Abstract
The outer membrane-directed lipoproteins are released from the inner membrane of Escherichia coli as a complex with LolA, a periplasmic chaperone. The LolA-dependent release of lipoproteins is critical for lipoprotein sorting as it depends on the outer membrane-specific sorting signal. To clarify molecular events involved in the LolA-dependent lipoprotein release, we attempted to establish an in vitro assay system. The major outer membrane lipoprotein (Lpp) was found to lose its release competence soon after being processed to mature Lpp in the inner membrane and therefore could not be used as a substrate for an in vitro system. An Lpp derivative, L10P, was constructed and found to retain the release competence long after its maturation. L10P was synthesized and radiolabeled in spheroplasts in the absence of LolA; therefore, it remained anchored to the inner membrane of spheroplasts. Right-side out membrane vesicles containing L10P were then prepared and used to examine the release of L10P. In addition to LolA, L10P release absolutely required nucleoside triphosphate (NTP). A non-hydrolyzable NTP analogue strongly inhibited the NTP-dependent release. The outer membrane-specific sorting signal was essential for the in vitro release of L10P. Furthermore, L10P released in vitro was specifically incorporated into the outer membrane. These results indicate that the in vitro release of L10P represents an in vivo reaction and requires energy.
Highlights
Lipid-modified proteins such as Rab in eukaryotes (1) and lipoproteins in bacteria (2, 3) have been studied with respect to post-translational modification pathways, physiological significance, and membrane targeting mechanisms
Vesicular transport requires correct membrane targeting of Rab (1), and the mislocation of lipoprotein is lethal to Escherichia coli (4)
Three derivatives were found to be correctly localized to the outer membrane in vivo. These results indicate that all the mutations introduced into Lpp do not affect the interaction with LolA and LolB, both of which play critical roles in the outer membrane localization of lipoproteins
Summary
Lipid-modified proteins such as Rab in eukaryotes (1) and lipoproteins in bacteria (2, 3) have been studied with respect to post-translational modification pathways, physiological significance, and membrane targeting mechanisms. To examine the post-processing release of Lpp, labeling of spheroplasts was started and terminated as described above but in the absence of LolA.
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