A FRET-based approach for identification of proteasome catalytic subunit compositionElectronic supplementary information (ESI) available. See DOI: 10.1039/c3mb70471h

Mammalian cells have two main types of proteasomes, the constitutive proteasome and the immunoproteasome, each containing a distinct set of three catalytic subunits. Recently, additional proteasome subtypes containing a non-standard mixture of catalytic subunits have gained increasing attention, especially due to their presence in cancer settings. However, practical methods for identifying proteasome subtypes have been lacking. Here, we report the development of the first fluorescence resonance energy transfer (FRET)-based strategy that can be utilized to identify different proteasome subtypes present within cells. We have developed FRET donor- and acceptor-probes that are based on previously reported peptide epoxyketones and selectively target individual proteasome catalytic subunits. Using the purified proteasome and cancer cell lysates, we demonstrate the feasibility of a FRET-based approach for determining the catalytic subunit composition of individual 20S proteasome subtypes. Ultimately, this approach may be utilized to study the functions of individual proteasome subtypes in cells. Proteasome anatomy revealed: FRET pairs that target proteasome catalytic subunits have been developed. Upon excitation, FRET between a donor-acceptor pair bound to two different catalytic subunits within a single 20S proteasome occurs, revealing the catalytic subunit composition.