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This Article Full Text (Papers in Press[PDF]) All Versions of this Article: mic.0.027219-0v1 155/6/1847    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Masi, M. Articles by Misra, R. Search for Related Content PubMed PubMed Citation Articles by Masi, M. Articles by Misra, R. Agricola Articles by Masi, M. Articles by Misra, R.

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

¹ Institut Pasteur;
² University of Houston;
³ Arizona State University

ABSTRACT

TolC is a multifunctional outer membrane protein (OMP) of Escherichia coli that folds into a unique /β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independent of known periplasmic folding factors. Yet TolC's assembly, 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 (TolCG2-22). A significant amount of TolCG2-22 was found 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 TolCG2-22 were found to associate with membranes, but these failed to assume a wild-type-like folded conformation. The null phenotype of TolC2-22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCG2-22 resumed normal function and folded like wild type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCG2-22 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 TolC's unique folding and assembly pathways to its large soluble -helical domain.

⁴ E-mail: rajeev.misra{at}asu.edu