title: Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome.
abstract: The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-α mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.
authors.affiliation: Cellular Informatics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Chan-Gi
authors.initials: CG
authors.lastname: Pack
authors.affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Yukii
authors.affiliation: Medical Mycology Center, Chiba University, 1-8-1 Inohana, Chiba 260-8673, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Toh-e
authors.affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Tai
authors.initials: T
authors.lastname: Kudo
authors.affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Tsuchiya
authors.affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Kaiho
authors.affiliation: Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany.
authors.forename: Eri
authors.initials: E
authors.lastname: Sakata
authors.affiliation: Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Murata
authors.affiliation: Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan.
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Yokosawa
authors.affiliation: Cellular Informatics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Sako
authors.affiliation: Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanaka
authors.affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Saeki
mesh_terms: D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism; D020285:Cryoelectron Microscopy; D003593:Cytoplasm / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D013997:Time Factors; D059008:Time-Lapse Imaging / Q000379:methods; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D046988:Proteasome Endopeptidase Complex; D029701:Saccharomyces cerevisiae Proteins; D000097573:Red Fluorescent Protein; C519193:Rpn7 protein, S cerevisiae; C573322:Pre6 protein, S cerevisiae; C061553:ATP dependent 26S protease