Membrane-Associated 26S Proteasomes:Molecular Mechanisms And Biological Functions

The ubiquitin-proteasome system(UPS)is responsible for degrading the majority of cellular proteins in eukaryotes and is indispensable for almost every cellular process.The regulatory mechanisms and intracellular localization of the 26 S proteasome are fundamental questions that have not been well characterized.Commonly regarded as floating in the cytosol and nucleoplasm,the proteasome has also been found recently to localize to various membranous structures of the cell.However,the molecular nature,biological function and regulatory details of membrane-associated proteasomes have been elusive.In this thesis,I have employed a variety of approaches including immunofluorescence,immuno-gold electron microscopy,cell fractionation,click chemistry,gene editing and quantitative mass spectrometry,to demonstrate that Nmyristoylation of the proteasome subunit Rpt2 is a fundamental means by which the proteasome is anchored to the membrane.Rpt2 N-myristoylation is conserved from yeast to human and widely present in all cell and tissue types examined.Blocking this modification in cells leads to marked reduction of membrane-localized proteasomes.More importantly,homozygous mutation of the Rpt2 myristoylation site is embryonic lethal in mice,underscoring the critical importance of the membrane-associated proteasome.I then performed SILAC/MS with mouse embryonic fibroblasts isolated from these mice and uncovered hundreds of proteins with altered abundance.Two thirds of these differentially regulated proteins were membrane-related,participating in a number of cellular activities including cell adhesion.Serendipitously,we also found that N-myristoylation is also required for the phosphorylation of Rpt2-Y439,which strictly depends on the membrane localization of Rpt2.This can be attributed to the membrane-bound tyrosine kinase,Src,and the membrane-bound form of PTPN2,a protein tyrosine phosphatase,that together control the level of Rpt2-Y439 phosphorylation.Phosphorylation of Rpt2-Y439 inhibits the assembly of proteasome.As a result,the activity of membrane-associated proteasomes is suppressed in cancer cells with hyperactivated Src.In a mouse xenograft model,we found that Rpt2-Y439 phosphorylation plays an important role in determining cellular responses to the anticancer drug saracatinib,a selective Src inhibitor,as cancer cells expressing the Y439 F mutant showed weakened sensitivity to the drug.Together,this thesis has elucidated the molecular details and biological meaning of proteasome association with the membrane,deepened our understanding of compartmentalized protein degradation,and provided new insights into proteasome regulation and protein degradation.It also suggests new possibilities for improving the therapeutic efficacy of tyrosine kinase inhibitors in anti-cancer treatment.