Using siRNA Techniques to Dissect Proteasome Assembly Pathways in Mammalian Cells

Abstract

The 26S proteasome is an ATP-dependent protease known to collaborate with ubiquitin, the polymerization of which acts as a marker for protein degradation in eukaryotic cells, and is involved in a diverse array of biological processes, such as the cell-cycle progression, DNA repair, apoptosis, immune response, signal transduction, transcription, metabolism, protein quality control, and developmental program. The 26S proteasome is a huge protease complex and consists of one catalytic core called the 20S proteasome (or 20S core particle) and one or two 19S regulatory particles (19S RP), which include 14 and 19 different subunits, respectively. Recent studies have revealed that the proteasome formation requires multiple assembly factors and that the assembly pathways are highly conserved between yeast and mammalian cells. This chapter is focused on experimental approaches to reveal the assembly pathways of the proteasome using small interfering RNA techniques in mammalian cells. Knockdown of a proteasome subunit causes arrest of the assembly process before incorporation of the targeted subunit and accumulation of a specific intermediate.