Archaea encode a streamlined version of the eukaryotic ubiquitin-proteasome system. In archaea, 20S proteasomes associate with AAA+ ATPases of simple subunit composition in the energy-dependent degradation of proteins. More recently, we demonstrate that ubiquitin-fold proteins of archaea (named small archaeal modifier proteins or SAMPs) are linked by isopeptide bonds to protein targets. This archaeal mechanism of attachment (named sampylation) appears less complex than ubiquitylation with a single ubiquitin-activating E1 homolog (UbaA) functioning in the absence of ubiquitin-conjugating E2 or ubiquitin-ligase E3 enzymes. An archaeal isopeptidase composed of a single JAMM (JAB1/MPN/Mov34 metalloenzyme) domain is found to remove SAMPs from diverse protein conjugates, providing evidence that sampylation is reversible and likely regulated. Sites of sampylation, mapped to 36 lysines of 28 proteins by high-throughput MS/MS analysis, were modeled in relationship to 3D-structure and used to guide in vivo analysis and provide a basis for elucidating the biological function of sampylation. Based on this work, we propose that sampylation targets proteins for proteasome-mediated proteolysis, modifies the catalytic active site of enzymes, modulates DNA-binding protein interactions and regulates protein-protein interactions.