Alternative Assembly Pathways of the 20S Proteasome and Non-canonical Complexes

Indiana University-Purdue University Indianapolis (IUPUI)The 20S proteasome, a multi-subunit protease complex, present in all domains of life and some orders of bacteria, is involved in degradation of the majority of cellular proteins. Structurally, it is made of α and β subunits arranged in four heptameric rings, with inner two β-rings sandwiched between outer two α-rings. The 20S proteasome in prokaryotes usually has one type of α and one type of β subunits, whereas eukaryotes have seven distinct types of α and seven distinct types of β subunits. Unlike the highly conserved structure of proteasome, its assembly pathway is different across the domains. In archaea and eukaryotes, proteasome assembly begins with α subunit interactions leading to the α-ring formation. By contrast, bacterial proteasome assembly pathway bypasses the α-ring formation step by initiating assembly through an α and β subunit interaction first. These early interactions are not well understood due to their highly rapid and dynamic nature. This dissertation focused on understanding the early events in proteasome assembly and contributed three significant findings. First, the archaeal proteasome assembly can also begin without formation of α-rings, demonstrating the coexistence of a bacterial-like assembly pathway. Second, a novel assembly intermediate was identified in yeast, and its composition argues for the presence of a similar α-ring independent assembly pathway. Third, the assembly chaperone Pba3-Pba4 prevents the formation of high molecular weight complexes arising from spontaneous and non-productive interactions among the α subunits. These findings provide a broader understanding of proteasome biogenesis and suggest considering proteasome assembly event as a network of interactions rather than a linear pathway. The results also shed light on assembly chaperone’s contribution in increasing the efficiency of proteasome assembly by streamlining the productive interactions.2020-12-0

Alpha-ring independent assembly of the 20S proteasome

Archaeal proteasomes share many features with their eukaryotic counterparts and serve as important models for assembly. Proteasomes are also found in certain bacterial lineages yet their assembly mechanism is thought to be fundamentally different. Here we investigate α-ring formation using recombinant proteasomes from the archaeon Methanococcus maripaludis. Through an engineered disulfide cross-linking strategy, we demonstrate that double α-rings are structurally analogous to half-proteasomes and can form independently of single α-rings. More importantly, via targeted mutagenesis, we show that single α-rings are not required for the efficient assembly of 20S proteasomes. Our data support updating the currently held "α-ring first" view of assembly, initially proposed in studies of archaeal proteasomes, and present a way to reconcile the seemingly separate bacterial assembly mechanism with the rest of the proteasome realm. We suggest that a common assembly network underpins the absolutely conserved architecture of proteasomes across all domains of life

YPL260W, a high-copy suppressor of a copper-sensitive phenotype in yeast, is linked to DNA repair and proteasome function

The ubiquitin–proteasome system directly impacts the metabolism of heavy metals and yeast has become an important model in understanding this interplay. We demonstrate that yeast mutants with defects in proteasome function are able to tolerate elevated levels of copper. In the course of our analysis, we isolate a yeast mutant that not only negates this copper tolerance in proteasome mutants, but renders yeast exquisitely sensitive to this metal. To better understand the nature of the defect, we carry out a plasmid-based genetic screen to identify high-copy suppressors of this strong copper sensitivity. We identify four genes not previously known to be associated with copper metabolism: CDC53, PSP1, YNL200C, and YPL260W. The latter is a highly conserved fungal gene of no known function. Here, we undertake the first characterization of YPL260W. We demonstrate YPL260W to have a role in bleomycin tolerance with links to DNA repair and proteasome function

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric β subunit rings sandwiched between two heptameric α subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome α and β subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete α-ring and one complete β-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes

Alpha-ring independent assembly of the 20S proteasome

Archaeal proteasomes share many features with their eukaryotic counterparts and serve as important models for assembly. Proteasomes are also found in certain bacterial lineages yet their assembly mechanism is thought to be fundamentally different. Here we investigate α-ring formation using recombinant proteasomes from the archaeon Methanococcus maripaludis. Through an engineered disulfide cross-linking strategy, we demonstrate that double α-rings are structurally analogous to half-proteasomes and can form independently of single α-rings. More importantly, via targeted mutagenesis, we show that single α-rings are not required for the efficient assembly of 20S proteasomes. Our data support updating the currently held "α-ring first" view of assembly, initially proposed in studies of archaeal proteasomes, and present a way to reconcile the seemingly separate bacterial assembly mechanism with the rest of the proteasome realm. We suggest that a common assembly network underpins the absolutely conserved architecture of proteasomes across all domains of life

YPL260W, a high-copy suppressor of a copper-sensitive phenotype in yeast, is linked to DNA repair and proteasome function

The ubiquitin–proteasome system directly impacts the metabolism of heavy metals and yeast has become an important model in understanding this interplay. We demonstrate that yeast mutants with defects in proteasome function are able to tolerate elevated levels of copper. In the course of our analysis, we isolate a yeast mutant that not only negates this copper tolerance in proteasome mutants, but renders yeast exquisitely sensitive to this metal. To better understand the nature of the defect, we carry out a plasmid-based genetic screen to identify high-copy suppressors of this strong copper sensitivity. We identify four genes not previously known to be associated with copper metabolism: CDC53, PSP1, YNL200C, and YPL260W. The latter is a highly conserved fungal gene of no known function. Here, we undertake the first characterization of YPL260W. We demonstrate YPL260W to have a role in bleomycin tolerance with links to DNA repair and proteasome function