Mechanism of proteasome gate modulation by assembly chaperones Pba1 and Pba2.

The active sites of the proteasome are housed within its central core particle (CP), a barrel-shaped chamber of four stacked heptameric rings, and access of substrates to the CP interior is mediated by gates at either axial end. These gates are constitutively closed and may be opened by the regulatory particle (RP), which binds the CP and facilitates substrate degradation. We recently showed that the heterodimeric CP assembly chaperones Pba1/2 also mediate gate opening through an unexpected structural arrangement that facilitates the insertion of the N terminus of Pba1 into the CP interior; however, the full mechanism of Pba1/2-mediated gate opening is unclear. Here, we report a detailed analysis of CP gate modulation by Pba1/2. The clustering of key residues at the interface between neighboring α-subunits is a critical feature of RP-mediated gate opening, and we find that Pba1/2 recapitulate this strategy. Unlike RP, which inserts at six α-subunit interfaces, Pba1/2 insert at only two α-subunit interfaces. Nevertheless, Pba1/2 are able to regulate six of the seven interfacial clusters, largely through direct interactions. The N terminus of Pba1 also physically interacts with the center of the gate, disrupting the intersubunit contacts that maintain the closed state. This novel mechanism of gate modulation appears to be unique to Pba1/2 and therefore likely occurs only during proteasome assembly. Our data suggest that release of Pba1/2 at the conclusion of assembly is what allows the nascent CP to assume its mature gate conformation, which is primarily closed, until activated by RP.

Protein Degradation and the Pathologic Basis of Disease.

The abundance of any protein is determined by the balance of protein synthesis and protein degradation. Regulated protein degradation has emerged as a powerful means of precisely controlling individual protein abundance within cells and is largely mediated by the ubiquitin-proteasome system (UPS). By controlling the levels of key regulatory proteins, the UPS contributes to nearly every aspect of cellular function. The UPS also functions in protein quality control, rapidly identifying and destroying misfolded or otherwise aberrant proteins that may be toxic to cells. Increasingly, we understand that dysregulation of protein degradation pathways is critical for many human diseases. Conversely, the versatility and scope of the UPS provides opportunities for therapeutic intervention. In this review, we will discuss the basic mechanisms of protein degradation by the UPS. We will then consider some paradigms of human disease related to protein degradation using selected examples. Finally, we will highlight several established and emerging therapeutic strategies based on altering pathways of protein degradation.

A pathway of targeted autophagy is induced by DNA damage in budding yeast.

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

Dysregulation of ceramide metabolism causes phytoceramide-dependent induction of the unfolded protein response.

The unfolded protein response (UPR) detects and mitigates the harmful effects of dysregulated endoplasmic reticulum (ER) function. The UPR has been best characterized as a protein quality control response, and the sole UPR sensor in yeast, Ire1, is known to detect misfolded ER proteins. However, recent work suggests the UPR can also sense diverse defects within the ER membrane, including increased fatty acid saturation and altered phospholipid abundance. These and other lipid-related stimuli have been referred to as lipid bilayer stress and may be sensed independently through Ire1's transmembrane domain. Here, we show that the loss of Isc1, a phospholipase that catabolizes complex ceramides, causes UPR induction, even in the absence of exogenous stress. A series of chemical and genetic approaches identified a requirement for very long-chain fatty acid (VLCFA)-containing phytoceramides for UPR induction. In parallel, comprehensive lipidomics analyses identified large increases in the abundance of specific VLCFA-containing phytoceramides in the isc1Δ mutant. We failed to identify evidence of an accompanying defect in protein quality control or ER-associated protein degradation. These results extend our understanding of lipid bilayer stress in the UPR and provide a foundation for mechanistic investigation of this fascinating intersection between ceramide metabolism, membrane homeostasis, and the UPR.

Yeast PI31 inhibits the proteasome by a direct multisite mechanism.

Proteasome inhibitors are widely used as therapeutics and research tools, and typically target one of the three active sites, each present twice in the proteasome complex. An endogeneous proteasome inhibitor, PI31, was identified 30 years ago, but its inhibitory mechanism has remained unclear. Here, we identify the mechanism of Saccharomyces cerevisiae PI31, also known as Fub1. Using cryo-electron microscopy (cryo-EM), we show that the conserved carboxy-terminal domain of Fub1 is present inside the proteasome's barrel-shaped core particle (CP), where it simultaneously interacts with all six active sites. Targeted mutations of Fub1 disrupt proteasome inhibition at one active site, while leaving the other sites unaffected. Fub1 itself evades degradation through distinct mechanisms at each active site. The gate that allows substrates to access the CP is constitutively closed, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting that Fub1 may function to neutralize aberrant proteasomes, thereby ensuring the fidelity of proteasome-mediated protein degradation.

Dysregulation of very-long-chain fatty acid metabolism causes membrane saturation and induction of the unfolded protein response.

The unfolded protein response (UPR) senses defects in the endoplasmic reticulum (ER) and orchestrates a complex program of adaptive cellular remodeling. Increasing evidence suggests an important relationship between lipid homeostasis and the UPR. Defects in the ER membrane induce the UPR, and the UPR in turn controls the expression of some lipid metabolic genes. Among lipid species, the very-long-chain fatty acids (VLCFAs) are relatively rare and poorly understood. Here, we show that loss of the VLCFA-coenzyme A synthetase Fat1, which is essential for VLCFA utilization, results in ER stress with compensatory UPR induction. Comprehensive lipidomic analyses revealed a dramatic increase in membrane saturation in the fat1Δ mutant, likely accounting for UPR induction. In principle, this increased membrane saturation could reflect adaptive membrane remodeling or an adverse effect of VLCFA dysfunction. We provide evidence supporting the latter, as the fat1Δ mutant showed defects in the function of Ole1, the sole fatty acyl desaturase in yeast. These results indicate that VLCFAs play essential roles in protein quality control and membrane homeostasis and suggest an unexpected requirement for VLCFAs in Ole1 function.

Regulation of the unfolded protein response in yeast by oxidative stress.

In the unfolded protein response (UPR), Ire1 activates Hac1 to coordinate the transcription of hundreds of genes to mitigate ER stress. Recent work in Caenorhabditis elegans suggests that oxidative stress inhibits this canonical Ire1 signalling pathway, activating instead an antioxidant stress response. We sought to determine whether this novel mode of UPR function also existed in yeast, where Ire1 has been best characterized. We show that the yeast UPR is also subject to inhibition by oxidative stress. Inhibition is mediated by a single evolutionarily conserved cysteine, and affects both luminal and membrane pathways of Ire1 activation. In yeast, Ire1 appears dispensable for resistance to oxidative stress and, therefore, the physiological significance of this pathway remains to be demonstrated.

Thiol-based direct threat sensing by the stress-activated protein kinase Hog1.

The yeast stress-activated protein kinase Hog1 is best known for its role in mediating the response to osmotic stress, but it is also activated by various mechanistically distinct environmental stressors, including heat shock, endoplasmic reticulum stress, and arsenic. In the osmotic stress response, the signal is sensed upstream and relayed to Hog1 through a kinase cascade. Here, we identified a mode of Hog1 function whereby Hog1 senses arsenic through a direct physical interaction that requires three conserved cysteine residues located adjacent to the catalytic loop. These residues were essential for Hog1-mediated protection against arsenic, were dispensable for the response to osmotic stress, and promoted the nuclear localization of Hog1 upon exposure of cells to arsenic. Hog1 promoted arsenic detoxification by stimulating phosphorylation of the transcription factor Yap8, promoting Yap8 nuclear localization, and stimulating the transcription of the only known Yap8 targets, ARR2 and ARR3, both of which encode proteins that promote arsenic efflux. The related human kinases ERK1 and ERK2 also bound to arsenic in vitro, suggesting that this may be a conserved feature of some members of the mitogen-activated protein kinase (MAPK) family. These data provide a mechanistic basis for understanding how stress-activated kinases can sense distinct threats and perform highly specific adaptive responses.

Targeted Degradation of Glucose Transporters Protects against Arsenic Toxicity.

The abundance of cell surface glucose transporters must be precisely regulated to ensure optimal growth under constantly changing environmental conditions. We recently conducted a proteomic analysis of the cellular response to trivalent arsenic, a ubiquitous environmental toxin and carcinogen. A surprising finding was that a subset of glucose transporters was among the most downregulated proteins in the cell upon arsenic exposure. Here we show that this downregulation reflects targeted arsenic-dependent degradation of glucose transporters. Degradation occurs in the vacuole and requires the E2 ubiquitin ligase Ubc4, the E3 ubiquitin ligase Rsp5, and K63-linked ubiquitin chains. We used quantitative proteomic approaches to determine the ubiquitinated proteome after arsenic exposure, which helped us to identify the ubiquitination sites within these glucose transporters. A mutant lacking all seven major glucose transporters was highly resistant to arsenic, and expression of a degradation-resistant transporter restored arsenic sensitivity to this strain, suggesting that this pathway represents a protective cellular response. Previous work suggests that glucose transporters are major mediators of arsenic import, providing a potential rationale for this pathway. These results may have implications for the epidemiologic association between arsenic exposure and diabetes.