Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1–Pba2 chaperone

Kock, Malte; Nunes, Maria M.; Hemann, M.; Kube, Sebastian; Dohmen, R. Jürgen; Herzog, Franz; Ramos, Paula C.; Wendler, Petra

doi:10.1038/ncomms7123

Cited by 48 publications

(86 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several classical NLSs exist within the N-termini of distinct α subunits which were proposed to be either accessible rendering the CP in an import-competent conformation, or to be masked rendering the CP in an import-incompatible conformation ( Tanaka et al , 1990). Indeed, recent cryo-EM structure analysis revealed flexible and less structured α ring surfaces in Ump1-associated CP precursor complexes ( Kock et al , 2015), in compliance with our finding that importin α recognizes CP precursor complexes but not mature CP with closed α rings ( Lehmann et al , 2002). Our model upon which CP precursor complexes are imported into the nucleus was supported by the following observations ( Figure 1).…”

Section: Discussion/analysis Of the Literaturesupporting

confidence: 91%

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Chowdhury

Enenkel

2015

F1000Res

View full text Add to dashboard Cite

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.

show abstract

Section: Discussion/analysis Of the Literaturesupporting

confidence: 91%

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Chowdhury

Enenkel

2015

F1000Res

View full text Add to dashboard Cite

show abstract

“…We also cannot fully exclude the possibility that half-mers associate, bind ␤7 subunits, and then dissociate again; however, ␤7 addition should stabilize the dimer, so this idea seems unlikely. We propose that the addition of ␤7 to the 15S intermediate induces structural changes in the ␤ ring (30) and provides a key additional trans-␤-ring interaction through its conserved C-terminal tail to drive rapid dimerization. In cells lacking ␤5pro but expressing excess ␤7, a step downstream of ␤7 addition but before dimerization appears to become limiting, consistent with a distinct role for ␤5pro in half-mer joining.…”

Section: Discussionmentioning

confidence: 99%

“…A recent chemical cross-linking study of the 15S half-mer (-␤7) intermediate revealed two cross-links involving the ␤5 propeptide segment: one cross-linked Lys-69 of the propeptide to ␤4 and the other was between Lys-16 of ␤5pro and ␣6 (30). As noted by the authors, the latter cross-link suggested that the ␤5 propeptide at this assembly stage pointed toward the ␣ ring rather than toward the future interface with a second half-mer.…”

Section: Discussionmentioning

confidence: 99%

“…In yeast, it is believed that Ump1 associates along with or shortly after the first ␤ subunits (␤2, ␤3, and ␤4) have been added to a full ␣-subunit ring (26). This species has been called the 13S or 15S intermediate (26 -29); we will use the "13S" name here because the "15S" designation has also been used (30) to refer to a later, nearly complete half-proteasome species that lacks only ␤7, the "half-mer(-␤7) intermediate" (26). Yeast cells lacking Ump1 are viable but accumulate proteasomal particles with partially processed subunits (25).…”

mentioning

confidence: 99%

“…Pba1/Pba2 remains bound to the assembling proteasome all the way to formation of the "pre-holoproteasome," in which half-mers have associated but the ␤-subunit precursors are not yet fully processed. After ␤-subunit maturation, a conformational change in the ␣ rings is triggered, which leads to both release of Pba1/Pba2 and enhanced 20S affinity for the 19S regulatory particle (30,32,34). Degradation of the entrapped Ump1 factor accompanies 20S maturation (25).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly

Arendt

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (␤1, ␤2, and ␤5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, ␤5 (Doa3/Pre2) has a 75-residue propeptide, ␤5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the ␤5 propeptide nonetheless causes substantial defects in proteasome maturation. Sequences closer to the cleavage site have critical but redundant roles in both assembly and self-cleavage. A conserved histidine two residues upstream of the autocleavage site strongly promotes processing. Surprisingly, although ␤5pro is functionally linked to the Ump1 assembly factor, trans-expressed ␤5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed ␤5pro mutants; the strongest encoded the ␤7 proteasome subunit. Previous data suggested that ␤7 and ␤5pro have overlapping roles in bringing together two half-proteasomes, but the timing of ␤7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind ␤7 incorporation into the half-mer. Our results suggest that ␤7 insertion precedes half-mer dimerization, and the ␤7 tail and ␤5 propeptide have unequal roles in half-mer joining.

show abstract

Native Gel Approaches in Studying Proteasome Assembly and Chaperones

Roelofs

Suppahia

Waite

et al. 2018

Methods in Molecular Biology

View full text Add to dashboard Cite

Summary Proteasomes are complex molecular machines that consist of 66 subunits. The assembly of these complexes is highly coordinated in a process that requires at least ten proteasome-specific molecular chaperones. One of the challenges in studying assembly intermediates is their relatively low abundance as compared to the proteasome holoenzyme. Therefore, superior separating techniques are crucial for analyses of proteasomal complexes in general and studies in the assembly in particular. For this reason, native gel analyses have been abundantly used in studying proteasomes, as they provide a high resolution. Native gels are very versatile and can be used in various combinatorial approaches. In this chapter, we outline two approaches to prepare samples for native gels. The first is a yeast cryo-grinding method and the second a core particle (CP) base reconstitution approach. We describe the native gel electrophoresis, as well as various downstream analyses, including 2D native-SDS-PAGE. These techniques and approaches that can all be used, often in parallel, to gain a variety of information about activity and composition of the complexes separated by native gel. The potential combined approaches are highlighted in the summary flow chart in figure 1.

show abstract

Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1–Pba2 chaperone

Cited by 48 publications

References 53 publications

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Intracellular Dynamics of the Ubiquitin-Proteasome-System

Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly

Native Gel Approaches in Studying Proteasome Assembly and Chaperones

Contact Info

Product

Resources

About