Year-end Sale % OFF 
Abstract
Proteasomes are composed of 20S core particles (CPs) of α- and β-type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeon Haloferax volcanii as a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α1 and α2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α1 Thr147, α2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α1, thus, revealing a new type of proteasomal modification. Probing the biological role of α1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α1 variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α1. The α1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.
Highlights
Proteasomes are multicatalytic proteases found in all three domains of life and are essential for growth in many organisms, including haloarchaea and eukaryotes [1,2,3,4,5]
Treatment of these purified 20S core particles (CPs) with phosphatase resulted in a basic shift in pI of both α1 and α2 proteins signifying both α-type subunits were phosphorylated (Figure 1(c))
Similar to the Ser129 phosphosite that was mapped to the CP β subunit [35], analogous nonphosphorylated peptides mapped to α1 Thr147, α2 Thr13/Ser14, and proteasome activating nucleotidase (PAN)-A Ser340 residues. These results suggest that the phosphosites of PAN and CP complexes are not fully occupied by phosphoryl groups and, the phosphorylation of proteasomes may be regulated in the cell
Summary
Proteasomes are multicatalytic proteases found in all three domains of life and are essential for growth in many organisms, including haloarchaea and eukaryotes [1,2,3,4,5]. Accessory proteins, such as 19S cap and proteasome activating nucleotidase (PAN) complexes, and protein-modification pathways such as ubiquitination are important in regulating protein degradation by proteasomes. There is evidence that proteasomal phosphorylation is regulated by growth and/or signaling molecules such as γ-interferon [11, 12, 17, 22, 24, 26]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
