Biology subjects

Clifton, L. A.

Publications and source records attributed to Clifton, L. A..

4 recordsLinked to original sources

bioRxiv2026

PqiABC forms a membrane-bridging conduit and mediates bidirectional phospholipid transport across the Gram-negative bacterial envelope

The mechanism by which glycerophospholipids are transported between the inner and outer membranes in Gram-negative bacteria remains poorly understood. In Escherichia coli, the paraquat-inducible (Pqi) pathway, comprising the inner membrane protein PqiA, the periplasm-spanning MCE-family protein PqiB, and the outer membrane lipoprotein PqiC, has been implicated in this process. These components are proposed to assemble into a quaternary complex that forms a continuous channel bridging the inner and outer membranes. Here, using neutron reflectometry and quartz crystal microbalance with dissipation monitoring, we perform a dynamic structural analysis of PqiABC within a planar double bilayer membrane-mimetic system. This approach reveals that PqiABC assembles into a stable, envelope-spanning complex anchored to both membranes, consistent with its proposed conduit architecture. Furthermore, using neutron reflectometry in combination with complementary fluorescence-based assays, we demonstrate that PqiABC mediates passive glycerophospholipid transport, supporting bidirectional lipid exchange between membranes. Together, these findings establish PqiABC as a membrane-bridging lipid transport system and provide direct evidence for a mechanism of passive glycerophospholipid equilibration across the bacterial envelope.

biophysics↗

bioRxiv2025

Bcl-2 Oligomerizes Bax on the Mitochondrial Membrane Surface Preventing the Initial Stages of Apoptosis

The Bcl-2 family of proteins control mitochondrial outer membrane (MOM) pore formation, crucial to cellular clearance via apoptosis. However, the molecular principles by which opposing family members inhibit, mediate or promote MOM perforation remain elusive. Here, we demonstrate that cell-protecting Bcl-2 directly sequesters cell-killing Bax into a protein-protein complex at the membrane interface preventing Bax forming apoptotic pores. Neutron reflectometry showed Bax association with Bcl-2 occurs through the formation of Bax protein oligomers on the membrane surface. Bax binding to the membrane surface was proportional to the membrane-embedded Bcl-2 suggestive of protein-protein complex formation through both Bcl-2/Bax and Bax/Bax interactions. Bcl-2/Bax sequestration to prevent perforation was observed in membrane models with and without pro-apoptotic cardiolipin present. Our findings shed fundamental new light on the communication of Bcl-2 with its cell-killing relatives at the mitochondrias membraneous exterior to prevent cells from undergoing apoptosis. TeaserMembrane-embedded Bcl-2 sequesters Bax, to prevent the perforation of mitochondrial membranes by Bax which would initiate apoptosis.

biophysics↗

bioRxiv2022

Bax Forms a Membrane Surface Protein-Lipid Complex as it Initiates Apoptosis

Cellular clearance by apoptosis is essential in life. In its intrinsic (mitochondrial) pathway apoptotic members of the Bcl-2 (B cell CLL/lymphoma-2) protein family, such as Bax (Bcl-2-associated X) protein, perforate the mitochondrial outer membrane (MOM), which causes release of apoptotic factors and final cell death. How those apoptotic proteins mechanistically exert their action at the membrane level still however remains elusive. Upon internal stress signals Bax is massively recruited to the MOM, where it oligomerizes and partially penetrates into the membrane. Using neutron reflectometry (NR) and attenuated total reflection Fourier-Transform infrared spectroscopy (ATR-FTIR) spectroscopy we unraveled key molecular steps of this membrane-affiliation process of Bax on a spatial and temporal scale. By titrating intact human Bax to MOM-like bilayers containing cardiolipin, essential for protein recruitment, we could identify different functional phases. Initially, there is a fast adsorption event to the membrane surface with high affinity. Thereafter, a kinetically slower (minutes to hours) event occurs with Bax penetration, thereby triggering a major reorganizing of the mitochondrial bilayer. Finally, a membrane-Bax complex is generated, with a minor Bax population remaining membrane-inserted, while the main population is relocated to the membrane surface upon lipid redistribution into a complex with Bax; a process enabling membrane perforation. We propose a comprehensive molecular model of mitochondrial membrane penetration by formation of complex Bax/lipid clusters; a concept which provides a new foundation to understand the cell-killing activity of Bax and its apoptotic relatives in human cells. Significance StatementThe apoptotic Bax protein is a key player in the mitochondrial apoptotic pathway. Here, neutron reflectometry (NR) unravels the mechanism by which Bax is targeting and perforating mitochondria to release apoptotic factors for final cell death. We found that this cardiolipin driven process of the outer mitochondrial membrane system has two main phases. Upon a fast (10-20 min) phase of membrane association Bax initiates the formation of pores by removing lipids and depositing them as Bax/lipid complexes on top of the bilayer on a time scale of several hours similar to in vivo apoptotic cell death. Our results provide a mechanistic rationale for cell-killing processes driven by apoptotic Bcl-2 proteins; and their molecular inhibition in many cancers.

biophysics↗

bioRxiv2018

Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting {beta}-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

biophysics↗