Proteasome condensate formation is driven by multivalent interactions with shuttle factors and ubiquitin chains.

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1, show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin-binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome-Rpn1, Rpn10, and Rpn13-and the Ubl domains of Rad23 and Dsk2 are critical under different condensate-inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes.

Proteasome condensate formation is driven by multivalent interactions with shuttle factors and K48-linked ubiquitin chains.

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on long K48-linked ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1 , show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome (Rpn1, Rpn10, and Rpn13) are critical under different condensate inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes. Significance: Stress conditions can cause the relocalization of proteasomes to condensates in yeast as well as mammalian cells. Our work shows that the formation of proteasome condensates in yeast depends on long K48-linked ubiquitin chains, the proteasome binding shuttle factors Rad23 and Dsk2 and proteasome intrinsic ubiquitin receptors. Here, different receptors are critical for different condensate inducers. These results indicate distinct condensates can form with specific functionality. Our identification of key factors involved in the process is crucial for understanding the function of proteasome relocalization to condensates. We propose that cellular accumulation of substrates with long ubiquitin chains results in the formation of condensates comprising those ubiquitinated substrates, proteasomes, and proteasome shuttle factors, where the ubiquitin chains serve as the scaffold for condensate formation.

Proteasome granule formation is regulated through mitochondrial respiration and kinase signaling.

In the yeast Saccharomyces cerevisiae, proteasomes are enriched in cell nuclei, in which they execute important cellular functions. Nutrient stress can change this localization, indicating that proteasomes respond to the metabolic state of the cell. However, the signals that connect these processes remain poorly understood. Carbon starvation triggers a reversible translocation of proteasomes to cytosolic condensates known as proteasome storage granules. Surprisingly, we observed strongly reduced levels of proteasome granules when cells had active cellular respiration prior to starvation. This suggests that the mitochondrial activity of cells is a determining factor in the response of proteasomes to carbon starvation. Consistent with this, upon inhibition of mitochondrial function, we observed that proteasomes relocalize to granules. These links between proteasomes and metabolism involve specific signaling pathways, as we identified a mitogen-activated protein kinase (MAPK) cascade that is critical to the formation of proteasome granules after respiratory growth but not following glycolytic growth. Furthermore, the yeast homolog of AMP kinase, Snf1, is important for proteasome granule formation induced by mitochondrial inhibitors, but it is dispensable for granule formation following carbon starvation. We propose a model in which mitochondrial activity promotes nuclear localization of the proteasome. This article has an associated First Person interview with the first author of the paper.

Proteaphagy is specifically regulated and requires factors dispensable for general autophagy.

Changing physiological conditions can increase the need for protein degradative capacity in eukaryotic cells. Both the ubiquitin-proteasome system and autophagy contribute to protein degradation. However, these processes can be differently regulated depending on the physiological conditions. Strikingly, proteasomes themselves can be a substrate for autophagy. The signals and molecular mechanisms that govern proteasome autophagy (proteaphagy) are only partly understood. Here, we used immunoblots, native gel analyses, and fluorescent microscopy to understand the regulation of proteaphagy in response to genetic and small molecule-induced perturbations. Our data indicate that chemical inhibition of the master nutrient sensor TORC1 (inhibition of which induces general autophagy) with rapamycin induces a bi-phasic response where proteasome levels are upregulated after an autophagy-dependent reduction. Surprisingly, several conditions that result in inhibited TORC1, such as caffeinine treatment or nitrogen starvation, only induced proteaphagy (i.e., without any proteasome upregulation), suggesting a convergence of signals upstream of proteaphagy under different physiological conditions. Indeed, we found that several conditions that activated general autophagy did not induce proteaphagy, further distinguishing proteaphagy from general autophagy. Consistent with this, we show that Atg11, a selective autophagy receptor, as well as the MAP kinases Mpk1, Mkk1, and Mkk2 all play a role in autophagy of proteasomes, although they are dispensable for general autophagy. Taken together, our data provide new insights into the molecular regulation of proteaphagy by demonstrating that degradation of proteasome complexes is specifically regulated under different autophagy-inducing conditions.

Proteasome activator Blm10 levels and autophagic degradation directly impact the proteasome landscape.

The proteasome selectively degrades proteins. It consists of a core particle (CP), which contains proteolytic active sites that can associate with different regulators to form various complexes. How these different complexes are regulated and affected by changing physiological conditions, however, remains poorly understood. In this study, we focused on the activator Blm10 and the regulatory particle (RP). In yeast, increased expression of Blm10 outcompeted RP for CP binding, which suggests that controlling the cellular levels of Blm10 can affect the relative amounts of RP-bound CP. While strong overexpression of BLM10 almost eliminated the presence of RP-CP complexes, the phenotypes this should induce were not observed. Our results show this was due to the induction of Blm10-CP autophagy under prolonged growth in YPD. Similarly, under conditions of endogenous BLM10 expression, Blm10 was degraded through autophagy as well. This suggests that reducing the levels of Blm10 allows for more CP-binding surfaces and the formation of RP-CP complexes under nutrient stress. This work provides important insights into maintaining the proteasome landscape and how protein expression levels affect proteasome function.

Tagging the proteasome active site ß5 causes tag specific phenotypes in yeast.

The efficient and timely degradation of proteins is crucial for many cellular processes and to maintain general proteostasis. The proteasome, a complex multisubunit protease, plays a critical role in protein degradation. Therefore, it is important to understand the assembly, regulation, and localization of proteasome complexes in the cell under different conditions. Fluorescent tags are often utilized to study proteasomes. A GFP-tag on the ß5 subunit, one of the core particle (CP) subunits with catalytic activity, has been shown to be incorporated into proteasomes and commonly used by the field. We report here that a tag on this subunit results in aberrant phenotypes that are not observed when several other CP subunits are tagged. These phenotypes appear in combination with other proteasome mutations and include poor growth, and, more significantly, altered 26S proteasome localization. In strains defective for autophagy, ß5-GFP tagged proteasomes, unlike other CP tags, localize to granules upon nitrogen starvation. These granules are reflective of previously described proteasome storage granules but display unique properties. This suggests proteasomes with a ß5-GFP tag are specifically recognized and sequestered depending on physiological conditions. In all, our data indicate the intricacy of tagging proteasomes, and possibly, large complexes in general.

Native Gel Approaches in Studying Proteasome Assembly and Chaperones.

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 cryogrinding 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 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 discussed in this review.

Starvation Induces Proteasome Autophagy with Different Pathways for Core and Regulatory Particles.

The proteasome is responsible for the degradation of many cellular proteins. If and how this abundant and normally stable complex is degraded by cells is largely unknown. Here we show that in yeast, upon nitrogen starvation, proteasomes are targeted for vacuolar degradation through autophagy. Using GFP-tagged proteasome subunits, we observed that autophagy of a core particle (CP) subunit depends on the deubiquitinating enzyme Ubp3, although a regulatory particle (RP) subunit does not. Furthermore, upon blocking of autophagy, RP remained largely nuclear, although CP largely localized to the cytosol as well as granular structures within the cytosol. In all, our data reveal a regulated process for the removal of proteasomes upon nitrogen starvation. This process involves CP and RP dissociation, nuclear export, and independent vacuolar targeting of CP and RP. Thus, in addition to the well characterized transcriptional up-regulation of genes encoding proteasome subunits, cells are also capable of down-regulating cellular levels of proteasomes through proteaphagy.