Direct Observation of Protein Translocation by the 26S Proteasome

Abstract

As with the ribosome, the proteasome is a motor that shapes the lives of other proteins in the cell. It handles all degradation flux from the ubiquitin proteasome system. The precise interplay between degradation signal (ubiquitin code) and motor function has remained elusive. Here we employ single molecule FRET to observe the interactions between an acceptor labeled proteasome and a donor labeled substrate. The proteasome is reconstituted from a heterologous expression system with non-standard amino acid reprogramming for fluorescent dye labeling. Ubiquitin chains of defined length and linkage type are enzymatically loaded onto fluorescently labeled substrates that we then observe engage with and translocate through the proteasome. We find that differential ubiquitin linkage types confer variance in the kinetics of interaction with the proteasome, which we call an engagement profile. These engagement profiles offer us the opportunity to begin to decipher the ubiquitin code in addition to understanding how the motor operates in reading this code. They also give us glimpses of force production by the 26S proteasome, a molecular machine tuned by billions of years of evolution.