Reversible 26S proteasome disassembly upon mitochondrial stress

In eukaryotic cells, proteasomes exist primarily as 26S holoenzymes, the most efficient configuration for ubiquitinated protein degradation. Here, we show that acute oxidative stress caused by environmental insults or mitochondrial defects results in rapid disassembly of 26S proteasomes into intact 20S core and 19S regulatory particles. Consequently, polyubiquitinated substrates accumulate, mitochondrial networks fragment, and cellular reactive oxygen species (ROS) levels increase. Oxidation of cysteine residues is sufficient to induce proteasome disassembly, and spontaneous reassembly from existing components is observed both in vivo and in vitro upon reduction. Ubiquitin-dependent substrate turnover also resumes after treatment with antioxidants. Reversible attenuation of 26S proteasome activity induced by acute mitochondrial or oxidative stress may be a short-term response distinct from adaptation to long-term ROS exposure or changes during aging

UBQLN2 mediates autophagy-independent protein aggregate clearance by the proteasome

Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice

A Perturbed Ubiquitin Landscape Distinguishes Between Ubiquitin in Trafficking and in Proteolysis*

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery—the ubiquitin-proteasome system and the ubiquitin trafficking system—were unevenly perturbed by expression of K0 ubiquitin

Regulated Endoplasmic Reticulum-associated Degradation of a Polytopic Protein: p97 RECRUITS PROTEASOMES TO Insig-1 BEFORE EXTRACTION FROM MEMBRANES*

Polytopic membrane proteins subjected to endoplasmic reticulum (ER)-associated degradation are extracted from membranes and targeted to proteasomes for destruction. The extraction mechanism is poorly understood. One polytopic ER protein subjected to ER-associated degradation is Insig-1, a negative regulator of cholesterol synthesis. Insig-1 is rapidly degraded by proteasomes when cells are depleted of cholesterol, and its degradation is inhibited when sterols accumulate in cells. Insig-2, a functional homologue of Insig-1, is degraded slowly, and its degradation is not regulated by sterols. Here, we report that a single amino acid substitution in Insig-2, Insig-2(L210A), causes Insig-2 to be degraded in an accelerated and sterol-regulated manner similar to Insig-1. In seeking an explanation for the accelerated degradation, we found that proteasomes bind to wild type Insig-1 and mutant Insig-2(L210A) but not to wild type Insig-2, whereas the proteins are still embedded in cell membranes. This binding depends on at least two factors, ubiquitination of Insig and association with the ATPase p97/VCP complex. These data suggest that p97 recruits proteasomes to polytopic ER proteins even before they are extracted from membranes

The Yeast E4 Ubiquitin Ligase Ufd2 Interacts with the Ubiquitin-like Domains of Rad23 and Dsk2 via a Novel and Distinct Ubiquitin-like Binding Domain*

Proteins containing ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains interact with various binding partners and function as hubs during ubiquitin-mediated protein degradation. A common interaction of the budding yeast UBL-UBA proteins Rad23 and Dsk2 with the E4 ubiquitin ligase Ufd2 has been described in endoplasmic reticulum-associated degradation among other pathways. The UBL domains of Rad23 and Dsk2 play a prominent role in this process by interacting with Ufd2 and different subunits of the 26 S proteasome. Here, we report crystal structures of Ufd2 in complex with the UBL domains of Rad23 and Dsk2. The N-terminal UBL-interacting region of Ufd2 exhibits a unique sequence pattern, which is distinct from any known ubiquitin- or UBL-binding domain identified so far. Residue-specific differences exist in the interactions of these UBL domains with Ufd2, which are coupled to subtle differences in their binding affinities. The molecular details of their differential interactions point to a role for adaptive evolution in shaping these interfaces