title: Subcellular distribution and dynamics of active proteasome complexes unraveled by a workflow combining in vivo complex cross-linking and quantitative proteomics.
abstract: Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins-which modulate proteasome activity, stability, localization, or substrate uptake-rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells.
authors.affiliation: CNRS/Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, Toulouse, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Fabre
authors.forename: Thomas
authors.initials: T
authors.lastname: Lambour
authors.forename: Julien
authors.initials: J
authors.lastname: Delobel
authors.forename: François
authors.initials: F
authors.lastname: Amalric
authors.forename: Bernard
authors.initials: B
authors.lastname: Monsarrat
authors.forename: Odile
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Bousquet-Dubouch
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D042541:Intracellular Space / Q000201:enzymology* / Q000378:metabolism; D007700:Kinetics; D007938:Leukemia / Q000378:metabolism / Q000473:pathology; D013058:Mass Spectrometry / Q000379:methods; D018613:Microscopy, Confocal; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D020298:U937 Cells
chemical_list: D003432:Cross-Linking Reagents; D009097:Multienzyme Complexes; D021122:Protein Subunits; D007371:Interferon-gamma; D046988:Proteasome Endopeptidase Complex