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Indecisive M13 procoat protein mutants bind to SecA but do not activate the translocation ATPase
T Roos1, D Kiefer, S Hugenschmidt
1Institute of Microbiology and Molecular Biology, University of Hohenheim, D-70593 Stuttgart, Germany.
Abstract:
The M13 procoat protein serves as the paradigm for the Sec-independent membrane insertion pathway. This protein is inserted into the inner membrane of Escherichia coli with two hydrophobic regions and a central periplasmic loop region of 20 amino acid residues. Extension of the periplasmic loop region renders M13 procoat membrane insertion Sec-dependent. Loop regions with 118 or more residues required SecA and SecYEG and were efficiently translocated in vivo. Two mutants having loop regions of 80 and 100 residues, respectively, interacted with SecA but failed to activate the membrane translocation ATPase of SecA in vitro. Similarly, a procoat mutant with two additional glutamyl residues in the loop region showed binding to SecA but did not stimulate the ATPase. The three mutants were also defective for precursor-stimulated binding of SecA to the membrane surface. Remarkably, the mutant proteins act as competitive inhibitors of the Sec translocase. This suggests that the region to be translocated is sensed by SecA but the activation of the SecA translocation ATPase is only successful for substrates with a minimum length of the translocated region.
Insights
M13 procoat protein insertion into Escherichia coli membranes becomes Sec-dependent with longer periplasmic loops. Longer loops are required for SecA activation, suggesting a minimum substrate length is essential for translocation.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Transport
Background:
- The M13 procoat protein exemplifies Sec-independent membrane insertion in E. coli.
- It possesses two hydrophobic regions and a 20-amino acid periplasmic loop.
- Loop length critically influences the protein's membrane insertion pathway.
Purpose of the Study:
- To investigate the effect of elongating the M13 procoat protein's periplasmic loop on its membrane insertion pathway.
- To determine the role of loop length in Sec-dependent translocation and SecA ATPase activation.
Main Methods:
- Construction and analysis of M13 procoat mutants with extended periplasmic loop regions.
- In vitro assays to assess SecA binding and ATPase activity.
- In vivo translocation efficiency studies.
Main Results:
- Extension of the periplasmic loop (≥118 residues) shifted M13 procoat insertion to a Sec-dependent pathway, requiring SecA and SecYEG.
- Mutants with loop lengths of 80 and 100 residues, and a mutant with two extra glutamyl residues, bound SecA but failed to activate its ATPase.
- These mutants also showed defective precursor-stimulated SecA binding to the membrane and acted as competitive inhibitors of the Sec translocase.
Conclusions:
- A minimum length of the translocated region is necessary for efficient SecA ATPase activation and successful translocation.
- SecA senses the region to be translocated, but activation is substrate-length dependent.
- Mutant M13 procoat proteins can inhibit the Sec translocase system.