Determination\nof Initial Rates of Lipopolysaccharide\nTransport

Nonvesicular lipid trafficking pathways are an important\nprocess\nin every domain of life. The mechanisms of these processes are poorly\nunderstood in part due to the difficulty in kinetic characterization.\nOne important class of glycolipids, lipopolysaccharides (LPS), are\nthe primary lipidic component of the outer membrane of Gram-negative\nbacteria. LPS are synthesized in the inner membrane and then trafficked\nto the cell surface by the <b>l</b>ipo<b>p</b>olysaccharide <b>t</b>ransport proteins, <u>Lpt</u>B<sub>2</sub>FGCADE. By characterizing the interaction of a fluorescent probe\nand LPS, we establish a quantitative assay to monitor the flux of\nLPS between proteoliposomes on the time scale of seconds. We then\nincorporate photocaged ATP into this system, which allows for light-based\ncontrol of the initiation of LPS transport. This control allows us\nto measure the initial rate of LPS transport (3.0 min<sup>–1</sup> per LptDE). We also find that the rate of LPS transport by the Lpt\ncomplex is independent of the structure of LPS. In contrast, we find\nthe rate of LPS transport is dependent on the proper function of the\nLptDE complex. Mutants of the outer membrane Lpt components, LptDE,\nthat cause defective LPS assembly in live cells display attenuated\ntransport rates and slower ATP hydrolysis compared to wild type proteins.\nAnalysis of these mutants reveals that the rates of ATP hydrolysis\nand LPS transport are correlated such that 1.2 ± 0.2 ATP are\nhydrolyzed for each LPS transported. This correlation suggests a model\nwhere the outer membrane components ensure the coupling of ATP hydrolysis\nand LPS transport by stabilizing a transport-active state of the Lpt\nbridge.