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

Abstract

Proteasomes are composed of 20S core particles (CPs) of $\alpha$- and $\beta$-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 of $\alpha 1$and $\alpha 2$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 $\alpha 1$Thr147, $\alpha$2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to $\alpha 1$, thus, revealing a new type of proteasomal modification. Probing the biological role of $\alpha 1$and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for $\alpha 1$variants including Thr147Ala, Thr158Ala and Ser58Ala. AnH. volcaniiRio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to $\alpha 1$. The $\alpha 1$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.

Authors:

Humbard, Matthew A. ^[1]; Reuter, Christopher J. ^[1]; Zuobi-Hasona, Kheir ^[1]; Zhou, Guangyin ^[1]; Maupin-Furlow, Julie A. ^[1]

---

+ Show Author Affiliations

1. Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA

Publication Date:

Fri Jan 01 00:00:00 EST 2010

Research Org.:

Univ. of Florida, Gainesville, FL (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

OSTI Identifier:

1198438

Alternate Identifier(s):

OSTI ID: 1626200

Grant/Contract Number:  

FG02-05ER15650

Resource Type:

Published Article

Journal Name:

Archaea

Additional Journal Information:

Journal Name: Archaea Journal Volume: 2010; Journal ID: ISSN 1472-3646

Publisher:

Hindawi

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Microbiology