Browsing by Author "Ramamurthy, Aishwarya"

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    Alpha-ring independent assembly of the 20S proteasome
    (Nature Publishing Group, 2015-08-19) Panfair, Dilrajkaur; Ramamurthy, Aishwarya; Kusmierczyk, Andrew R.; Department of Biology, School of Science
    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.
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    Investigating the early events in proteasome assembly
    (2014) Ramamurthy, Aishwarya; Kusmierczyk, Andrew; Atkinson, Simon; Randall, Stephen Karl, 1953-
    Proteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.
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    YPL260W, a high-copy suppressor of a copper-sensitive phenotype in yeast, is linked to DNA repair and proteasome function
    (Elsevier, 2015-11-27) Firestone, Kyle; Awonusi, Damilola; Panfair, Dilrajkaur; Roland, Derrick; Ramamurthy, Aishwarya; Kusmierczyk, Andrew R.; Department of Biology, School of Science
    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.