Related Experiment Videos
Module swaps between related translocator proteins pIV(f1), pIV(IKe) and PulD: identification of a specificity domain
Abstract:
In Gram-negative bacteria, type II and type III secretion and filamentous phage assembly systems use related outer membrane proteins for substrate-specific transport across the outer membrane. We show here that the specificity domain of the phage f1 outer membrane protein pIV is contained within the 149 N-terminal amino acid residues. When the pIV(f1) specificity domain is fused to the translocator domain of the related pIV of phage IKe, the chimeric construct supports f1 but not IKe assembly. Functional coupling between the two domains in this chimeric construct is poor and is improved by a single amino acid change in the translocator domain of the pIV(IKe). In native pIV(IKe), two amino acid changes within its specificity domain are both necessary and sufficient to change the specificity from IKe to f1 assembly. Analysis of 39 chimeric constructs between pIV(f1) and the outer membrane protein PulD of the pullulanase secretion system failed to identify a comparable exchangeable specificity domain. These results indicate that the two domains may not function autonomously, and suggest that tertiary and quarternary changes of the entire translocator component rather than of an autonomous functional domain are required for specific translocation across the outer membrane.
Insights
Researchers identified the specificity domain of phage f1 outer membrane protein pIV. Specificity changes in bacterial secretion systems may involve broader protein structural alterations rather than isolated domains.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- Type II and type III secretion systems and filamentous phage assembly in Gram-negative bacteria rely on outer membrane proteins for substrate-specific transport.
- Outer membrane proteins facilitate the translocation of substrates across the bacterial outer membrane.
Purpose of the Study:
- To investigate the role of specific domains within the phage f1 outer membrane protein pIV in determining substrate specificity.
- To understand the functional relationship between specificity and translocator domains in outer membrane protein function.
Main Methods:
- Construction and functional analysis of chimeric proteins between phage f1 pIV and phage IKe pIV.
- Site-directed mutagenesis to identify key amino acid residues affecting protein function and specificity.
- Analysis of chimeric constructs between phage f1 pIV and the outer membrane protein PulD.
Main Results:
- The N-terminal 149 amino acids of phage f1 pIV contain its specificity domain.
- A chimeric protein with the f1 specificity domain and IKe translocator domain supported f1 assembly, but not IKe assembly.
- Specific amino acid changes in the IKe pIV specificity domain were sufficient to confer f1 assembly specificity.
- No exchangeable specificity domain was identified when comparing f1 pIV and PulD, suggesting domain non-autonomy.
Conclusions:
- The specificity and translocator domains of outer membrane proteins may not function independently.
- Specific substrate translocation across the outer membrane likely requires coordinated tertiary and quaternary structural changes in the entire translocator component, not just an autonomous specificity domain.