MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress.

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.

Dynamic Second Harmonic Imaging of Proton Translocation Through Water Needles in Lipid Membranes.

Proton translocation through lipid membranes is a fundamental process in the field of biology. Several theoretical models have been developed and presented over the years to explain the phenomenon, yet the exact mechanism is still not well understood. Here, we show that proton translocation is directly related to membrane potential fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we report apparently universal transmembrane potential fluctuations in lipid membrane systems. Molecular simulations and free energy calculations suggest that H+ permeation proceeds predominantly across a thin, membrane-spanning water needle and that the transient transmembrane potential drives H+ ions across the water needle. This mechanism differs from the transport of other cations that require completely open pores for transport and follows naturally from the well-known Grotthuss mechanism for proton transport in bulk water. Furthermore, SH imaging and conductivity measurements reveal that the rate of proton transport depends on the structure of the hydrophobic core of bilayer membranes.

Molecular Simulations Reveal the Free Energy Landscape and Transition State of Membrane Electroporation.

The formation of pores over lipid membranes by the application of electric fields, termed membrane electroporation, is widely used in biotechnology and medicine to deliver drugs, vaccines, or genes into living cells. Continuum models for describing the free energy landscape of membrane electroporation were proposed decades ago, but they have never been tested against spatially detailed atomistic models. Using molecular dynamics (MD) simulations with a recently proposed reaction coordinate, we computed potentials of mean force of pore nucleation and pore expansion in lipid membranes at various transmembrane potentials. Whereas the free energies of pore expansion are compatible with previous continuum models, the experimentally important free energy barrier of pore nucleation is at variance with established models. The discrepancy originates from different geometries of the transition state; previous continuum models assumed the presence of a membrane-spanning defect throughout the process, whereas, according to the MD simulations, the transition state of pore nucleation is typically passed before a transmembrane defect has formed. A modified continuum model is presented that qualitatively agrees with the MD simulations. Using kinetics of pore opening together with transition state theory, our free energies of pore nucleation are in excellent agreement with previous experimental data.

Phosphopeptide binding to the N-SH2 domain of tyrosine phosphatase SHP2 correlates with the unzipping of its central ß-sheet.

SHP2 is a tyrosine phosphatase that plays a regulatory role in multiple intracellular signaling cascades and is known to be oncogenic in certain contexts. In the absence of effectors, SHP2 adopts an autoinhibited conformation with its N-SH2 domain blocking the active site. Given the key role of N-SH2 in regulating SHP2, this domain has been extensively studied, often by X-ray crystallography. Using a combination of structural analyses and molecular dynamics (MD) simulations we show that the crystallographic environment can significantly influence the structure of the isolated N-SH2 domain, resulting in misleading interpretations. As an orthogonal method to X-ray crystallography, we use a combination of NMR spectroscopy and MD simulations to accurately determine the conformation of apo N-SH2 in solution. In contrast to earlier reports based on crystallographic data, our results indicate that apo N-SH2 in solution primarily adopts a conformation with a fully zipped central ß-sheet, and that partial unzipping of this ß-sheet is promoted by binding of either phosphopeptides or even phosphate/sulfate ions.

Hairpin protein partitioning from the ER to lipid droplets involves major structural rearrangements.

Lipid droplet (LD) function relies on proteins partitioning between the endoplasmic reticulum (ER) phospholipid bilayer and the LD monolayer membrane to control cellular adaptation to metabolic changes. It has been proposed that these hairpin proteins integrate into both membranes in a similar monotopic topology, enabling their passive lateral diffusion during LD emergence at the ER. Here, we combine biochemical solvent-accessibility assays, electron paramagnetic resonance spectroscopy and intra-molecular crosslinking experiments with molecular dynamics simulations, and determine distinct intramembrane positionings of the ER/LD protein UBXD8 in ER bilayer and LD monolayer membranes. UBXD8 is deeply inserted into the ER bilayer with a V-shaped topology and adopts an open-shallow conformation in the LD monolayer. Major structural rearrangements are required to enable ER-to-LD partitioning. Free energy calculations suggest that such structural transition is unlikely spontaneous, indicating that ER-to-LD protein partitioning relies on more complex mechanisms than anticipated and providing regulatory means for this trans-organelle protein trafficking.

Atomistic ensemble of active SHP2 phosphatase.

SHP2 phosphatase plays an important role in regulating several intracellular signaling pathways. Pathogenic mutations of SHP2 cause developmental disorders and are linked to hematological malignancies and cancer. SHP2 comprises two tandemly-arranged SH2 domains, a catalytic PTP domain, and a disordered C-terminal tail. Under physiological, non-stimulating conditions, the catalytic site of PTP is occluded by the N-SH2 domain, so that the basal activity of SHP2 is low. Whereas the autoinhibited structure of SHP2 has been known for two decades, its active, open structure still represents a conundrum. Since the oncogenic mutant SHP2E76K almost completely populates the active, open state, this mutant has been extensively studied as a model for activated SHP2. By molecular dynamics simulations and accurate explicit-solvent SAXS curve predictions, we present the heterogeneous atomistic ensemble of constitutively active SHP2E76K in solution, encompassing a set of conformational arrangements and radii of gyration in agreement with experimental SAXS data.