Putting it all together: intrinsic and extrinsic mechanisms governing proteasome biogenesis

Background The 26S proteasome is at the heart of the ubiquitin-proteasome system, which is the key cellular pathway for the regulated degradation of proteins and enforcement of protein quality control. The 26S proteasome is an unusually large and complicated protease comprising a 28-subunit core particle (CP) capped by one or two 19-subunit regulatory particles (RP). Multiple activities within the RP process incoming ubiquitinated substrates for eventual degradation by the barrel-shaped CP. The large size and elaborate architecture of the proteasome have made it an exceptional model for understanding mechanistic themes in macromolecular assembly. Objective In the present work, we highlight the most recent mechanistic insights into proteasome assembly, with particular emphasis on intrinsic and extrinsic factors regulating proteasome biogenesis. We also describe new and exciting questions arising about how proteasome assembly is regulated and deregulated in normal and diseased cells. Methods A comprehensive literature search using the PubMed search engine was performed, and key findings yielding mechanistic insight into proteasome assembly were included in this review. Results Key recent studies have revealed that proteasome biogenesis is dependent upon intrinsic features of the subunits themselves as well as extrinsic factors, many of which function as dedicated chaperones. Conclusion Cells rely on a diverse set of mechanistic strategies to ensure the rapid, efficient, and faithful assembly of proteasomes from their cognate subunits. Importantly, physiological as well as pathological changes to proteasome assembly are emerging as exciting paradigms to alter protein degradation in vivo

The polySUMOylation axis promotes nucleolar release of Tof2 for mitotic exit

Summary: In budding yeast, the nucleolus serves as the site to sequester Cdc14, a phosphatase essential for mitotic exit. Nucleolar proteins Tof2, Net1, and Fob1 are required for this sequestration. Although it is known that these nucleolar proteins are SUMOylated, how SUMOylation regulates their activity remains unknown. Here, we show that Tof2 exhibits cell-cycle-regulated nucleolar delocalization and turnover. Depletion of the nuclear small ubiquitin-like modifier (SUMO) protease Ulp2 not only causes Tof2 polySUMOylation, nucleolar delocalization, and degradation but also leads to Cdc14 nucleolar release and activation. This outcome depends on polySUMOylation and the activity of downstream enzymes, including SUMO-targeted ubiquitin ligase and Cdc48/p97 segregase. We further developed a system to tether SUMO machinery to Tof2 and generated a SUMO-deficient tof2 mutant, and the results indicate that Tof2 polySUMOylation is necessary and sufficient for its nucleolar delocalization and degradation. Together, our work reveals a polySUMO-dependent mechanism that delocalizes Tof2 from the nucleolus to facilitate mitotic exit

An Allosteric Interaction Network Promotes Conformation State-Dependent Eviction of the Nas6 Assembly Chaperone from Nascent 26S Proteasomes

Summary: The 26S proteasome is the central ATP-dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation-specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP binding by the ATPase subunit Rpt3 promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly-coupled allosteric signals promote chaperone eviction and provides a framework for understanding the eviction of other chaperones from this biomedically important molecular machine. : Nemec et al. report how the evolutionarily conserved Nas6 assembly chaperone is evicted from nascent 26S proteasomes. Nucleotide binding events within the nascent proteasome trigger formation of conformation-specific intersubunit contacts that expel Nas6. This mechanism may serve a quality control function by blocking formation of 26S proteasomes from defective components. Keywords: proteasome, macromolecular complex, ubiquitin, proteolysis, gankyrin, Nas6, assembly, oncogen

A Conserved Protein with AN1 Zinc Finger and Ubiquitin-like Domains Modulates Cdc48 (p97) Function in the Ubiquitin-Proteasome Pathway

Regulated protein degradation mediated by the ubiquitin-proteasome system (UPS) is critical to eukaryotic protein homeostasis. Often vital to degradation of protein substrates is their disassembly, unfolding, or extraction from membranes. These processes are catalyzed by the conserved AAA-ATPase Cdc48 (also known as p97). Here we characterize the Cuz1 protein (Cdc48-associated UBL/zinc finger protein-1), encoded by a previously uncharacterized arsenite-inducible gene in budding yeast. Cuz1, like its human ortholog ZFAND1, has both an AN1-type zinc finger (Zf_AN1) and a divergent ubiquitin-like domain (UBL). We show that Cuz1 modulates Cdc48 function in the UPS. The two proteins directly interact, and the Cuz1 UBL, but not Zf_AN1, is necessary for binding to the Cdc48 N-terminal domain. Cuz1 also associates, albeit more weakly, with the proteasome, and the UBL is dispensable for this interaction. Cuz1-proteasome interaction is strongly enhanced by exposure of cells to the environmental toxin arsenite, and in a proteasome mutant, loss of Cuz1 enhances arsenite sensitivity. Whereas loss of Cuz1 alone causes only minor UPS degradation defects, its combination with mutations in the Cdc48(Npl4-Ufd1) complex leads to much greater impairment. Cuz1 helps limit the accumulation of ubiquitin conjugates on both the proteasome and Cdc48, suggesting a possible role in the transfer of ubiquitylated substrates from Cdc48 to the proteasome or in their release from these complexes