Substrate Insolubility Dictates Hsp104-Dependent Endoplasmic-Reticulum-Associated Degradation.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by ER-associated degradation (ERAD). Although the retrotranslocation of misfolded proteins from the ER has been reconstituted, how a polypeptide is initially selected for ERAD remains poorly defined. To address this question while controlling for the diverse nature of ERAD substrates, we constructed a series of truncations in a single ER-tethered domain. We observed that the truncated proteins exhibited variable degradation rates and discovered a positive correlation between ERAD substrate instability and detergent insolubility, which demonstrates that aggregation-prone species can be selected for ERAD. Further, Hsp104 facilitated degradation of an insoluble species, consistent with the chaperone's disaggregase activity. We also show that retrotranslocation of the ubiquitinated substrate from the ER was inhibited in the absence of Hsp104. Therefore, chaperone-mediated selection frees the ER membrane of potentially toxic, aggregation-prone species.

Differential sensitivity of the yeast Lon protease Pim1p to impaired mitochondrial respiration.

Mitochondria are essential organelles whose proteome is well protected by regulated protein degradation and quality control. While the ubiquitin-proteasome system can monitor mitochondrial proteins that reside at the mitochondrial outer membrane or are not successfully imported, resident proteases generally act on proteins within mitochondria. Herein, we assess the degradative pathways for mutant forms of three mitochondrial matrix proteins (mas1-1HA, mas2-11HA, and tim44-8HA) in Saccharomyces cerevisiae. The degradation of these proteins is strongly impaired by loss of either the matrix AAA-ATPase (m-AAA) (Afg3p/Yta12p) or Lon (Pim1p) protease. We determine that these mutant proteins are all bona fide Pim1p substrates whose degradation is also blocked in respiratory-deficient "petite" yeast cells, such as in cells lacking m-AAA protease subunits. In contrast, matrix proteins that are substrates of the m-AAA protease are not affected by loss of respiration. The failure to efficiently remove Pim1p substrates in petite cells has no evident relationship to Pim1p maturation, localization, or assembly. However, Pim1p's autoproteolysis is intact, and its overexpression restores substrate degradation, indicating that Pim1p retains some functionality in petite cells. Interestingly, chemical perturbation of mitochondria with oligomycin similarly prevents degradation of Pim1p substrates. Our results demonstrate that Pim1p activity is highly sensitive to mitochondrial perturbations such as loss of respiration or drug treatment in a manner that we do not observe with other proteases.

A structurally unique E2-binding domain activates ubiquitination by the ERAD E2, Ubc7p, through multiple mechanisms.

Cue1p is an integral component of yeast endoplasmic reticulum (ER)-associated degradation (ERAD) ubiquitin ligase (E3) complexes. It tethers the ERAD ubiquitin-conjugating enzyme (E2), Ubc7p, to the ER and prevents its degradation, and also activates Ubc7p via unknown mechanisms. We have now determined the crystal structure of the Ubc7p-binding region (U7BR) of Cue1p with Ubc7p. The U7BR is a unique E2-binding domain that includes three α-helices that interact extensively with the "backside" of Ubc7p. Residues essential for E2 binding are also required for activation of Ubc7p and for ERAD. We establish that the U7BR stimulates both RING-independent and RING-dependent ubiquitin transfer from Ubc7p. Moreover, the U7BR enhances ubiquitin-activating enzyme (E1)-mediated charging of Ubc7p with ubiquitin. This demonstrates that an essential component of E3 complexes can simultaneously bind to E2 and enhance its loading with ubiquitin. These findings provide mechanistic insights into how ubiquitination can be stimulated.

Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B.

RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.

RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination.

RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

A protein quality control pathway at the mitochondrial outer membrane.

Maintaining the essential functions of mitochondria requires mechanisms to recognize and remove misfolded proteins. However, quality control (QC) pathways for misfolded mitochondrial proteins remain poorly defined. Here, we establish temperature-sensitive (ts-) peripheral mitochondrial outer membrane (MOM) proteins as novel model QC substrates in Saccharomyces cerevisiae. The ts- proteins sen2-1HAts and sam35-2HAts are degraded from the MOM by the ubiquitin-proteasome system. Ubiquitination of sen2-1HAts is mediated by the ubiquitin ligase (E3) Ubr1, while sam35-2HAts is ubiquitinated primarily by San1. Mitochondria-associated degradation (MAD) of both substrates requires the SSA family of Hsp70s and the Hsp40 Sis1, providing the first evidence for chaperone involvement in MAD. In addition to a role for the Cdc48-Npl4-Ufd1 AAA-ATPase complex, Doa1 and a mitochondrial pool of the transmembrane Cdc48 adaptor, Ubx2, are implicated in their degradation. This study reveals a unique QC pathway comprised of a combination of cytosolic and mitochondrial factors that distinguish it from other cellular QC pathways.

Proteins are molecules that need to fold into the right shape to do their job. If proteins lose that shape, not only do they stop working but they risk clumping together and becoming toxic, potentially leading to disease. Fortunately, the cell has quality control systems that normally detect and remove misfolded proteins before they can cause damage to the cell. First, sets of proteins known as chaperones recognize the misfolded proteins, and then another class of proteins attaches a molecular tag, known as ubiquitin, to the misshapen proteins. When several ubiquitin tags are attached to a protein, forming chains of ubiquitin, it is transported to a large molecular machine within the cell called the proteasome. The proteasome unravels the protein and breaks it down into its constituent building blocks, which can then be used to create new proteins. Proteins are found throughout the different compartments of the cell and quality control processes have been well-studied in some parts of the cell but not others. Metzger et al. have now revealed how the process works on the surface of mitochondria, the compartment that provides the cell with most of its energy. To do this, they used baker's yeast, a model laboratory organism that shares many fundamental properties with animal cells, but which is easier to manipulate genetically. The quality control process was studied using two mitochondrial proteins that had been mutated to make them sensitive to changes in temperature. This meant that, when the temperature increased from 25°C to 37°C, these proteins would begin to unravel and trigger the clean-up operation. This approach has been used previously to understand the quality control processes in other parts of the cell. By removing different quality control machinery in turn from the yeast cells, Metzger et al. could detect which were necessary for the process on mitochondria. This showed that there were many similarities with how this process happen in other parts of the cell but that the precise combination of chaperones and enzymes involved was distinct. Furthermore, when the proteasome was not working, the misfolded proteins remained on the mitochondria, showing that they are not transported to other parts of the cell to be broken down. In the future, understanding this process could help to find potential drug targets for mitochondrial diseases. The next steps will be to see how well these findings apply to human and other mammalian cells.

Crystal structure of the Schizosaccharomyces pombe U7BR E2-binding region in complex with Ubc7.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality-control pathway in eukaryotes in which misfolded ER proteins are polyubiquitylated, extracted and ultimately degraded by the proteasome. This process involves ER membrane-embedded ubiquitin E2 and E3 enzymes, as well as a soluble E2 enzyme (Ubc7 in Saccharomyces cerevisiae and UBE2G2 in mammals). E2-binding regions (E2BRs) that recruit these soluble ERAD E2s to the ER have been identified in humans and S. cerevisiae, and structures of E2-E2BR complexes from both species have been determined. In addition to sequence and structural differences between the human and S. cerevisiae E2BRs, the binding of E2BRs also elicits different biochemical outcomes with respect to E2 charging by E1 and E2 discharge. Here, the Schizosaccharomyces pombe E2BR was identified and purified with Ubc7 to resolve a 1.7 Šresolution co-crystal structure of the E2BR in complex with Ubc7. The S. pombe E2BR binds to the back side of the E2 as an α-helix and, while differences exist, it exhibits greater similarity to the human E2BR. Structure-based sequence alignments reveal differences and conserved elements among these species. Structural comparisons and biochemistry reveal that the S. pombe E2BR presents a steric impediment to E1 binding and inhibits E1-mediated charging, respectively.

The Ubiquitin Ligase (E3) Psh1p Is Required for Proper Segregation of both Centromeric and Two-Micron Plasmids in Saccharomyces cerevisiae.

Protein degradation by the ubiquitin-proteasome system is essential to many processes. We sought to assess its involvement in the turnover of mitochondrial proteins in Saccharomyces cerevisiae We find that deletion of a specific ubiquitin ligase (E3), Psh1p, increases the abundance of a temperature-sensitive mitochondrial protein, mia40-4pHA, when it is expressed from a centromeric plasmid. Deletion of Psh1p unexpectedly elevates the levels of other proteins expressed from centromeric plasmids. Loss of Psh1p does not increase the rate of turnover of mia40-4pHA, affect total protein synthesis, or increase the protein levels of chromosomal genes. Instead, psh1Δ appears to increase the incidence of missegregation of centromeric plasmids relative to their normal 1:1 segregation. After generations of growth with selection for the plasmid, ongoing missegregation would lead to elevated plasmid DNA, mRNA, and protein, all of which we observe in psh1Δ cells. The only known substrate of Psh1p is the centromeric histone H3 variant Cse4p, which is targeted for proteasomal degradation after ubiquitination by Psh1p However, Cse4p overexpression alone does not phenocopy psh1Δ in increasing plasmid DNA and protein levels. Instead, elevation of Cse4p leads to an apparent increase in 1:0 plasmid segregation events. Further, 2 µm high-copy yeast plasmids also missegregate in psh1Δ, but not when Cse4p alone is overexpressed. These findings demonstrate that Psh1p is required for the faithful inheritance of both centromeric and 2 µm plasmids. Moreover, the effects that loss of Psh1p has on plasmid segregation cannot be accounted for by increased levels of Cse4p.

Working on a chain: E3s ganging up for ubiquitylation.

Substrate specificity in ubiquitylation is conferred by ubiquitin ligases (E3s). Now, several ways that E3s can interact to mediate ubiquitylation are illustrated for Ubr1 (a RING finger E3) and Ufd4 (a HECT domain E3), in Saccharomyces cerevisiae. These interactions and the related concept of E4 activity are discussed.