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Title: Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

Proteasomes are composed of 20S core particles (CPs) ofα- andβ-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 archaeonHaloferax volcaniias a model. Indicative of phosphorylation, phosphatase-sensitive isoforms ofα1andα2were 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 includingα1Thr147,α2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped toα1, thus, revealing a new type of proteasomal modification. Probing the biological role ofα1and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation forα1variants including Thr147Ala, Thr158Ala and Ser58Ala. AnH. volcaniiRio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer toα1. Theα1variants 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.
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [1]
  1. Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA
Publication Date:
OSTI Identifier:
1198438
Grant/Contract Number:
FG02-05ER15650
Type:
Published Article
Journal Name:
Archaea
Additional Journal Information:
Journal Volume: 2010; Related Information: CHORUS Timestamp: 2016-08-11 11:11:23; Journal ID: ISSN 1472-3646
Publisher:
Hindawi
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES