Ecm29 Fulfils Quality Control Functions in Proteasome Assembly

Lehmann, Andrea; Niewienda, Agathe; Jechow, Katharina; Janek, Katharina; Enenkel, Cordula

doi:10.1016/j.molcel.2010.06.016

Cited by 71 publications

(95 citation statements)

References 30 publications

(81 reference statements)

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“…At the same time, polyubiquitinated proteins accumulate. This may seem contradictory, but the same phenotype has recently been described for the ecm29⌬ mutant (35). Furthermore, it has been reported that RP-CP complexes are stabilized when the active sites of CP are inhibited (29), and polyubiquitinated substrates obviously accumulate.…”

Section: Discussionmentioning

confidence: 80%

“…The capacity of the proteasome to degrade proteins depends crucially on the RP-CP interaction. There is a wide range of factors affecting this interaction, including metabolites (4), proteasome-associated proteins (35,49), the metabolic state of the cell (5), and salt concentrations (33). One of these factors is Ecm29, a protein first identified in a screen for yeast displaying cell wall defects (39).…”

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confidence: 99%

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Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29

Panasenko

Collart

2011

Molecular and Cellular Biology

131

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In this study we determine that the Not4 E3 ligase is important for proteasome integrity. Consequently, deletion of Not4 leads to an accumulation of polyubiquitinated proteins and reduced levels of free ubiquitin. In the absence of Not4, the proteasome regulatory particle (RP) and core particle (CP) form salt-resistant complexes, and all other forms of RPs are unstable. Not4 can associate with RP species present in purified proteasome holoenzyme but not with purified RP. Additionally, Not4 interacts with Ecm29, a protein that stabilizes the proteasome. Interestingly, Ecm29 is identified in RP species that are inactive and not detectable in cells lacking Not4. In the absence of Not4, Ecm29 interacts less well with the proteasome and becomes ubiquitinated and degraded. Our results characterize Ecm29 as a proteasome chaperone whose appropriate interaction with the proteasome requires Not4.In eukaryotes, short-lived proteins are degraded primarily by the ubiquitin-proteasome system (UPS) (25). The multicatalytic protease, the 26S proteasome, is responsible for this degradation. Most proteasome substrates are modified by polyubiquitin chains that are recognized by the proteasome. The UPS controls a diverse array of biologically important processes, including cell cycle progression, DNA repair, signal transduction, and protein quality control.The 26S proteasome consists of 2 major subcomplexes: a proteolytically active 20S core particle (CP) bound at one or both ends by a 19S regulatory particle (RP; also called PA700 in mammals). The CP has a hollow cylindrical shape and consists of a stack of 4 heptameric rings. The 2 outer rings contain ␣-type subunits, and the 2 inner rings contain ␤-type subunits. The proteolytic sites of the proteasome are located in its central cavity on specific ␤ subunits (19). Free CP exists in an autoinhibited state in which the N termini of ␣ subunits form a gate to block substrate entry. Activation of CP occurs upon opening of this gate by a proteasome activator, 19S. In mammals, two additional activators have been identified: the PA28 (or PA26)/11S regulator and PA200. In Saccharomyces cerevisiae, Blm10, which is similar to mammalian PA200, can function as an alternative activator (49).RP consists of 2 subcomplexes: the base, which binds directly to CP, and a peripheral lid. The base includes 6 ATPase subunits (Rpt1 to -6) that facilitate gate opening, substrate unfolding, and translocation into CP using ATP (47). The lid consists of 9 non-ATPase subunits (Rpn3, -5 to -9, -11, and -12 and Sem1) and is required for the recognition and deubiquitination of substrates (50).Several recent investigations have focused on how this highly abundant complex of about 2,500 kDa is assembled. CP assembly requires the assistance of CP chaperones (23). Similarly RP assembly is realized by several RP chaperones: Nas2, Hsm3, Nas6, and Rpn14 (36,46). Once RP is assembled, the base binds to the lid and all chaperones are released prior to, or during, RP-CP association (12,48). Recent data suggest that lid,...

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Section: Discussionmentioning

confidence: 80%

mentioning

confidence: 99%

Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29

Panasenko

Collart

2011

Molecular and Cellular Biology

131

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“…Supernatants were applied to a Superose 6B column, and peptidase and proteasome activity was determined in cytosolic fractions using 100 mM of the following substrates: H-Pro-AMC, H-Arg-AMC, H-Leu-AMC, and Suc-Leu-Leu-Val-Tyr-AMC (Bachem). Active fractions were separated on SDS-PAGE, and protein bands were Coomassie stained, tryptic ingel digested, and measured by mass spectrometry using a MALDI-TOF/ TOF instrument (4700 Proteomics Analyzer; Applied Biosystems) (20). Data were analyzed using Swiss-Prot 57.9 and NCBInr 20090513 databases.…”

Section: Identification Of Cytosolic Peptidasesmentioning

confidence: 99%

The Efficiency of Human Cytomegalovirus pp65495–503 CD8+ T Cell Epitope Generation Is Determined by the Balanced Activities of Cytosolic and Endoplasmic Reticulum-Resident Peptidases

Urban

Textoris-Taube

Reimann

et al. 2012

The Journal of Immunology

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Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8+ CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65495–503 is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65495–503 generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65495–503 epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65495–503 epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65495–503-specific T cell activation, indicating the importance of ER aminopeptidases in pp65495–503 generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65495–503 epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.

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“…The dynamics of proteasomes and their substrates can be thoroughly studied in yeast, since yeast division occurs through closed mitosis, where the nuclear envelope remains intact. Evidence is found in the literature that nuclear substrates exit the nucleus and cytoplasmic substrates enter the nucleus for proteasomal degradation [13][14][15]. The polyubiquitination of misfolded cytosolic proteins is mainly achieved by two ubiquitin ligases, the cytoplasmic Ubr1 ligase and the nuclear San1 ligase.…”

Section: Short Communicationmentioning

confidence: 99%

Ecm29 Fulfils Quality Control Functions in Proteasome Assembly

Cited by 71 publications

References 30 publications

Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29

Not4 E3 Ligase Contributes to Proteasome Assembly and Functional Integrity in Part through Ecm29

The Efficiency of Human Cytomegalovirus pp65495–503 CD8+ T Cell Epitope Generation Is Determined by the Balanced Activities of Cytosolic and Endoplasmic Reticulum-Resident Peptidases

Proteasome Dynamics: Will the Territory of Proteasomes Be Claimed By Mitochondrial Proteases Under Stress Conditions?

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