Abstract:
: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.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Fu X,Adams Z,Maupin-Furlow Jdoi
10.1016/bs.mie.2018.12.036subject
Has Abstractpub_date
2019-01-01 00:00:00pages
161-178eissn
0076-6879issn
1557-7988pii
S0076-6879(18)30533-0journal_volume
619pub_type
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