Every living cell relies on a molecular shredder called the proteasome to chop up damaged or unneeded proteins, and when this shredder breaks down, faulty proteins pile up in ways linked to ageing, neurodegeneration, and cancer. Scientists know that stress can disrupt proteasome assembly, but they do not understand the basic mechanisms that control how the proteasome is built from its 33 component parts. This project uses baker’s yeast—a simple but powerful stand-in for human cells—to answer three fundamental questions: how cells regulate the abundance of proteasome parts, how the cell’s internal skeleton (actin) controls the transport of the messenger RNAs that encode those parts, and where inside the cell the proteasome actually gets assembled. The researchers will engineer the proteasome itself to create new tools that report on assembly in living cells. This is fundamental science with no immediate practical application. However, understanding how cells coordinate the construction of large protein complexes under stress could eventually point toward new strategies for restoring proteasome function in age-related diseases—much as earlier work on yeast protein quality control unexpectedly revealed the machinery behind Parkinson’s and Huntington’s diseases.
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The proteasome is an essential proteolytic complex made up of 33 distinct proteins that need to be coordinately assembled by proteasome assembly chaperones (PACs) in cells. Proteosomes degrade many cellular proteins thereby impacting virtually all cellular processes. The proteasome is essential in all organisms, and cells with defective proteasomal degradation accumulate faulty, misfolded, and damaged proteins as insoluble aggregates. This failure to maintain a functional proteome is associated with a broad range of human diseases, including neurodegeneration and cancer. While the contribution of proteasome dysfunction to age-related diseases is well-appreciated, the underlying mechanisms are often unknown. Thus, understanding the regulation of proteasome homeostasis and how it is affected by stress and disease is of paramount importance. Using budding yeast as a model system, we recently gained the first mechanistic insights into how stress increases proteasome assembly and activity. We discovered that PAC mRNAs travel along actin cables, but under different stresses, these actin cables are disrupted, causing the mRNA to relocalise to cortical actin patches for translation. This results in the coordinated upregulation of PACs, promoting the assembly of functional proteasomes and supporting cell survival. While this pioneering work provides the first clues into how proteasome homeostasis is regulated by stress, it also raises three fundamental questions that are the focus of this application: (1) What are the mechanisms regulating the abundance of proteasome components? (2) How does actin control PAC mRNAs transport and translation to ensure proper proteasome assembly? (3) How and where is the proteasome assembled in cells? Here, we will employ genome editing, biochemistry, proteomics, cell biology and proteasome engineering in budding yeast to address these questions and unravel the regulation of proteasome homeostasis during stress. We will obtain a comprehensive view of how the abundance of different proteasome components is regulated in cells and identify new intrinsic and extrinsic elements important for PAC mRNA regulation (Aim 1). We will define the machinery responsible for RP-PAC mRNA transport and translation during stress, including the identification of key actin cytoskeletal proteins involved (Aim 2). Finally, we will engineer the proteasome to develop new tools reporting on the regulation of proteasome assembly in living cells. We will leverage these tools to understand how and where the proteasome assembles in cells (Aim 3). This bold work has the potential to significantly advance our understanding of proteasome biology and localised mRNA translation, while also generating new tools for the scientific community. This will pave the way for future studies on the regulation of higher-order complex assembly, such as the proteasome, and how this process is coupled to mRNA localisation and translation. This aligns directly with the research priorities of the BBSRC, including understanding the rules of life and developing transformative technologies.
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