Unlike Ub, Pup is not processed proteolytically from a larger precursor protein. Pup appears to be de-amidated at the C-terminal Gln. From this point, it has been proposed that PafA phosphorylates the γ-carboxylate of the C-terminus of Pup, but this has not been established. The attachment of Pup to the substrate Lys can potentially be via either the α- or γ-carboxylate. It is not known if poly-pupylation occurs, nor is it known if Pup is removed by a de-pupylase (“DPUP”) prior to degradation, and recycled like Ub.