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Sorting of lipids and proteins is a key process allowing eukaryotic cells to execute efficient and accurate intracellular transport and to maintain membrane homeostasis. It occurs during the formation of highly curved transport intermediates that shuttle between cell compartments. Protein sorting is reasonably well described, but lipid sorting is much less understood. Lipid sorting has been proposed to be mediated by a physical mechanism based on the coupling between membrane composition and high curvature of the transport intermediates. To test this hypothesis, we have performed a combination of fluorescence and force measurements on membrane tubes of controlled diameters pulled from giant unilamellar vesicles. A model based on membrane elasticity and nonideal solution theory has also been developed to explain our results. We quantitatively show, using 2 independent approaches, that a difference in lipid composition can build up between a curved and a noncurved membrane. Importantly, and consistent with our theory, lipid sorting occurs only if the system is close to a demixing point. Remarkably, this process is amplified when even a low fraction of lipids is clustered upon cholera toxin binding. This can be explained by the reduction of the entropic penalty of lipid sorting when some lipids are bound together by the toxin. Our results show that curvature-induced lipid sorting results from the collective behavior of lipids and is even amplified in the presence of lipid-clustering proteins. In addition, they suggest a generic mechanism by which proteins can facilitate lipid segregation in vivo. [ABSTRACT FROM AUTHOR]

TRPC and Orai proteins have both been proposed to form Ca2+- selective, store-operated calcium entry (SOCE) channels that are activated by store-depletion with Ca2+ chelators or calcium pump inhibitors. In contrast, only TRPC proteins have been proposed to form nonselective receptor-operated calcium entry (ROCE) cation channels that are activated by Gq/Gi-PLCβ signaling, which is the physiological stimulus for store depletion. We reported previously that a dominant negative Orai1 mutant, R91W. inhibits Ca2+ entry through both SOCE and ROCE channels, implicating Oral participation in both channel complexes. However, the argument for Orai participating in ROCE independently of store depletion is tenuous because store depletion is an integral component of the ROCE response, which includes formation of lP3, a store-depleting agent. Here we show that the R91W mutant also blocks diacylglycerol (DAG)-activated Ca2+ entry into cells that stably, or transiently, express DAG-responsive TRPC proteins. This strongly suggests that Oral and TRPC proteins form complexes that participate in Ca2+ entry with or without activation of store depletion. To integrate these results with recent data linking SOCE with recruitment of Orai and TRPCs to lipid rafts by STIM, we develop the hypothesis that Orai:TRPC complexes recruited to lipid rafts mediate SOCE, whereas the same complexes mediate ROCE when they are outside of lipid rafts. It remains to be determined whether the molecules forming the permeation pathway are the same when Orai:TRPC complexes mediate ROCE or SOCE. [ABSTRACT FROM AUTHOR]

Misfolding and subsequent aggregation of endogeneous proteins constitute essential steps in many human disorders, including Alzheimer and prion diseases. In most prion protein-folding studies, the posttranslational modifications, the lipid anchor in particular, were lacking. Here, we studied a fully posttranslationally modified cellular prion protein, carrying two N-glycosylations and the natural GPI anchor. We used time-resolved FTIR to study the prion protein secondary structure changes when binding to a raft-like lipid membrane via its GPI anchor. We observed that membrane anchoring above a threshold concentration induced refolding of the prion protein to intermolecular β-sheets. Such transition is not observed in solution and is membrane specific. Excessive membrane anchoring, analyzed with molecular sensitivity. is thought to be a crucial event in the development of prion diseases. [ABSTRACT FROM AUTHOR]

Misfolding and subsequent aggregation of endogeneous proteins constitute essential steps in many human disorders, including Alzheimer and prion diseases. In most prion protein-folding studies, the posttranslational modifications, the lipid anchor in particular, were lacking. Here, we studied a fully posttranslationally modified cellular prion protein, carrying two N-glycosylations and the natural GPI anchor. We used time-resolved FTIR to study the prion protein secondary structure changes when binding to a raft-like lipid membrane via its GPI anchor. We observed that membrane anchoring above a threshold concentration induced refolding of the prion protein to intermolecular β-sheets. Such transition is not observed in solution and is membrane specific. Excessive membrane anchoring, analyzed with molecular sensitivity. is thought to be a crucial event in the development of prion diseases. [ABSTRACT FROM AUTHOR]

The dynamical coupling between proteins and their hydration water is important for the understanding of macromolecular function in a cellular context. In the case of membrane proteins, the environment is heterogeneous, composed of lipids and hydration water, and the dynamical coupling might be more complex than in the case of the extensively studied soluble proteins. Here, we examine the dynamical coupling between a biological membrane, the purple membrane (PM), and its hydration water by a combination of elastic incoherent neutron scattering, specific deuteration, and molecular dynamics simulations. Examining completely deuterated PM, hydrated in H2O, allowed the direct experimental exploration of water dynamics. The study of natural abundance PM in D2O focused on membrane dynamics. The temperature-dependence of atomic mean-square displacements shows inflections at 120 K and 260 K for the membrane and at 200 K and 260 K for the hydration water. Because transition temperatures are different for PM and hydration water, we conclude that ps-ns hydration water dynamics are not directly coupled to membrane motions on the same time scale at temperatures <260 K. Molecular-dynamics simulations of hydrated PM in the temperature range from 100 to 296 K revealed an onset of hydration-water translational diffusion at 200 K, but no transition in the PM at the same temperature. Our results suggest that, in contrast to soluble proteins, the dynamics of the membrane protein is not controlled by that of hydration water at temperatures <260 K. Lipid dynamics may have a stronger impact on membrane protein dynamics than hydration water. [ABSTRACT FROM AUTHOR]

Single biomolecules in free solution have long been of interest for detailed study by optical methods, but Brownian motion prevents the observation of one single molecule for extended periods. We have used an anti-Brownian electrokinetic (ABEL) trap to trap individual protein molecules in free solution, under ambient conditions, without requiring any attachment to beads or surfaces. We also demonstrate trapping and manipulation of single virus particles, lipid vesicles, and fluorescent semiconductor nanocrystals. [ABSTRACT FROM AUTHOR]

Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, synaptobrevin, and synaptosome-associatacl protein of 25 kDa on deposited lipid bilayers. Surprisingly, we found a mixture of parallel as well as antiparallel configurations involving the SNARE motifs of syntaxin and synaptobrevin as well as those of syntaxin and synaptosome-associated protein of 25 kDa. The subpopulation with the parallel four-helix bundle configuration could be greatly enriched by an additional purification step in the presence of denaturant, indicating that the parallel configuration is the energetically most favorable state. Interconversion between the configurations was not observed. From this observation, we infer the conversion rate to be <1.5 h[sup-1. The existence of antiparallel configurations suggests a regulatory role of chaperones, such as N-ethylmaleimide-sensitive factor, or the membrane environment during SNARE complex assembly in vivo, and it could be a partial explanation for the relatively slow rates of vesicle fusion observed by reconstituted fusion experiments in vitro. [ABSTRACT FROM AUTHOR]

The article discusses the result of a study conducted by C.S. Ejsing and colleagues which identified lipid molecular species in the yeast lipidome and quantified them. In choosing yeast, the researchers have simplified the challenge because these cells can be grown on a miminal medium lacking lipids. They also found that genes and transcripts do not always predict precise levels of active proteins/enzymes.