Trigger Factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli

Area of Science:

• Molecular Biology
• Protein Folding and Chaperones
• Bacterial Protein Targeting

Background:

• Nascent polypeptides require molecular chaperones and targeting factors for proper cellular localization (cytoplasmic, membrane, or exported).
• In Escherichia coli, SecB, Trigger Factor (TF), and DnaK are crucial for polypeptide processing post-translation.
• SecB deficiency leads to a cold-sensitive (Cs) phenotype and protein aggregation.

Purpose of the Study:

• To investigate the role of Trigger Factor (TF) in the cold-sensitive phenotype observed in SecB-deficient Escherichia coli.
• To elucidate the functional relationship between TF, SecB, and the DnaK/DnaJ chaperone system in protein targeting and folding.
• To understand TF's contribution to cotranslational protein targeting to the Sec translocon.

Main Methods:

• Analysis of cold-sensitive (Cs) phenotypes in various Escherichia coli mutant strains (secB, dnaK, dnaJ, tig).
• Suppressor analyses to identify genetic interactions between TF and other chaperone/targeting factors.
• Assessment of protein aggregation levels in mutant strains.
• Biochemical assays to determine the involvement of TF's ribosome-binding, chaperone, and PPIase activities.
• Cellular fractionation to analyze the association of SecA and ribosomes with the inner membrane.

Main Results:

• Inactivating mutations in the tig gene (encoding TF) fully suppressed the Cs phenotype and protein aggregation in secB null mutants.
• TF's antagonistic effect on SecB function is dependent on its ribosome-binding and chaperone activities, not its PPIase activity.
• TF inactivation partially suppressed the Cs phenotype of a compromised DnaK/DnaJ chaperone system.
• The secB dnaJ double mutant is viable only when TF is absent, highlighting TF's antagonistic role.
• Absence of TF leads to increased association of SecA and ribosomes with the inner membrane, suggesting interference with cotranslational targeting.

Conclusions:

• Trigger Factor (TF) plays an antagonistic role to SecB in protein folding and targeting in Escherichia coli, particularly under cold stress.
• TF's chaperone and ribosome-binding activities are critical for its antagonistic function, independent of its PPIase activity.
• TF appears to interfere with the efficient cotranslational targeting of polypeptides to the Sec translocon, impacting protein secretion pathways.