Nonvesicular lipid trafficking pathways are an important
process
in every domain of life. The mechanisms of these processes are poorly
understood in part due to the difficulty in kinetic characterization.
One important class of glycolipids, lipopolysaccharides (LPS), are
the primary lipidic component of the outer membrane of Gram-negative
bacteria. LPS are synthesized in the inner membrane and then trafficked
to the cell surface by the lipopolysaccharide transport proteins, LptB2FGCADE. By characterizing the interaction of a fluorescent probe
and LPS, we establish a quantitative assay to monitor the flux of
LPS between proteoliposomes on the time scale of seconds. We then
incorporate photocaged ATP into this system, which allows for light-based
control of the initiation of LPS transport. This control allows us
to measure the initial rate of LPS transport (3.0 min–1 per LptDE). We also find that the rate of LPS transport by the Lpt
complex is independent of the structure of LPS. In contrast, we find
the rate of LPS transport is dependent on the proper function of the
LptDE complex. Mutants of the outer membrane Lpt components, LptDE,
that cause defective LPS assembly in live cells display attenuated
transport rates and slower ATP hydrolysis compared to wild type proteins.
Analysis of these mutants reveals that the rates of ATP hydrolysis
and LPS transport are correlated such that 1.2 ± 0.2 ATP are
hydrolyzed for each LPS transported. This correlation suggests a model
where the outer membrane components ensure the coupling of ATP hydrolysis
and LPS transport by stabilizing a transport-active state of the Lpt
bridge.