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Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
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Charged amino acids in a preprotein inhibit SecA-dependent protein translocation

Nico Nouwen1, Greetje Berrelkamp, Arnold J M Driessen

  • 1Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands.

Journal of Molecular Biology
|February 27, 2009
PubMed

Insights

Charged amino acid residues impede bacterial protein translocation by blocking the Sec translocase machinery. Positively charged residues are stronger inhibitors, preventing SecA ATPase activity and protein insertion into the membrane.

Area of Science:

  • Molecular Biology
  • Protein Biochemistry
  • Bacterial Cell Biology

Background:

  • The Sec translocase system is essential for inserting and translocating proteins across bacterial membranes.
  • Understanding the factors that regulate protein translocation is crucial for bacterial physiology and potential therapeutic targets.

Purpose of the Study:

  • To investigate the impact of charged amino acid residues on protein translocation mediated by the Sec translocase.
  • To analyze the effect of charged residues on the interaction between preproteins and the SecA ATPase.

Main Methods:

  • In vitro translocation assays using modified proOmpA preproteins with charged amino acid stretches.
  • Analysis of protein translocation into Escherichia coli inner membrane vesicles.
  • Assay of preprotein-stimulated SecA ATPase activity.

Main Results:

  • Both negatively and positively charged amino acid residues inhibit proOmpA translocation, causing partial translocation and blocking the translocation site.
  • Positively charged residues exhibit stronger inhibition of translocation and SecA ATPase activity compared to negatively charged residues.
  • Charged amino acid clusters render preproteins poor substrates for the Sec translocase and reduce SecA stimulation.

Conclusions:

  • Charged amino acid residues significantly impede the Sec translocase machinery's function.
  • The Sec translocase exhibits differential sensitivity to positive versus negative charges, with positive charges being more inhibitory.
  • Inhibition of translocation by charged residues correlates with a lack of SecA ATPase stimulation, highlighting the charge-dependent substrate recognition.

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