The N-Terminal Penultimate Residue of 20S Proteasome {alpha}1 Influences its N{alpha} Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

doi:10.1128/JB.00090-09

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Journal of Bacteriology, June 2009, p. 3794-3803, Vol. 191, No. 12
0021-9193/09/$08.00+0 doi:10.1128/JB.00090-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The N-Terminal Penultimate Residue of 20S Proteasome ${alpha}$ 1 Influences its N Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii ^,

Matthew A. Humbard, Guangyin Zhou, and Julie A. Maupin-Furlow^*

University of Florida, Department of Microbiology and Cell Science, Gainesville, Florida 32611-0700

Received 23 January 2009/ Accepted 3 April 2009

Proteasomes are energy-dependent proteolytic machines. We elaboratehere on the previously observed N acetylation of the initiatormethionine of the ${alpha}$ 1 protein of 20S core particles (CPs) of Haloferaxvolcanii proteasomes. Quantitative mass spectrometry revealedthis was the dominant N-terminal form of ${alpha}$ 1 in H. volcanii cells.To further examine this, ${alpha}$ 1 proteins with substitutions in theN-terminal penultimate residue as well as deletion of the CP"gate" formed by the ${alpha}$ 1 N terminus were examined for their Nacetylation. Both the "gate" deletion and Q2A substitution completelyaltered the N-acetylation pattern of ${alpha}$ 1, with the deletion rendering ${alpha}$ 1 unavailable for N acetylation and the Q2A modification apparentlyenhancing cleavage of ${alpha}$ 1 by methionine aminopeptidase (MAP),resulting in acetylation of the N-terminal alanine. Cells expressingthese two ${alpha}$ 1 variants were less tolerant of hypoosmotic stressthan the wild type and produced CPs with enhanced peptidaseactivity. Although ${alpha}$ 1 proteins with Q2D, Q2P, and Q2T substitutionswere N acetylated in CPs similar to the wild type, cells expressingthese variants accumulated unusually high levels of ${alpha}$ 1 as ringsin N-acetylated, unmodified, and/or MAP-cleaved forms. Moredetailed examination of this group revealed that while CP peptidaseactivity was not impaired, cells expressing these ${alpha}$ 1 variantsdisplayed higher growth rates and were more tolerant of hypoosmoticand high-temperature stress than the wild type. Overall, theseresults suggest that N acetylation of ${alpha}$ 1 is important in CP assemblyand activity, high levels of ${alpha}$ 1 rings enhance cell proliferationand stress tolerance, and unregulated opening of the CP "gate"impairs the ability of cells to overcome salt stress.

* Corresponding author. Mailing address: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700. Phone: (352) 392-4095. Fax: (352) 392-5922. E-mail: jmaupin{at}ufl.edu

Published ahead of print on 17 April 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

The N-Terminal Penultimate Residue of 20S Proteasome 1 Influences its N Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii ,

The N-Terminal Penultimate Residue of 20S Proteasome ${alpha}$ 1 Influences its N Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii ^,