In vitro Analysis of Ubiquitin-like Protein Modification in Archaea

Abstract

The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of Ubl protein modification and following analysis of this modification in Haloferax volcanii, a halophilic archaeon serving as the model organism.

Multidomain ribosomal protein trees and the planctobacterial origin of neomura (eukaryotes, archaebacteria)

“…Planctobacteria, the more ancient Armatimonadetes, some Proteobacteria, and one archaebacterium, have a Ubl protein differing from Pup, so it will be important to see if it mediates a more eukaryote-like tagging mechanism or is yet another prokaryotic variant of tagging machinery likely to extend back to LUCA. Archaebacteria additionally use Ubl tagging (sampylation) both for Stransfer and for identifying metabolic proteins for proteolysis by proteasomes, best studied in euryarchaeotes (Fu et al 2016(Fu et al , 2018Hepowit et al 2016;Maupin-Furlow 2013a). Involvement of Cdc48 type AAA+ ATPases in halobacterial sampylation for proteasomal targeting (Fu et al 2016) as in eukaryotic ERAD proteolytic digestion show that link had already evolved at least as early as the neomuran stem, consistent with ERAD-like proteolysis perhaps having preceded phagotrophy as a way of feeding on external proteins (Cavalier-Smith 2009).…”