Dissecting the Molecular Properties of Prokaryotic Flotillins

Abstract

Flotillins are universally conserved proteins that are present in all kingdoms of life. Recently it was demonstrated that the B. subtilis flotillin YuaG (FloT) has a direct influence on membrane domain formation by orchestrating lipid domains. Thereby it allocates a proper environment for diverse cellular machineries. YuaG creates platforms for signal transduction, processes crucial for biofilm formation, sporulation, competence, secretion, and others. Even though, flotillins are an emerging topic of research in the field of microbiology little is known about the molecular architecture of prokaryotic flotillins. All flotillins share common structural elements and are tethered to the membrane N’- terminally, followed by a so called PHB domain and a flotillin domain. We show here that prokaryotic flotillins are, similarly to eukaryotic flotillins, tethered to the membrane via a hairpin loop. Further it is demonstrated by sedimentation assays that B. subtilis flotillins do not bind to the membrane via their PHB domain contrary to eukaryotic flotillins. Size exclusion chromatography experiments, blue native PAGE and cross linking experiments revealed that B. subtilis YuaG can oligomerize into large clusters via the PHB domain. This illustrates an important difference in the setup of prokaryotic flotillins compared to the organization of eukaryotic flotillins.

Flotillin mediated membrane fluidity controls peptidoglycan synthesis and MreB movement

“…Order parameters of POPC-d31 acyl chain were derived from experimental quadrupolar splittings and plotted as a function of the labeled carbon position. Insert : schematic depiction of a liposome with added FloT which attaches to the membrane via a hairpin loop 45 .…”

Non-classical Protein Excretion Is Boosted by PSMα-Induced Cell Leakage

“…Deletions were generated in the accessory secretory system ( secA2-Y2 ), the twin arginine translocation system ( tatAC ) (Biswas et al, 2009), the ESAT-6 secretion system (Burts et al, 2005), the holin ( cidAB ) (Rice et al, 2003) and anti-holin system ( lrgAB ) (Ranjit et al, 2011), and the competence system ( comEC ) (Draskovic and Dubnau, 2005). In addition we also created mutants in flotillin genes ( floA and floT ) that are potentially involved in lipid raft formation (Bach and Bramkamp, 2015). Finally, we also inactivated the global regulator agr , which controls a large number of secreted S. aureus toxins (Koenig et al, 2004; Peng et al, 1988).…”

“…One of the first questions we addressed was whether their abundance in the supernatant was dependent on one of the known alternative protein secretion systems such as Tat, accessory Sec-system (SacA2-Y2), ESAT-6 (Type VII), the holin-antiholin systems Cid and Lrg, or the competence system (com). We also investigated floA and floB mutants as these genes are involved in lipid raft-associated cellular processes, including membrane sorting, trafficking, cell polarization, and signal transduction (Bach and Bramkamp, 2015; Bramkamp and Lopez, 2015). However, none of the mutations showed a conspicuous decrease in FbaA excretion.…”

Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus

“…Bacterial FMM appear to facilitate efficient oligomerization of FMM-associated protein complexes (FMM act as “oligomerization factories”) [ 12 ], and flotillin scaffold activity has a key role in facilitating interaction of FMM-associated membrane protein partners. Biochemical approaches such as pull-down experiments or BN-PAGE coupled with immunodetection demonstrated flotillin interaction with FMM-associated proteins, as well as its importance for correct oligomerization of protein partners in various bacterial species [ 9 , 14 , 25 , 55 , 57 , 77 ].…”