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Polypeptide binding of Escherichia coli FtsH (HflB)
Y Akiyama1, M Ehrmann, A Kihara
1Department of Cell Biology, Institute for Virus Research, Kyoto University, Japan. yakiyama@virus.kyoto-u.ac.jp
Molecular Microbiology
|June 27, 1998
Summary
Escherichia coli FtsH protease undergoes ATP-dependent conformational changes and binds denatured proteins like alkaline phosphatase (PhoA). This binding is uncoupled from proteolysis, suggesting in vivo relevance.
Area of Science:
- Molecular biology
- Protein biochemistry
- Cell biology
Background:
- Escherichia coli FtsH is a membrane-bound, ATP-dependent protease.
- Understanding FtsH's function requires characterizing its conformational dynamics and substrate interactions.
Purpose of the Study:
- To investigate ATP-dependent conformational changes in FtsH.
- To identify polypeptide binding partners of FtsH.
- To determine if FtsH binding to substrates leads to proteolysis.
Main Methods:
- Protease protection assays to detect conformational changes.
- Affinity chromatography to identify binding partners.
- Sucrose gradient sedimentation to assess protein complex formation.
- Analysis of MalF-PhoA hybrid protein localization.
Main Results:
- ATP binding induced trypsin resistance in a 33 kDa FtsH segment.
- ATP and ATPgammaS inhibited self-aggregation of solubilized FtsH.
- FtsH associated with denatured alkaline phosphatase (PhoA) but not native PhoA.
- Denatured PhoA prevented FtsH aggregation and co-sedimented with FtsH.
- FtsH also bound to detergent-solubilized SecY.
- FtsH-bound SecY underwent proteolysis, but FtsH-bound PhoA did not.
- FtsH overproduction increased cytoplasmic localization of PhoA in a MalF-PhoA hybrid protein.
Conclusions:
- ATP binding induces significant conformational changes in FtsH.
- FtsH binds denatured polypeptides, including PhoA and SecY.
- FtsH-mediated binding of denatured PhoA is uncoupled from proteolysis.
- FtsH's interaction with denatured PhoA likely occurs in vivo.