Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli

Abstract

TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique α/β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of β-barrel OMPs, is dependent on BamA and BamD, two essential components of the β-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCΔ2–22). A significant amount of TolCΔ2–22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCΔ2–22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCΔ2–22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCΔ2–22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCΔ2–22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble α-helical domain.

tRNA Methylation Is a Global Determinant of Bacterial Multi-drug Resistance

“…(F) Cells expressing C37-tRNA Pro/UGG (red) died faster than cells expressing G37-tRNA Pro/UGG (blue) after exposure to gentamicin (Gen) or vancomycin (Van). Data and error bars are mean ± SEM, n = 3. and substitution of Pro with Ala by mutating the CCC codon to GCG reduced the protein to undetectable levels (data not shown), probably because of membrane mistargeting and destabilization (Masi et al, 2009). These data suggest that the conservation of Pro at the 6 th position is critical for TolC structure and function and that its incorporation into the protein is regulated at the codon level by m 1 G37.…”

Dissection of β-barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli

“…It is possible that the presence of the large soluble domains and their intrinsic folding properties render the folding of these two proteins independent of known periplasmic folding factors. In support of this notion, we recently showed that a small quantity of signal sequence‐less mature TolC can fold and trimerize in the cytoplasm, but was unable to properly insert into the inner membrane (Masi et al ., 2009).…”

Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members

“…The OMP assembly pathway as it pertains to multimerization of oligomers, and how the Bam complex participates, is still not fully elucidated (5,26,27). It has been suggested that some multimeric OMPs, such as PhoE and TolC, do not proceed through a folded monomeric intermediate form, but rather, trimerization occurs concomitantly with folding and assembly and may help precipitate folding in the first place (28,29), while other OMPs, like LamB, may proceed though folded monomeric intermediates that then trimerize upon insertion in the OM (30). If multimeric OMPs (e.g., LamB) indeed multimerize before or during OM integration, this would necessitate a mechanism by which individual neighboring Bam complexes could act coordinately to simultaneously integrate multiple monomers of compatible OMP species.…”