title: Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system.
abstract: The proper functioning of any biological system depends on the coordinated activity of its components. Regulation at the genetic level is, in many cases, effective in determining the cellular levels of system components. However, in cases where regulation at the genetic level is insufficient for attaining harmonic system function, posttranslational regulatory mechanisms are often used. Here, we uncover posttranslational regulatory mechanisms in the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), the bacterial equivalent of the eukaryotic ubiquitin-proteasome system. Pup, a ubiquitin analog, is conjugated to proteins through the activities of two enzymes, Dop (deamidase of Pup) and PafA (proteasome accessory factor A), the Pup ligase. As Dop also catalyzes depupylation, it was unclear how PPS function could be maintained without Dop and PafA canceling the activity of the other, and how the two activities of Dop are balanced. We report that tight Pup binding and the limited degree of Dop interaction with high-molecular-weight pupylated proteins results in preferred Pup deamidation over protein depupylation by this enzyme. Under starvation conditions, when accelerated protein pupylation is required, this bias is intensified by depletion of free Dop molecules, thereby minimizing the chance of depupylation. We also find that, in contrast to Dop, PafA presents a distinct preference for high-molecular-weight protein substrates. As such, PafA and Dop act in concert, rather than canceling each other's activity, to generate a high-molecular-weight pupylome. This bias in pupylome molecular weight distribution is consistent with the proposed nutritional role of the PPS under starvation conditions.
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel;
authors.forename: Yifat
authors.initials: Y
authors.lastname: Elharar
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel;
authors.forename: Ziv
authors.initials: Z
authors.lastname: Roth
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel;
authors.forename: Nir
authors.initials: N
authors.lastname: Hecht
authors.affiliation: Department of Biological Services, Weizmann Institute of Science, Rehovot 76100, Israel;
authors.forename: Ron
authors.initials: R
authors.lastname: Rotkopf
authors.affiliation: The Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
authors.forename: Isam
authors.initials: I
authors.lastname: Khalaila
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; gure@bgu.ac.il.
authors.forename: Eyal
authors.initials: E
authors.lastname: Gur
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D008970:Molecular Weight; D020102:Mycobacterium smegmatis / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D046988:Proteasome Endopeptidase Complex
keywords: Dop; PafA; Pup; mycobacteria; proteasome