Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

Chan-Gi Pack; Haruka Yukii; Akio Toh-e; Tai Kudo; Hikaru Tsuchiya; Ai Kaiho; Eri Sakata; Shigeo Murata; Hideyoshi Yokosawa; Yasushi Sako; Wolfgang Baumeister; Keiji Tanaka; Yasushi Saeki

doi:10.1038/ncomms4396

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

Nat Commun. 2014 Mar 6:5:3396. doi: 10.1038/ncomms4396.

Authors

Affiliations

¹ Cellular Informatics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
² Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
³ Medical Mycology Center, Chiba University, 1-8-1 Inohana, Chiba 260-8673, Japan.
⁴ Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany.
⁵ Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
⁶ Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan.

PMID: 24598877
DOI: 10.1038/ncomms4396

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Active Transport, Cell Nucleus
Cell Nucleus / metabolism
Cryoelectron Microscopy
Cytoplasm / metabolism*
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism
Kinetics
Luminescent Proteins / genetics
Luminescent Proteins / metabolism
Microscopy, Confocal
Microscopy, Fluorescence
Mutation
Proteasome Endopeptidase Complex / genetics
Proteasome Endopeptidase Complex / metabolism*
Proteasome Endopeptidase Complex / ultrastructure
Red Fluorescent Protein
Saccharomyces cerevisiae / genetics
Saccharomyces cerevisiae / metabolism*
Saccharomyces cerevisiae Proteins / genetics
Saccharomyces cerevisiae Proteins / metabolism*
Saccharomyces cerevisiae Proteins / ultrastructure
Time Factors
Time-Lapse Imaging / methods

Substances

Luminescent Proteins
Rpn7 protein, S cerevisiae
Saccharomyces cerevisiae Proteins
Green Fluorescent Proteins
Pre6 protein, S cerevisiae
Proteasome Endopeptidase Complex
ATP dependent 26S protease