Understanding the Regulation of 26S Proteasome Assembly

The 26S proteasome is involved in protein degradation in eukaryotic cells and is the last step for the ubiquitin-proteasome system (UPS). It is involved in the degradation of cellular proteins and as a result, is responsible for most cellular processes including the cell cycle, DNA replication, transcription, signal transduction, and stress responses. The evolutionary conservation of the proteasome in most species signifies that any inhibition of the proteasome is lethal and can cause significant ripple effects to any cellular processes involved. In this report, two approaches are used to aid the understanding of proteasomal regulation further. A quantitative approach was utilised to measure the assembly of RP base subunits which involved engineering the proteasome using CRISPR gene-editing (chapter 1). This further highlighted the difficulty of tagging proteasome genes and how critical endogenous Rpt6 regulation is to cellular health. Furthermore, the second approach used was a physiological approach to studying the role of actin in proteasome homeostasis through the cell cycle (chapter 2). This aim is achieved through the establishment of the cell synchronisation protocol and is used to study how proteasome mRNA localisation relative to actin is regulated throughout the cell cycle. The dynamic relationship between the activation of the mitogen-activated protein (MAP) kinase Mpk1 and the level of the actin nucleation-promoting factor Vrp1 was also explored and found to have an inverted correlation. Future work will entail integrating these two chapters by focusing on the localisation of the RP via microscopy by swapping the split-NanoLuc with the split-GFP technology.