Can An Autotransporter Protein Truly Transport Itself Across A Lipid Bilayer?

Abstract

Autotransporter (AT) proteins are synthesized with an N-terminal signal sequence, which is cleaved after secretion through the inner membrane, a passenger domain (the mature protein), and a C-terminal porin domain. The porin domain in inserted in the outer membrane (OM) and the passenger domain is secreted through this pore. OM secretion does not require ATP nor a proton gradient, therefore the driving force for efficient secretion remains unknown, nor is it known what prevents premature folding of the passenger in the periplasm. There is an ongoing debate in the literature about the role, if any, of interactions between AT proteins and periplasmic chaperones and/or OM proteins like Omp85 on AT secretion. We are developing an in vitro system to test the autonomy of AT secretion, using the model AT pertactin from Bordetella pertussis. By mixing unfolded pertactin passenger+porin domains with lipid vesicles, we will test whether the purified protein is competent for secretion across a membrane. While this does not exclude the possibility of chaperone interactions and the participation of other proteins in vivo, it would show that ATs have the capacity to cross a membrane independently, strengthening the importance of β-sheet formation as a potential driving force for OM secretion. Vesicles consisting of PC, 10% PG, and 1% NBD-PC were made; the fluorophore NBD is spread equally in the inner and outer membrane of the vesicles. Vesicle formation was confirmed by fluorescence microscopy, and quenching experiments. We have successfully expressed the isolated pertactin porin domain, and refolded it. Future work will determine the insertion of passenger+porin into vesicles, and the extent of transport, if any.