Dissection of the assembly pathway of the proteasome lid in Saccharomyces cerevisiae

Fukunaga, Kimitoshi; Kudo, Tai; Toh‐e, Akio; Tanaka, Keiji; Saeki, Yasushi

doi:10.1016/j.bbrc.2010.05.061

Cited by 59 publications

(59 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This requirement for an additional contact might explain why we were not able to identify a second PCI binding partner of Rpn6. Rpn5, Rpn6, and Rpn9 form a subcomplex together with Rpn8 and Rpn11 (33), suggesting that one of the latter non-PCI subunits is required for the attachment of Rpn6 to Rpn5 in addition to the subunit II-III interface. The assembly pathway of the lid suggests that the PCI subunits Rpn3 and Rpn7 form a dimer, and PCI subunit Rpn12 attaches to the Rpn3/Rpn7 dimer after its binding to the Rpn6/Rpn5/Rpn8/Rpn9/Rpn11 pentamer (33).…”

Section: Resultsmentioning

confidence: 99%

“…Rpn5, Rpn6, and Rpn9 form a subcomplex together with Rpn8 and Rpn11 (33), suggesting that one of the latter non-PCI subunits is required for the attachment of Rpn6 to Rpn5 in addition to the subunit II-III interface. The assembly pathway of the lid suggests that the PCI subunits Rpn3 and Rpn7 form a dimer, and PCI subunit Rpn12 attaches to the Rpn3/Rpn7 dimer after its binding to the Rpn6/Rpn5/Rpn8/Rpn9/Rpn11 pentamer (33). Thus, Rpn6 and Rpn7 followed by Rpn3 and Rpn12 could form the right end of the horseshoe, perhaps stabilized by coiled-coil interactions of their C-terminal helices; the interaction of Rpn6 with Rpn5 would require Rpn8 and Rpn11, resulting in the second subcomplex.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

Pathare

Nagy

Bohn

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

141

162

View full text Add to dashboard Cite

Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

Pathare

Nagy

Bohn

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

141

162

View full text Add to dashboard Cite

show abstract

“…Standard protocols were used for yeast manipulation 48 . The proteasome subunits RPN1, RPN7 and PRE6 were chromosomally tagged with yeast enhanced green fluorescent protein (GFP) or monomeric Cherry fluorescent protein (mCherry), as described previously 21,40,49 . Genetically stabilized 26S proteasomes in which the CP subunit PRE6/a4 was fused to the RP subunit RPT1 or RPT2 were constructed as follows.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2014

Self Cite

View full text Add to dashboard Cite

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-a 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.

show abstract

“…Third, the ensemble is consistent with the structural data not used to compute it, including 21 residue-specific intersubunit cross-links from S. cerevisiae, the PCI domain contacts implied by the Rpn6 crystal contacts (26), the high-confidence fits of the atomic models of the leucine rich repeat (LRR) region of Rpn2 (40) (Fig. S4), the peripheral position of Rpn12 from the whole complex mass spectrometry (41), the Rpn9 interaction with the Ub receptor Rpn10 from a two-hybrid assay (42), and the subcomplexes formed during lid assembly (43). Moreover, the ensemble is consistent with >95% of the XL/MS data at the residue level, although cross-links were imposed only at the subunit-level (Fig.…”

Section: Stage 2: System Representation and Translation Of Informatiomentioning

confidence: 99%

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

Lasker

Förster

Bohn

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

426

513

View full text Add to dashboard Cite

The 26S proteasome is at the executive end of the ubiquitinproteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The "lid" of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAAATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.coiled coils | mass spectrometry | proteasome-COP9-eIF3 domain | proteasome-cyclosome repeats I n eukaryotes, the ubiquitin-proteasome pathway (UPP) is essential for proteostasis: Misfolded proteins or otherwise defective proteins as well as short-lived regulatory proteins are eliminated by degradation (1). The UPP regulates many fundamental cellular processes, such as protein quality control, DNA repair, and signal transduction (for review see ref.2). The 26S proteasome is the most downstream element of the UPP, executing the degradation of polyubiquitylated substrates (3-5). It consists of the barrelshaped core particle (CP; approximately 700 kDa), which sequesters the proteolytically active site in its central cavity, and the regulatory particle (RP; approximately 900 kDa), which is attached at either one or both ends of the CP and prepares substrates for degradation (6).The RP consists of 19 different canonical subunits, including six regulatory particle AAA-ATPase subunits (Rpt1-6) and 13 regulatory particle non-ATPase subunits (Rpn1-3, Rpn5-13, and Rpn15). The integral ubiquitin (Ub) receptors Rpn10 and Rpn13 recognize polyubiquitylated substrates (7-9). Alternatively, polyubiquitylated substrates can be recruited by shuttling Ub-receptors (Dsk2, Rad23, Ddi2), which bind to substrates with their Ub-associated domain, and to Rpn1, Rpn10, or Rpn13 at their Ub-like domain (5). The metalloprotease Rpn11 deubiquitylates substrates prior to their degradation (10, 11). The functions of the other Rpn subunits remain to be established. The AAA-ATPases form a hexameric ring that unfolds substrates, opens the gate to the CP, and eventually translocates the substrates to the CP.Electron microscopy (EM) (12) and X-...

show abstract

Dissection of the assembly pathway of the proteasome lid in Saccharomyces cerevisiae

Cited by 59 publications

References 27 publications

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

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

Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

Contact Info

Product

Resources

About