The Effect of the Osmotically Active Compound Concentration Difference on the Passive Water and Proton Fluxes across a Lipid Bilayer

The molecular details of the passive water flux across the hydrophobic membrane interior are still a matter of debate. One of the postulated mechanisms is the spontaneous, water-filled pore opening, which facilitates the hydrophilic connection between aqueous phases separated by the membrane. In the paper, we provide experimental evidence showing that the spontaneous lipid pore formation correlates with the membrane mechanics; hence, it depends on the composition of the lipid bilayer and the concentration of the osmotically active compound. Using liposomes as an experimental membrane model, osmotically induced water efflux was measured with the stopped-flow technique. Shapes of kinetic curves obtained at low osmotic pressure differences are interpreted in terms of two events: the lipid pore opening and water flow across the aqueous channel. The biological significance of the dependence of the lipid pore formation on the concentration difference of an osmotically active compound was illustrated by the demonstration that osmotically driven water flow can be accompanied by the dissipation of the pH gradient. The application of the Helfrich model to describe the probability of lipid pore opening was validated by demonstrating that the probability of pore opening correlates with the membrane bending rigidity. The correlation was determined by experimentally derived bending rigidity coefficients and probabilities of lipid pores opening.     

Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide

One of the reported substrates for the endoplasmic reticulum (ER) translocation inhibitor cyclotriazadisulfonamide (CADA) is DNAJC3, a chaperone of the unfolded protein response during ER stress. In this study, we investigated the impact of altered DNAJC3 protein levels on the inhibitory activity of CADA. By comparing WT DNAJC3 with a CADA-resistant DNAJC3 mutant, we observed the enhanced sensitivity of human CD4, PTK7 and ERLEC1 for CADA when DNAJC3 was expressed at high levels. Combined treatment of CADA with a proteasome inhibitor resulted in synergistic inhibition of protein translocation and in the rescue of a small preprotein fraction, which presumably corresponds to the CADA affected protein fraction that is stalled at the Sec61 translocon. We demonstrate that DNAJC3 enhances the protein translation of a reporter protein that is expressed downstream of the CADA-stalled substrate, suggesting that DNAJC3 promotes the clearance of the clogged translocon. We propose a model in which a reduced DNAJC3 level by CADA slows down the clearance of CADA-stalled substrates. This results in higher residual translocation into the ER lumen due to the longer dwelling time of the temporarily stalled substrates in the translocon. Thus, by directly reducing DNAJC3 protein levels, CADA attenuates its net down-modulating effect on its substrates.