Abstract: The present invention relates to prokaryotic ubiquitin-like protein (Pup) nucleic acid sequences and Pup amino acid sequences encoded therefrom. Also encompassed are antibodies that are immunologically specific for Pup. Methods directed to isolation and identification of pupylated substrates that utilize the conjugated Pup as an affinity tag are also included. Pupylated substrates that are identified using the method and reagents of the present invention provide tools useful for the identification of additional components of prokaryotic proteasomal machinery. Modulators of Pup activity and methods for identifying such modulators are also encompassed herein.