Proteasomes are composed of 20S core particles (CPs) of - and -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 and 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 Thr147, 2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to , thus, revealing a new type of proteasomal modification. Probing the biological role of and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to . The 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. 1. Introduction Proteasomes are multicatalytic proteases found in all three domains of life and are essential for growth in many organisms, including haloarchaea and eukaryotes [1–5]. 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. Proteasomal proteins are also subject to co- and posttranslational modification (PTM) including N-terminal acetylation [6–9], phosphorylation [10–27], S-glutathionation [28], N-myristoylation [29], O-linked glycosylation [30], and processing of N-terminal propeptides including exposure of the Thr active site residues of 20S core particles (CPs) [31]. Despite decades of study, the mechanisms and biological significance of many of the posttranslational modifications of proteasomes are poorly understood. In particular, our knowledge of the phosphorylation of proteasomes remains limited. Polo-kinase, casein kinase II and calcium/calmodulin-dependent protein kinase II are known to mediate the phosphorylation of 26S proteasomes in vitro [11, 13, 14, 16–19, 27, 32,
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