Biology subjects

Humpolickova, J.

Publications and source records attributed to Humpolickova, J..

4 recordsLinked to original sources

bioRxiv2026

A Kiss-and-Ride Mechanism for Lipid Exchange at Membrane Contact Sites

Lipid transfer proteins (LTPs) mediate non-vesicular lipid transport at membrane contact sites, yet how transient membrane tethering contributes to lipid transfer remains poorly understood. Here, we use the single-domain yeast phosphatidylserine transporter Osh6, a member of the oxysterol-binding protein-related protein family, to investigate tethering-dependent lipid transport. We show that Osh6 can access two transport modes depending on membrane composition: conventional shuttling between membranes and a tethering-dependent mode in which the opposing membrane facilitates cargo extraction from the donor membrane. Combining lipid transport and membrane-tethering measurements with kinetic analysis, lipid partitioning, and in vivo complementation, we provide evidence for a transient membrane-bridged intermediate during lipid transfer. Productive phosphatidylinositol 4-phosphate (PI4P) extraction destabilizes this bridged state, allowing cargo-loaded Osh6 to disengage and continue the transport cycle. We term this mechanism kiss-and-ride, in which transient membrane contact facilitates cargo extraction ("kiss") before the cargo-loaded protein disengages ("ride"). Consistent with this model, PI4P transport is preserved in a lidless Osh6 mutant with impaired membrane dissociation, whereas phosphatidylserine transport is strongly reduced, revealing distinct mechanistic requirements for the two cargoes. Together, our findings reveal a tethering-dependent transport mode in a cup-like LTP and suggest that membrane contact sites can actively shape the mechanism of lipid transport rather than merely bringing donor and acceptor membranes into proximity.

biophysics↗

bioRxiv2023

Coordination of transporter, cargo, and membrane properties during non-vesicular lipid transport.

Homeostasis of cellular membranes is maintained by fine-tuning their lipid composition. Yeast lipid transporter Osh6, belonging to the oxysterol-binding protein-related proteins family, was found to participate in the transport of phosphatidylserine (PS). PS synthesized in the endoplasmic reticulum is delivered to the plasma membrane, where it is exchanged for phosphatidylinositol-4 phosphate (PI4P). PI4P provides the driving force for the directed PS transport against its concentration gradient. In this study, we employed an in vitro approach to reconstitute the transport process into the minimalistic system of large unilamellar vesicles to reveal its fundamental biophysical determinants. Our study draws a comprehensive portrait of the interplay between the structure and dynamics of Osh6, the carried cargo lipid, and the physical properties of the involved membranes, with particular attention to their charge and fluidity. Specifically, we address the role of the cargo lipid, which, by occupying the transporter, imposes changes in its dynamics and, consequently, predisposes the cargo to disembark in the correct target membrane.

biophysics↗

bioRxiv2018

Dimerization of rhomboid protease RHBDL2 in lipid membranes addressed by FRET with MC simulations

Many membrane proteins are thought to function as oligomers, but measuring membrane protein dimerization in native lipid membranes is particularly challenging. Forster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are non-invasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. The effects inherent to such two-dimensional systems, such as excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and FCS we introduce methods to measure surface protein density and to estimate kappa squared, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined 2D environments. We then use FRET and FCS to analyze the dimerization of human rhomboid protease RHBDL2 in its native lipid membranes. While previous reports have proposed that rhomboid proteases dimerize and this allosterically activates them, we find no evidence for stable oligomers of RHBDL2 in lipid membranes of human cells. This indicates that the rhomboid transmembrane core may be intrinsically monomeric. Finally, our findings will find use in the application of FRET and FCS for the analysis of oligomerization of transmembrane proteins in lipid membranes.

biophysics↗

bioRxiv2018

iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires ADAM17/TACE, which cleaves TNF from its transmembrane tether, releasing it for signalling. The trafficking of ADAM17/TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. Here we report a novel protein, that we name iTAP (iRhom tail-associated protein) that binds to iRhoms, enhancing the stability of iRhoms and TACE, preventing their degradation in lysosomes. iTAP-null primary human macrophages, or tissues from iTAP KO mice, are dramatically depleted in the levels of iRhom2 and active TACE, and are, consequently, profoundly impaired in TNF production. Our work illustrates iTAP as a physiological rheostat controlling TNF signalling and a novel target for the control of inflammation.

biochemistry↗