Premium
Illuminating the Proteasome: Fluorescence Assays for Investigating the Kinetics of Proteasome Substrate Processing
Author(s) -
Bard Jared Abraham,
Jonsson Erik,
Martin Andreas
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.597.2
Subject(s) - proteasome , ubiquitin , substrate (aquarium) , microbiology and biotechnology , chemistry , degradation (telecommunications) , biophysics , biochemistry , computational biology , biology , computer science , gene , ecology , telecommunications
Degradation of ubiquitinated substrates by the 26S proteasome is a tightly regulated multi‐step process. The 33 unique subunits of the proteasome must coordinate to bind the substrate's ubiquitin targeting signal, engage its unstructured region, translocate and unfold the polypeptide chain while concurrently removing the ubiquitin chains, and finally hydrolyze the peptide bonds. Many of the details of how the proteasome performs these tasks are still unknown. A better understanding of the timing and coordination of these events would provide critical insight into how the cell regulates the rate or extent of protein degradation by the proteasome. In order to gain molecular understanding of these processes, we have developed a fluorescent assay for directly studying the binding, engagement, and translocation of substrates by the heterohexameric AAA+ motor that feeds these substrates into the proteasome core for degradation. This was achieved by incorporating unnatural amino acids into the proteasome, which allowed for the site‐specific attachment of fluorescent dyes. Combined with previously developed tools for the heterologous expression, purification, and in‐vitro reconstitution of proteasome subcomplexes, this assay allows for a kinetic dissection of the substrate‐processing pathway. It will also allow us to test the effects of manipulating the substrate geometry or mutating proteasome components on the rate of individual processing steps. In addition to ensemble experiments, single‐molecule studies will reveal how individual processing events are timed and coordinated. Support or Funding Information J.B acknowledges support from the US National Science Foundation Graduate Research Fellowship. A.M. acknowledges support from the Searle Scholars Program, the US National Institutes of Health (grant R01‐GM094497), the US National Science Foundation CAREER Program (NSF‐MCB‐1150288), and A.M. and E.J. acknowledges support from the Howard Hughes Medical Institute.