Amphipathic Lipid Packing Sensor Research Articles

SNARE chaperone Sly1 directly mediates close-range vesicle tethering.

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.

Tumor protein D54 binds intracellular nanovesicles via an extended amphipathic region

Tumor protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54, and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical (e.g., COPI and COPII) transport vesicles. Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by four amphipathic helices (AH1-4), which could fold upon binding to lipid membranes. Limited proteolysis, CD spectroscopy, tryptophan fluorescence, and cysteine mutagenesis coupled to covalent binding of a membrane-sensitive probe showed that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. Furthermore, site-directed mutagenesis indicated that AH2 and AH3 have a predominant role in TPD54 binding to membranes both in cells and using model liposomes. We found that AH3 has the physicochemical features of an amphipathic lipid packing sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Accordingly, we observed that binding of TPD54 to liposomes is very sensitive to membrane curvature and lipid unsaturation. We conclude that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and ALPS-independent mechanisms.

Computational study of membrane binding and free energy of amphipathic helix (ALPS) on Osh4 to varied lipid bilayers

The Osh4 Amphipathic Lipid Packing Sensor (ALPS) peptide plays an important role in membrane binding to facilitate lipid transport. This work focuses on molecular simulations but is supplemented with wet lab experiments to probe the ALPS peptide binding to varied lipid bilayers for both the wildtype and selected mutations. The wet lab experiments are conducted in collaboration with Dr. Karlsson's Lab at U. Maryland. Our main findings are that the penetration depth of the peptide is enhanced by lower area per lipid membranes (DMPS and possibly DMPC vs.

Experimental investigation of the mechanism of lipid-binding of the ALPS-like motif of Osh4 protein

Intracellular lipid traffic between organelles occurs primarily through a non-vesicular pathway via lipid transport proteins (LTPs). The oxysterol-binding homologue (Osh) proteins in the yeast Saccharomyces cerevisiae play an important role in the transportation of signaling lipids and are examples of LTPs. At its N-terminus, the Osh4 protein has an amphipathic lipid-packing sensor (ALPS)-like motif. To understand the kinetics of the binding of the ALPS peptide, we utilized isothermal titration calorimetry, which showed the ALPS peptide bound to liposomes containing 75% POPC-25% POPA with a KD of 129 μM.

ArfGAP1 inhibits mTORC1 lysosomal localization and activation.

The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid‐deficient conditions is not completely understood. Here, we identify ADP‐ribosylation factor GTPase‐activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature‐sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.

Yeast Nup84-Nup133 complex structure details flexibility and reveals conservation of the membrane anchoring ALPS motif

The hallmark of the eukaryotic cell is the complex endomembrane system that compartmentalizes cellular functions. Transport into and out of the nucleus occurs through the nuclear pore complex (NPC). The heptameric Nup84 or Y complex is an essential scaffolding component of the NPC. Here we report two nanobody-bound structures: the full-length Nup84-Nup133 C-terminal domain complex and the Nup133 N-terminal domain, both from S. cerevisiae. Together with previously published structures, this work enables the structural description of the entire 575 kDa Y complex from one species. The structure of Nup84-Nup133CTD details the high flexibility of this dimeric unit of the Y complex. Further, the Nup133NTD contains a structurally conserved amphipathic lipid packing sensor motif, confirmed by liposome interaction studies. The presented structures reveal important details about the function of the Y complex that affect our understanding of NPC structure and assembly.

Membrane Thinning Induces Sorting of Lipids and the Amphipathic Lipid Packing Sensor (ALPS) Protein Motif.

Heterogeneities (e.g., membrane proteins and lipid domains) and deformations (e.g., highly curved membrane regions) in biological lipid membranes cause lipid packing defects that may trigger functional sorting of lipids and membrane-associated proteins. To study these phenomena in a controlled and efficient way within molecular simulations, we developed an external field protocol that artificially enhances packing defects in lipid membranes by enforcing local thinning of a flat membrane region. For varying lipid compositions, we observed strong thinning-induced depletion or enrichment, depending on the lipid's intrinsic shape and its effect on a membrane's elastic modulus. In particular, polyunsaturated and lysolipids are strongly attracted to regions high in packing defects, whereas phosphatidylethanolamine (PE) lipids and cholesterol are strongly repelled from it. Our results indicate that externally imposed changes in membrane thickness, area, and curvature are underpinned by shared membrane elastic principles. The observed sorting toward the thinner membrane region is in line with the sorting expected for a positively curved membrane region. Furthermore, we have demonstrated that the amphipathic lipid packing sensor (ALPS) protein motif, a known curvature and packing defect sensor, is effectively attracted to thinner membrane regions. By extracting the force that drives amphipathic molecules toward the thinner region, our thinning protocol can directly quantify and score the lipid packing sensing of different amphipathic molecules. In this way, our protocol paves the way toward high-throughput exploration of potential defect- and curvature-sensing motifs, making it a valuable addition to the molecular simulation toolbox.

Regulation of LC3 lipidation by the autophagy-specific class III phosphatidylinositol-3 kinase complex.

Autophagy is a conserved eukaryotic pathway critical for cellular adaptation to changes in nutrition levels and stress. The class III phosphatidylinositol (PI)3-kinase complexes I and II (PI3KC3-C1 and -C2) are essential for autophagosome initiation and maturation, respectively, from highly curved vesicles. We used a cell-free reaction that reproduces a key autophagy initiation step, LC3 lipidation, as a biochemical readout to probe the role of autophagy-related gene (ATG)14, a PI3KC3-C1-specific subunit implicated in targeting the complex to autophagy initiation sites. We reconstituted LC3 lipidation with recombinant PI3KC3-C1, -C2, or various mutant derivatives added to extracts derived from a CRISPR/Cas9-generated ATG14-knockout cell line. Both complexes C1 and C2 require the C-terminal helix of VPS34 for activity on highly curved membranes. However, only complex C1 supports LC3 lipidation through the curvature-targeting amphipathic lipid packing sensor (ALPS) motif of ATG14. Furthermore, the ALPS motif and VPS34 catalytic activity are required for downstream recruitment of WD-repeat domain phosphoinositide-interacting protein (WIPI)2, a protein that binds phosphatidylinositol 3-phosphate and its product phosphatidylinositol 3, 5-bisphosphate, and a WIPI-binding protein, ATG2A, but do not affect membrane association of ATG3 and ATG16L1, enzymes contributing directly to LC3 lipidation. These data reveal the nuanced role of the ATG14 ALPS in membrane curvature sensing, suggesting that the ALPS has additional roles in supporting LC3 lipidation.

Effect of Membrane Lipid Packing on Stable Binding of the ALPS Peptide.

The amphipathic lipid packing sensor (ALPS) motif, originally discovered on the ArfGAP1 membrane-binding protein, binds to pre-existing large packing defects in a membrane (spontaneous or due to membrane curvature), though a more precise relationship between the ALPS peptide and packing defect characteristics of a membrane remains unclear. We developed an image processing technique for identifying packing defects to quantify the relationship between the ALPS peptide of the Osh4 protein in yeast and packing defects on a membrane model using molecular dynamics simulations. We used the highly mobile membrane mimetic (HMMM) model to create very large packing defects and expedite the binding time scale. Most prominently, we show that the probability of the ALPS peptide moving toward the membrane increases when it is near a large packing defect. Deviations from this trend exist for very large packing defects (≳115 Å2), which we propose is due to an overwhelming hydrophobic effect and a reduced electrostatic effect when large portions of the nonpolar core are exposed and the peptide is oriented unfavorably. Furthermore, we compared our HMMM results to similar simulations using all-atom lipid membranes. The binding time scales of the ALPS peptide can be reduced by roughly 1 order of magnitude when HMMM is used, while still maintaining many of the important physical characteristics of the binding process observed when using an all-atom lipid membrane.

Preferred Binding Mechanism of Osh4's Amphipathic Lipid-Packing Sensor Motif, Insights from Molecular Dynamics.

Amphipathic helices are key domains of peripheral membrane proteins, targeting specific membranes to enable proper protein function as well as changing the local topology and lipid dynamics of the membranes they bind. Here, we use extended all-atom molecular dynamics to study, in detail, the binding mechanism and conformation of the N-terminus of the lipid-transport protein Osh4 in yeast, that is, the amphipathic lipid-packing sensor (ALPS) motif. We identified two binding conformations: (i) a vertical one with the N-terminus of the peptide embedded into the hydrophobic core and (ii) a horizontal, and energetically favored, conformation in which the hydrophobic side chains of ALPS are fully embedded into the membrane hydrophobic core. From extensive analysis on 21 trajectories of 2 μs each, we describe peptide binding in terms of the structural changes that both the peptide and the membrane undergo upon binding as well as energetics of this interaction. The membrane models in this study include a simple binary lipid mixture, with a neutral and a charged lipid (1,2-dioleoyl- sn-glycero-3-phosphocholine-1,2-dioleoyl- sn-glycero-3-phospho-l-serine) and complex mixtures with lipid compositions characteristic of two organelles in yeast (each with more than six lipid types and an accurate sterol content). Our conclusions are in agreement with available literature, showing that the ALPS peptide is more likely to bind membrane surfaces with packing defects and higher anionic character. In addition, we show that there is an interplay between ALPS binding an existing packing defect and creating or enhancing one as the peptide binds to the membrane, which was previously suggested in the literature.

Detecting phospholipase activity with the amphipathic lipid packing sensor motif of ArfGAP1

The amphipathic lipid packing sensor (ALPS) motif of ArfGAP1 brings this GTPase activating protein to membranes of high curvature. Phospholipases are phospholipid-hydrolyzing enzymes that generate different lipid products that alter the lateral organization of membranes. Here, we evaluate by fluorescence microscopy how in-situ changes of membrane lipid composition driven by the activity of different phospholipases promotes the binding of ALPS. We show that the activity of phospholipase A2, phospholipase C and phospholipase D drastically enhances the binding of ALPS to the weakly-curved membrane of giant liposomes. Our results suggest that the enzymatic activity of phospholipases can modulate the ArfGAP1-mediated intracellular traffic and that amphiphilic peptides such as the ALPS motif can be used to study lipolytic activities at lipid membranes.

Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol 3-Kinase Signal Transduction by Altering Plasma Membrane Packing

Psoralen and ultraviolet A light (PUVA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft-versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC50), 8.4 ± 1.1 versus 4.3 ± 1.1 μm) and glycoprotein VI (EC50, 1.61 ± 0.85 versus 0.26 ± 0.21 μg/ml) but not PAR4 (EC50, 50 ± 1 versus 58 ± 1 μm) signal transduction. Our findings were confirmed in T-cells from graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases.

A filter at the entrance of the Golgi that selects vesicles according to size and bulk lipid composition

When small phosphatidylcholine liposomes are added to perforated cells, they bind preferentially to the Golgi suggesting an exceptional avidity of this organelle for curved membranes without stereospecific interactions. We show that the cis golgin GMAP-210 accounts for this property. First, the liposome tethering properties of the Golgi resembles that of the amphipathic lipid-packing sensor (ALPS) motif of GMAP-210: both preferred small (radius < 40 nm) liposomes made of monounsaturated but not saturated lipids. Second, reducing GMAP-210 levels or redirecting its ALPS motif to mitochondria decreased liposome capture by the Golgi. Extensive mutagenesis analysis suggests that GMAP-210 tethers authentic transport vesicles via the same mechanism whereby the ALPS motif senses lipid-packing defects at the vesicle surface through its regularly spaced hydrophobic residues. We conclude that the Golgi uses GMAP-210 as a filter to select transport vesicles according to their size and bulk lipid composition.

The HOPS/Class C Vps Complex Tethers High‐Curvature Membranes via a Direct Protein–Membrane Interaction

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low-curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p-bearing low-curvature liposomes even when the high-curvature liposomes are protein-free. Phosphorylation of the curvature-sensing amphipathic lipid-packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high-curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high-curvature liposomes and Ypt7p-bearing low-curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein-membrane interaction. Such high-curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole-vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane.

Protein sorting to intracellular compartments.

The organization of eukaryotic cells depends on correct targeting of proteins and lipids to the organelles at which they function, but how organelle identity is maintained in the face of a constant flux of material is a major problem for the cell. New mechanisms addressing this question were revealed at the Minisymposium on “Membrane Traffic: Dynamic and Regulation.” Starting with the endoplasmic reticulum (ER), Silvere Pagant (Miller lab) showed that some plasma membrane (PM) proteins require multiple signals to drive capture into COPII vesicles. One signal comes from the cargo protein itself, and the second comes signal from a cargo receptor; both signals simultaneously engage the cargo adaptor for the COPII coat, Sec24. Prasanna Satpute-Krishnan (Lippincott-Schwartz and Hegde labs), recipient of the 2014 Merton Bernfield Award, presented her work on a novel quality-control mechanism for glycosylphosphatidylinositol (GPI)-anchored proteins called RESET (for rapid ER stress-induced export). ER stress causes receptor-mediated exit of GPI-anchored proteins from the ER through the secretory pathway to the PM, from which they are endocytosed and degraded in lysosomes. Only misfolded GPI-anchored proteins bind tightly to the cargo receptor, Tmp21, providing a new paradigm in ER stress clearance. Moving from the ER to the Golgi, Mie Wong (Munro lab) presented an elegant system to test the function of a family of diverse Golgi-localized coiled-coil proteins. When different golgins were ectopically attached to mitochondria, specific classes of vesicles were redirected to mitochondria, clearly demonstrating a vesicle capture role for these proteins. A possible mechanism of lipid transfer from the ER to the PM was revealed by a structural study presented by Chris Schauder (Reinisch and de Camilli groups). The synaptotagmin-like mitochondrial-lipid binding protein (SMP) domain of the ER–PM tether E-Syt2 forms a long hydrophobic channel that can harbor multiple phospholipid molecules. Two SMP domains form a stacked dimer to double the length of the channel. Continuing the theme of lipid composition in organelle identity, Cathy Jackson presented her group's results showing how amphipathic helices (AHs) of distinct chemistries specifically target different organelles through recognition of organelle lipid composition. Regulation of a lipid-binding AH of the Arf guanine nucleotide exchange factor (GEF) GBF1 mediates its localization to either the Golgi or lipid droplets. Looking at a related Arf GEF, yeast Sec7, Brian Richardson (Fromme lab) reported the first crystal structure of a noncatalytic region of these proteins. The N-terminal region is made up of α-helices forming a series of armadillo folds that form an extended platform. This structure provides insight into how this region is involved in dimerization and protein interactions. New vesicle tethering and targeting mechanisms were reported, with Chris Stroupe presenting how the homotypic fusion and protein sorting (HOPS)/class C Vps tethering complex connects specific membrane compartments. This complex binds to a Rab GTPase in one membrane and directly to a second, highly curved membrane through a curvature-sensing amphipathic lipid packing sensor (ALPS) motif. Margaret Heider (Munson lab) provided the most comprehensive view to date of the architecture of the exocyst complex, a multisubunit tether that targets secretory vesicles to the PM. The session ended with a talk by Christine Insinna-Kettenhofen (Westlake group) reporting on a new mechanism for targeting of vesicles to cilia mediated by the epidermal growth factor receptor substrate 15 homology domain–containing (EHD) proteins. These proteins are localized to vesicles, where they cooperate with the Rab8-Rab11 cascade and soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) to regulate morphological changes to the vesicle membrane important for fusion.

Interaction of the Spo20 membrane-sensor motif with phosphatidic acid and other anionic lipids, and influence of the membrane environment.

The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.

O-Linked β-N-Acetylglucosamine (O-GlcNAc) Site Thr-87 Regulates Synapsin I Localization to Synapses and Size of the Reserve Pool of Synaptic Vesicles

O-GlcNAc is a carbohydrate modification found on cytosolic and nuclear proteins. Our previous findings implicated O-GlcNAc in hippocampal presynaptic plasticity. An important mechanism in presynaptic plasticity is the establishment of the reserve pool of synaptic vesicles (RPSV). Dynamic association of synapsin I with synaptic vesicles (SVs) regulates the size and release of RPSV. Disruption of synapsin I function results in reduced size of the RPSV, increased synaptic depression, memory deficits, and epilepsy. Here, we investigate whether O-GlcNAc directly regulates synapsin I function in presynaptic plasticity. We found that synapsin I is modified by O-GlcNAc during hippocampal synaptogenesis in the rat. We identified three novel O-GlcNAc sites on synapsin I, two of which are known Ca(2+)/calmodulin-dependent protein kinase II phosphorylation sites. All O-GlcNAc sites mapped within the regulatory regions on synapsin I. Expression of synapsin I where a single O-GlcNAc site Thr-87 was mutated to alanine in primary hippocampal neurons dramatically increased localization of synapsin I to synapses, increased density of SV clusters along axons, and the size of the RPSV, suggesting that O-GlcNAcylation of synapsin I at Thr-87 may be a mechanism to modulate presynaptic plasticity. Thr-87 is located within an amphipathic lipid-packing sensor (ALPS) motif, which participates in targeting of synapsin I to synapses by contributing to the binding of synapsin I to SVs. We discuss the possibility that O-GlcNAcylation of Thr-87 interferes with folding of the ALPS motif, providing a means for regulating the association of synapsin I with SVs as a mechanism contributing to synapsin I localization and RPSV generation.

Identification of a second amphipathic lipid-packing sensor-like motif that contributes to Gcs1p function in the early endosome-to-TGN pathway

ArfGAPs, GTPase-activating proteins for Arf small GTPases, are involved in multiple steps of vesicle formation of various transport pathways. Amphipathic lipid-packing sensor (ALPS) motif was first identified in the C-terminal regions of ArfGAP1 and its yeast homologue Gcs1p as a region that adsorbs preferentially onto highly curved membranes by folding into an amphipathic α-helix (AH). We previously showed that Gcs1p functionally interacted with the phospholipid flippase Cdc50p-Drs2p in the early endosome-to-TGN retrieval pathway. In this study, we performed functional analyses of the C-terminal region of Gcs1p containing ALPS. Hydrophobic cluster analysis suggested that there is another potential AH-forming region downstream of ALPS in Gcs1p. Mutational analysis suggested that the ALPS motif is important for the Gcs1p function in the Golgi-to-ER retrograde pathway, whereas ALPS and the predicted AH region redundantly function in the post-Golgi pathways including the early endosome-to-TGN pathway. Liposome flotation assay indicated that this downstream region preferentially interacted with liposomes of smaller size. The region containing the ALPS motif was also required for the interaction with SNARE proteins including Snc1p and Tlg1p. These results suggest that ALPS and the predicted AH region are involved in the regulation and function of Gcs1p by interacting with membrane phospholipids and vesicle proteins.

Amphipathic Lipid Packing Sensor Motifs: Probing Bilayer Defects with Hydrophobic Residues

Sensing membrane curvature allows fine-tuning of complex reactions that occur at the surface of membrane-bound organelles. One of the most sensitive membrane curvature sensors, the Amphipathic Lipid Packing Sensor (ALPS) motif, does not seem to recognize the curved surface geometry of membranes per se; rather, it recognizes defects in lipid packing that arise from membrane bending. In a companion paper, we show that these defects can be mimicked by introducing conical lipids in a flat lipid bilayer, in agreement with experimental observations. Here, we use molecular-dynamics (MD) simulations to characterize ALPS binding to such lipid bilayers. The ALPS motif recognizes lipid-packing defects by a conserved mechanism: peptide partitioning is driven by the insertion of hydrophobic residues into large packing defects that are preformed in the bilayer. This insertion induces only minor modifications in the statistical distribution of the free packing defects. ALPS insertion is severely hampered when monounsaturated lipids are replaced by saturated lipids, leading to a decrease in packing defects. We propose that the hypersensitivity of ALPS motifs to lipid packing defects results from the repetitive use of hydrophobic insertions along the monotonous ALPS sequence.

New and Notable: Key New Insights into Membrane Targeting by Proteins

New and Notable: Key New Insights into Membrane Targeting by Proteins