also showed that ribosomal proteins accumulate more quickly than other components. Photobleaching and mass-spectrometry experiments suggest that only a subset of newly synthesized ribosomal proteins are assembled into ribosomes and exported to the cytoplasm. Inhibition of the proteasome caused an accumulation of ribosomal proteins in the nucleus but not in the cytoplasm. Inhibition of rRNA transcription prior to proteasomal inhibition further increased the accumulation of ribosomal proteins in the nucleoplasm.


CONCLUSIONS
Ribosomal proteins are expressed at high levels beyond that required for the typical rate of ribosome-subunit production and accumulate in the nucleolus more quickly than all other nucleolar components. This is balanced by continual degradation of unassembled ribosomal proteins in the nucleoplasm, thereby providing a mechanism for mammalian cells to ensure that ribosomal protein levels are never rate limiting for the efficient assembly of ribosome subunits. The dual time-lapse strategy used in this study, combining proteomics and imaging, provides a powerful approach for the quantitative analysis of the flux of newly synthesized proteins through a cell organelle.
Lam
Yun Wah
YW
Division of Gene Regulation and Expression, Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Lamond
Angus I
AI
Mann
Matthias
M
Andersen
Jens S
JS
D002466:Cell Nucleolus / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D046988:Proteasome Endopeptidase Complex / Q000502:physiology; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D012335:RNA, Ribosomal / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D012335:RNA, Ribosomal; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D046988:Proteasome Endopeptidase Complex