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Applied and Environmental Microbiology, December 2004, p. 7530-7538, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7530-7538.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Analysis of Proteasome-Dependent Proteolysis in Haloferax volcanii Cells, Using Short-Lived Green Fluorescent Proteins{dagger}

Christopher J. Reuter and Julie A. Maupin-Furlow*

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida

Received 9 April 2004/ Accepted 6 August 2004

Proteasomes are energy-dependent proteases that are central to the quality control and regulated turnover of proteins in eukaryotic cells. Dissection of this proteolytic pathway in archaea, however, has been hampered by the lack of substrates that are easily detected in whole cells. In the present study, we developed a convenient reporter system by functional expression of a green fluorescent protein variant with C-terminal fusions in the haloarchaeon Haloferax volcanii. The levels of this reporter protein correlated with whole-cell fluorescence that was readily detected in culture. Accumulation of the reporter protein was dependent on the sequence of the C-terminal amino acid fusion, as well as the presence of an irreversible, proteasome-specific inhibitor (clasto-lactacystin ß-lactone). This inhibitor was highly specific for H. volcanii 20S proteasomes, with a Ki of ~40 nM. In contrast, phenylmethanesulfonyl fluoride did not influence the levels of fluorescent reporter protein or inhibit 20S proteasomes. Together, these findings provide a powerful tool for the elucidation of protein substrate recognition motifs and the identification of new genes which may be involved in the proteasome pathway of archaea.

* 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.

{dagger} Florida Agricultural Experiment Station Journal Series no. R-10375.

Applied and Environmental Microbiology, December 2004, p. 7530-7538, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7530-7538.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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