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Journal of Bacteriology, August 2004, p. 4960-4971, Vol. 186, No. 15
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.15.4960-4971.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of the Protease and the Turnover Signal Responsible for Cell Cycle-Dependent Degradation of the Caulobacter FliF Motor Protein

Björn Grünenfelder, Sherif Tawfilis, Stefanie Gehrig, Magne Østerås, Daniel Eglin, and Urs Jenal^*

Division of Molecular Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland

Received 12 January 2004/ Accepted 23 April 2004

Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms.

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* Corresponding author. Mailing address: Division of Molecular Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland. Phone: +41-61-267-21-35. Fax: +41-61-267-21-18. E-mail: urs.jenal{at}unibas.ch.

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

Present address: The Scripps Research Institute, La Jolla, CA 92037.

Present address: Department of Plant Sciences, University of Oxford, Oxford, OX1 3RB, Great Britain.

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Journal of Bacteriology, August 2004, p. 4960-4971, Vol. 186, No. 15
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.15.4960-4971.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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