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The assembly pathway of outer membrane protein PhoE of Escherichia coli
C Jansen1, M Heutink, J Tommassen
1Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, The Netherlands.
Abstract:
The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.
Insights
Investigating Escherichia coli PhoE porin assembly revealed intermediates and a C-terminal phenylalanine sorting signal. Wild-type PhoE assembles rapidly, while mutants show slower kinetics, highlighting key assembly steps.
Area of Science:
- Molecular Biology
- Biochemistry
- Microbiology
Background:
- Outer membrane porins are essential for bacterial cell envelope function.
- The assembly pathway of porins into the outer membrane is complex and not fully understood.
- Escherichia coli PhoE porin serves as a model system for studying outer membrane protein assembly.
Purpose of the Study:
- To investigate the in vitro and in vivo assembly process of wild-type and mutant PhoE porins from Escherichia coli.
- To identify and characterize intermediates in the PhoE assembly pathway.
- To elucidate the role of specific protein regions, such as the C-terminal phenylalanine, in PhoE assembly and localization.
Main Methods:
- In vivo pulse-chase experiments to track protein assembly kinetics.
- In vitro refolding studies using detergents like N,N-dimethyldodecylamine-N-oxide (LDAO).
- Analysis of mutant proteins (DeltaF330 and a beta-strand mutant) to assess assembly defects.
- SDS-PAGE and trypsin sensitivity assays to characterize protein intermediates.
Main Results:
- Wild-type PhoE exhibits rapid assembly, with half assembling within 30 seconds, while the other half follows slower kinetics involving soluble monomers, cell envelope-associated monomers, and metastable trimers.
- The C-terminal phenylalanine residue in PhoE (DeltaF330 mutant) appears to function as a sorting signal, influencing assembly kinetics but not intrinsic folding ability.
- A mutant with a hydrophilic residue in a beta-strand showed altered in vitro folding in a lipopolysaccharide/Triton system, suggesting folded monomers may not be true in vivo assembly intermediates.
Conclusions:
- The assembly of PhoE porin is a multi-step process involving distinct intermediates.
- The C-terminal phenylalanine residue is crucial for efficient sorting of PhoE to the outer membrane.
- The role of folded monomers as assembly intermediates requires further investigation, potentially depending on the specific in vitro system used.