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

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

doi:10.1038/ncomms4396

Cited by 113 publications

(141 citation statements)

References 61 publications

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“…Recent fluorescence correlation spectroscopy studies also support the conclusion that proteasomes can be imported into the nucleus as holo-enzymes ( Pack et al , 2014; Figure 3). However, the maturation state of the GFP-labelled proteasomes was unclear.…”

Section: Discussion/analysis Of the Literaturementioning

confidence: 60%

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Chowdhury

Enenkel

2015

F1000Res

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The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus.Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm.Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body’s cells.

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Section: Discussion/analysis Of the Literaturementioning

confidence: 60%

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Chowdhury

Enenkel

2015

F1000Res

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“…We did not detect intact proteasomes in the nucleus, and virtually all peptidase activity was present in the cytosol. These findings diverge from the general opinion, which maintains that proteasomes degrade proteins inside the nucleus (4,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Our results predict an important regulatory role for export in the degradation of nuclear proteins.…”

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confidence: 56%

“…All proteasome activity that was applied to the column was recovered in these fractions, and none was detected in the trailing column fractions. Because proteasome subunits are present predominantly in intact complexes in vivo (18), the weak 20S, specific activity observed in fractions 16 and 17, could reflect post-lysis dissociation of the 26S proteasome. The fractionated proteins were examined by immunoblotting, and 20S subunits (␣4, ␤7) were found in all fractions that contained peptidase activity (Fig.…”

Section: Resultsmentioning

confidence: 99%

Catalytically Active Proteasomes Function Predominantly in the Cytosol

Dang

Chen

Madura

2016

Journal of Biological Chemistry

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The ubiquitin/proteasome pathway is a well characterized system for degrading intracellular proteins, although many aspects remain poorly understood. There is, for instance, a conspicuous lack of understanding of the site(s) where nuclear proteins are degraded because the subcellular distribution of peptidase activity has not been investigated systematically. Although nuclear proteins could be degraded by importing proteasomes into the nucleus, it is also evident that some nuclear proteins are degraded only after export to cytosolic proteasomes. Proteasomes and substrates are mobile, and consequently, the sites of degradation might not be static. We sought to identify the location of proteasomes to provide more conclusive evidence on the sites of protein degradation. We report that catalytically active proteasomes exist almost exclusively in the cytosol. The resulting lack of nuclear peptidase activity suggests that little, if any, degradation occurs in the nucleus. These and other studies suggest that the export of proteolytic substrates could define an important regulatory step in the degradation of nuclear proteins by cytosolic proteasomes.The ubiquitin/proteasome pathway is a major mechanism for eliminating regulatory and damaged proteins. The key enzymology is well understood, and many targeting factors that attach ubiquitin to proteolytic substrates have been identified and characterized (1). Despite these advances, there remain areas of ambiguity. A detailed understanding of the assembly of multiubiquitin chains, the transport of proteolytic substrates, and sites of intracellular protein turnover remain unclear.Many proteins that are involved in cell cycle progression, DNA repair, and transcription are nuclear proteins that play a central role in cell growth and stress response. The stability of these proteins is controlled by the proteasome, and it is generally assumed that they are degraded inside the nucleus. This view is fostered by the detection of proteasome subunits in the nucleus (2, 3) and enrichment in the nuclear envelope (4). However, many of these studies examined the distribution of GFPtagged proteins, which does not ensure that the tagged subunits are present in intact proteasomes. This is an important consideration because certain proteasome subunits and subcomplexes perform non-proteolytic roles in the nucleus (5, 6). We also note that certain GFP-tagged proteasome subunits are not efficiently assembled into intact complexes, and in some instances the fluorescence is reduced after assembly into the proteasome (7). Consequently, the signal observed could arise predominantly from the free form of proteasome subunits. Critically, there is no convincing evidence that peptidase activity is present in the nucleus.We and others reported that the yeast DNA repair protein Rad4, DNA polymerase subunit Cdc17 (2), and HO endonuclease (8) are stabilized in nuclear export mutants, although proteasome assembly and catalytic activity are unaffected. The stabilizing effect of blocking export is not restri...

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“…Although it was shown that CP and RP components are imported separately into the nucleus, a recent study demonstrated that the 26S proteasome completes its assembly process in the cytoplasm and is able to enter the nucleus as a holocomplex (Pack et al, 2014). Thus, although XopJ itself is not a nuclear protein, its interference with proteasome assembly outside the nucleus eventually inhibits proteasome activity in the cytoplasm as well as in the nucleus, because it prevents the nuclear import of functional proteasome complexes.…”

Section: Discussionmentioning

confidence: 99%

The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT6

Üstün

Börnke

2015

Plant Physiol.

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Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities.

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

Cited by 113 publications

References 61 publications

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Catalytically Active Proteasomes Function Predominantly in the Cytosol

The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT6

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