In vitro Reconstitution Assays of Arabidopsis 20S Proteasome

Abstract

The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously reported methods. The main strategy to obtain the 20S core proteasome here is to strip away the 19S regulatory subunits from the 26S proteasome. The protocol has two major parts: 1) Affinity purification of 20S proteasomes from stable transgenic lines expressing epitope-tagged PAG1, an essential component of the 20S proteasome (Procedures A-D) and 2) an in vitro 20S proteasome degradation assay (Procedure E). We anticipate that these protocols will provide simple and effective approaches to study in vitro degradation by the 20S proteasome and advance the study of protein metabolism in plants.