The asymmetric outer membrane (OM) of gram-negative bacteria consists of phospholipids (PL) as well as lipopolysaccharides (LPS), with LPS exclusively located in the outer leaflet. LPS levels in Escherichia coli are strictly controlled by post-translational regulation of LpxC, the key enzyme of LPS synthesis. LpxC is degraded by the membrane-bound protease FtsH. This process is regulated by the adaptor protein LapB (YciM), which binds LpxC and directs it towards FtsH. Further, based on periplasmic LPS levels, the activity of LapB itself is controlled by the anti-adaptor protein YejM. Our previous studies demonstrated that LapB interacts with other proteins in the lipopolysaccharide synthesis pathway, namely LpxA and LpxD (Möller et al., 2023). In addition, we found that LapB binds FabZ, the first enzyme in the phospholipid synthesis (PL) pathway. Since the proper balance between LPS and PL is mandatory for bacterial survival, LapB is believed to be a central player in cell envelope synthesis by serving as an anchor for the involved cytosolic enzymes. We now investigate, how the interaction between those proteins is navigated.

We study the formation of protein complexes with purified LapB and its interaction partners to understand if the protein-protein interactions of LapB can occur simultaneously. Therefore, we use different in vitro approaches, such as size exclusion chromatography, blue native PAGE and pull downs. In addition, we employ targeted mutagenesis to investigate the interaction sites where LapB binds to its interaction partners, since it harbors nine tetratricopeptide repeat motifs (TPR) in its cytosolic domain. The results will provide insights into how LapB coordinates the assembly of a dynamic protein complex to orchestrate cell envelope biogenesis.