Assays for ubiquitin-like protein ligation and proteasome function in archaea

Ubiquitin-like protein (Ubl) ligation is common to diverse archaea and targets many cellular pathways, including those associated with sulfur mobilization, and also tags proteins as substrates for degradation by the proteasome. Here we highlight protocols to assay proteasome function and Ubl ligation in archaea. A chase assay is described to monitor the impact of proteasome function on the stability of Ubl-modified proteins in the cell. A method to reconstitute Ubl ligation using a purified E1-like enzyme (UbaA), Ubl (SAMP2), methionine sulfoxide reductase A (MsrA), and cell lysate of an ΔmsrA ΔubaA Δsamp1-3 mutant is also described. MsrA is found to have the surprising ability to stimulate the formation of Ubl bonds. Haloferax volcanii, a halophilic archaeon originally isolated from the Dead Sea, serves as the model organism for these protocols.