The understanding of the sorting and extraction of lipoproteins through the LolCDE ABC-transporter from the inner membrane to the periplasmic chaperone LolA

Details

Supervisors

Jean-François Collet ; François Chaumont

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

The cell envelope of Gram-negative bacteria is composed of the inner membrane (IM) and an outer membrane (OM). The IM and OM are separated by the periplasm. About one third of proteins of Escherichia coli (E. coli) are targeted to the envelope. An important group of envelope proteins are the lipoproteins, which are globular proteins anchored to one of the two membranes by a lipid moiety (triacyls). They carry out a variety of important functions. Lipoproteins are synthesized in the cytoplasm as precursors. After their transport to the periplasmic side of the IM, three well-conserved enzymes (Lgt, Lsp and Lnt) are involved in their maturation process. The mature lipoprotein either remains anchored in the IM or is transported to the OM. In Gram-negative bacteria, approximately 95% of lipoproteins are predicted to be targeted to the OM. Lipoprotein sorting between the IM and OM is achieved by the lipoprotein trafficking system (the Lol system), which is essential for the survival of the cell and therefore an attractive target for the design of new antibiotics. In addition, it is responsible for transporting the lipoproteins to the OM across the hydrophilic periplasm. OM lipoproteins are extracted from the IM by the LolCDE ATP binding cassette (ABC) transporter and transferred to the chaperone LolA. LolA shuttles lipoproteins across the periplasm and delivers them LolB, an OM lipoprotein itself, which anchors lipoproteins to the inner leaflet of the OM. The LolCDE complex comprises a heterodimer of LolC and LolE and a homodimer of LolD. LolD is present on the cytoplasmic side of the IM and has a nucleotide binding domain that enables ATP hydrolysis and provides the energy for the extraction of the lipoproteins from the IM to LolA. When LolA binds on the top of LolCDE, it is located ~60 Å away from the IM. This suggests that lipoproteins, with their hydrophobic lipid moiety, need to travel a considerable distance from the inner membrane to LolA. The goal of my master thesis is to better understand how the LolCDE complex can sort and extract lipoproteins from the IM and transfers them to LolA. To investigate how lipoproteins can cross this ~60 Å distance, in vivo site-directed photocrosslinking will be used to capture interactions between LolCDE and RcsF, a well-studied OM lipoprotein. E. coli will serve as our bacterial model. However, the Lol system is conserved in Gram-negative bacteria, including major pathogens. Our model suggests that only the lipid moiety of RcsF enters the LolCDE complex, while the linker makes the connection with the globular hydrophilic domain located outside the complex in the periplasm. It is suggested that both IM and OM lipoproteins do enter the LolCDE complex and that their sorting occurs within the LolCDE complex. In fact, IM lipoproteins, according to our results, are not avoided by LolCDE as the previous models suggested. Only OM lipoproteins are transported to LolA probably through the inside of the periplasmic region of LolC, while IM lipoproteins enter the cavity before exiting to remain anchored in the IM.