Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Nature Publishing Group full text link Nature Publishing Group Free PMC article
Full text links

Actions

Share

.2009 Jun 11;459(7248):861-5.
doi: 10.1038/nature08063.

Chaperone-mediated pathway of proteasome regulatory particle assembly

Affiliations

Chaperone-mediated pathway of proteasome regulatory particle assembly

Jeroen Roelofs et al. Nature..

Abstract

The proteasome is a protease that controls diverse processes in eukaryotic cells. Its regulatory particle (RP) initiates the degradation of ubiquitin-protein conjugates by unfolding the substrate and translocating it into the proteasome core particle (CP) to be degraded. The RP has 19 subunits, and their pathway of assembly is not understood. Here we show that in the yeast Saccharomyces cerevisiae three proteins are found associated with RP but not with the RP-CP holoenzyme: Nas6, Rpn14 and Hsm3. Mutations in the corresponding genes confer proteasome loss-of-function phenotypes, despite their virtual absence from the holoenzyme. These effects result from deficient RP assembly. Thus, Nas6, Rpn14 and Hsm3 are RP chaperones. The RP contains six ATPases-the Rpt proteins-and each RP chaperone binds to the carboxy-terminal domain of a specific Rpt. We show in an accompanying study that RP assembly is templated through the Rpt C termini, apparently by their insertion into binding pockets in the CP. Thus, RP chaperones may regulate proteasome assembly by directly restricting the accessibility of Rpt C termini to the CP. In addition, competition between the RP chaperones and the CP for Rpt engagement may explain the release of RP chaperones as proteasomes mature.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Nas6, Hsm3 and Rpn14 bind to free RP
a, Proteasomes were affinity-purified from strains expressing HA-tagged Nas6. Samples were resolved by SDS-PAGE and immunoblotted for Nas6 (anti-HA), RP (anti-Rpn8 and anti-Rpn12) and CP (anti-α7).b, Proteasomes were affinity-purified from strains expressing HA-tagged Hsm3, eluted, and analyzed for the presence of Hsm3, RP, and CP.c, Proteasomes were affinity-purified from strains expressing HA-tagged Nas6 and Rpn14. After elution, samples were analyzed as ina.d, HA-specific immunoprecipitations were tested for RP and CP by immunoblotting.
Figure 2
Figure 2. Phenotypic analysis ofnas6Δ,hsm3Δ, andrpn14Δ mutants
a, Strains with indicated genes deleted were spotted on plates in four-fold dilutions and grown at the indicated temperature.b, Cell lysates (75µg) were resolved on native gels. Gels were stained for LLVY-AMC hydrolytic activity.c, Cell lysates of wild-type ornas6Δrpn14Δ strains were resolved by two-dimensional native-SDS/PAGE gel electrophoresis followed by immunoblotting.d, Cell lysates were resolved on 5.25% native gels and immunoblotted. BP1 assignment based on ref. . *, background band.
Figure 3
Figure 3. RP chaperones bind to the C-domains of Rpt proteins in proximity to the CP
a, RP, lid, base bound to IgG beads or untreated IgG beads were incubated with GST-Nas6, GST-Rpn14, or GST-Hsm3 purified fromE. coli. Beads were analyzed for bound proteins.b, Domain composition of Rpt3; other Rpt proteins have similar architecture. “cc” predicted coiled-coil region; “ATPase” ATPase domain; “C” C-domain; “aa” amino acids.c, His-tagged C-domains were co-expressed with GST or GST-tagged Nas6, Rpn14, or Hsm3 inE. coli. Glutathione-Sepharose purified samples were immunoblotted using His-tag antibody followed by Coomassie Blue staining.d, Modeling of the interaction of Rpt ring with CP in the presence (right) and absence (left) of Nas6: Nas6 binding to the ATPase ring appears to block the C-terminal tail of Rpt3 from docking into the CP. The CP α-ring (beige), was combined with the PAN hexameric ATPase (ATPase domain grey; C-domains blue). The Nas6-Rpt3 structure was mapped onto the ATPase ring by aligning the Rpt3 structure with the PAN C-domain (dark blue; C-terminus red), with R.M.S. 1.1 Å over 283 atoms. The last twelve residues of the PA26 C-terminus (magenta) are docked into CP–. The length corresponds to that of Rpt3 C-terminal tail that is absent from the Nas6-Rpt3 structure.e, Affinity-purified, resin-bound base was incubated with indicated RP-chaperone, washed, then loaded with purified CP. Resin-bound proteins were eluted and analyzed by immunoblotting.
Figure 4
Figure 4. Functional conservation between Nas6 and gankyrin
a,nas6Δ rpn14Δ strains expressingNAS6 or gankyrin from aGAL promoter were spotted onto YPD plates or YEP Raf/Gal plates in four-fold dilutions, and incubated at indicated temperatures.b, Proteasomes were purified from yeast strains expressing HA-tagged gankyrin and analyzed by immunoblotting.c, His-tagged C-domains of indicated human Rpt proteins were co-expressed inE. coli with GST-tagged gankyrin. Glutathione-Sepharose purified samples were separated by SDS-PAGE, and stained with Coomassie Blue. Band assignments were confirmed by immunoblotting (data not shown).d, Human proteasomes purified via HTBH-tagged Rpn11 (Ref. 5) were were resolved by two-dimensional native-SDS/PAGE gel electrophoresis. Proteins were silver stained or analyzed by immunoblotting.e, asc, only GST-gankyrin was replaced by GST-S5b.f, HeLa cell lysate was resolved on Blue native gels. Fractions were analyzed by mass spectrometry, and protein abundance estimated using spectral counting. Based on their abundance profiles, most proteasome subunits can be divided into four groups (left graph); CP (α1–7 and β1–7), lid (Rpn3,5,6,7,8,9,10,11), baseB (Rpt3,4,5,6 and Rpn2) and BaseA (Rpt1,2, and Rpn1). Interestingly, S5b is absent from proteasomes (fraction 1 and 2), but abundant in fraction 4. Right panel, S5b and individual profiles of BaseA members.g, HeLa cell lysates frome were separated by 2D native-SDS-PAGE and analyzed by immunoblotting.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Finley D. Recognition and Processing of Ubiquitin-Protein Conjugates by the Proteasome. Annu. Rev. Biochem. 2009;78 - PMC - PubMed
    1. Park S, et al. Hexameric Assembly of the Proteasomal ATPases is Templated through their C-termini. Nature. (Submitted; accompanying paper). - PMC - PubMed
    1. Leggett DS, et al. Multiple associated proteins regulate proteasome structure and function. Mol. Cell. 2002;10:495–507. - PubMed
    1. Verma R, et al. Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Biol. Cell. 2000;11:3425–3439. - PMC - PubMed
    1. Wang X, et al. Mass spectrometric characterization of the affinity-purified human 26S proteasome complex. Biochemistry (Mosc) 2007;46:3553–3565. - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
Nature Publishing Group full text link Nature Publishing Group Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp