SUMMARY Most available information on ER-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca2+ entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here we show that the three Extended-Synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca2+ in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca2+ regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca2+ entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast), mediate the formation of ER-PM contacts sites which are functionally distinct from those mediated by STIM1 and Orai1.

Snakes possess a unique sensory system for detecting infrared radiation, enabling them to generate a ‘thermal image’ of predators or prey. Infrared signals are initially received by the pit organ, a highly specialized facial structure that is innervated by nerve fibers of the somatosensory system. How this organ detects and transduces infrared signals into nerve impulses is not known. Here we use an unbiased transcriptional profiling approach to identify TRPA1 channels as infrared receptors on sensory nerve fibers that innervate the pit organ. TRPA1 orthologues from pit bearing snakes (vipers, pythons, and boas) are the most heat sensitive vertebrate ion channels thus far identified, consistent with their role as primary transducers of infrared stimuli. Thus, snakes detect infrared signals through a mechanism involving radiant heating of the pit organ, rather than photochemical transduction. These findings illustrate the broad evolutionary tuning of TRP channels as thermosensors in the vertebrate nervous system.

Spherical high density lipoproteins (HDL) † predominate in human plasma. However, little information exists on the structure of the most common HDL protein, apolipoprotein (apo) A-I, in spheres vs. better studied discoidal forms. We produced spherical HDL by incubating reconstituted discoidal HDL with physiological plasmaremodeling enzymes and compared apoA-I structure in discs and spheres of comparable diameter (79 -80 and 93-96 Å). Using cross-linking chemistry and mass spectrometry, we determined that the general structural organization of apoA-I was overall similar between discs and spheres, regardless of diameter. This was the case despite the fact that the 93 Å spheres contained three molecules of apoA-I per particle compared with only two in the discs. Thus, apoA-I adopts a consistent general structural framework in HDL particles-irrespective of shape, size and the number of apoA-Is present. Furthermore, a similar cross-linking pattern was demonstrated in HDL particles isolated from human serum. We propose the first experiment-based molecular model of apoA-I in spherical HDL particles. This model provides a new foundation for understanding how apoA-I structure modulates HDL function and metabolism.sphere ͉ disk G iven the inverse correlation between high density lipoprotein (HDL) levels and cardiovascular disease, a key question in vascular biology relates to how apolipoproteins modulate the metabolism and function of HDL. Significant evidence supports a role for HDL in the process of reverse cholesterol transport whereby lipids and cholesterol in the vessel wall are transported to the liver for catabolism. However, because of a lack of information on HDL structure and the molecular basis of its interactions with other proteins, our understanding of HDL metabolism and function is at a basic stage.The ''glue'' that holds most HDL particles together is apolipoprotein (apo)A-I, a highly ␣-helical, 28-kDa polypeptide. It comprises some 70% of HDL protein mass, making it the clear starting point for deriving a basic understanding of HDL structure. In humans, apoA-I is primarily present in two major spherical HDL species, HDL 2 (d ϭ 1.063-1.125 g/ml) and HDL 3 (d ϭ 1.125-1.210 g/ml) with diameters ranging from 70 to 120 Å. More minor, but clearly important, HDL subspecies include lipid-poor apoA-I and nascent discoidal particles (reviewed in ref. 1). Highly reactive but low abundance discoidal HDLs are critical intermediates between lipid-poor apoA-I and mature spherical HDL. Easily produced in vitro, they have been heavily used for structural studies (2). Despite some debates on details of certain regions of apoA-I in discs, the majority of recent theoretical and experimental data supports the so-called ''double belt'' model (3). In this scheme, each of two ring-shaped apoA-I molecules wrap around a leaflet of a disk-like patch of lipid bilayer in an anti-parallel orientation.Despite their abundance in plasma, much less is known about the structure of apoA-I in spherical particles. They contain a neutral lipid...

Steroids regulate cell proliferation, tissue development, and cell signaling via two pathways: a nuclear receptor mechanism and genome-independent signaling. Sperm activation, egg maturation, and steroid-induced anesthesia are executed via the latter pathway, the key components of which remain unknown. Here, we present characterization of the human sperm progesterone receptor that is conveyed by the orphan enzyme α/β hydrolase domain–containing protein 2 (ABHD2). We show that ABHD2 is highly expressed in spermatozoa, binds progesterone, and acts as a progesterone-dependent lipid hydrolase by depleting the endocannabinoid 2-arachidonoylglycerol (2AG) from plasma membrane. The 2AG inhibits the sperm calcium channel (CatSper), and its removal leads to calcium influx via CatSper and ensures sperm activation. This study reveals that progesterone-activated endocannabinoid depletion by ABHD2 is a general mechanism by which progesterone exerts its genome-independent action and primes sperm for fertilization.

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.

Synaptic vesicles undergo a maturation step, termed priming, in which they become competent to fuse with the plasma membrane. To morphologically define the site of vesicle priming and identify fusion-competent synaptic vesicles, we combined a rapid physical-fixation technique with immunogold staining and high-resolution morphometric analysis at Caenorhabditis elegans neuromuscular junctions. In these presynaptic terminals, a subset of synaptic vesicles contact the plasma membrane within ϳ100 nm of a presynaptic dense projection. UNC-13, a protein required for vesicle priming, localizes to this same region of the plasma membrane. In an unc-13 null mutant, few synaptic vesicles contact the plasma membrane, suggesting that membrane-contacting synaptic vesicles represent the morphological correlates of primed vesicles. Interestingly, a subpopulation of membrane-contacting vesicles, located within 30 nm of a dense projection, are unperturbed in unc-13 mutants. We show that UNC-10/Rim, a protein implicated in presynaptic plasticity, localizes to dense projections and that loss of UNC-10/Rim causes an UNC-13-independent reduction in membrane-contacting synaptic vesicles within 30 nm of the dense projections. Our data together identify a discrete domain for vesicle priming within 100 nm of dense projections and further suggest that UNC-10/Rim and UNC-13 separately contribute to the membrane localization of synaptic vesicles within this domain.

Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide array of physical and chemical stimuli, making them important components of sensory systems in both vertebrate and invertebrate organisms. Mammalian TRPA1 channels are activated by chemically reactive irritants, whereas snake and Drosophila TRPA1 orthologs are preferentially activated by heat. By comparing human and rattlesnake TRPA1 channels, we have identified two portable heat-sensitive modules within the ankyrin repeat-rich aminoterminal cytoplasmic domain of the snake ortholog. Chimeric channel studies further demonstrate that sensitivity to chemical stimuli and modulation by intracellular calcium also localize to the N-terminal ankyrin repeat-rich domain, identifying this region as an integrator of diverse physiological signals that regulate sensory neuron excitability. These findings provide a framework for understanding how restricted changes in TRPA1 sequence account for evolution of physiologically diverse channels, also identifying portable modules that specify thermosensitivity.chemosensation | pain | thermosensation | calcium modulation | somatosensation P rimary afferent (somatosensory) neurons detect a range of physical and chemical stimuli, including temperature, pressure, and noxious irritants (1). The transient receptor potential (TRP) channel family has been shown to play a predominant role in these processes, particularly in regard to thermosensitivity and chemosensitivity (2-5). TRPA1, otherwise known as the "wasabi receptor," plays a key role in somatosensation in evolutionarily diverse phyla, including vertebrate and invertebrate species. Mammalian TRPA1 is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of environmental and endogenous chemical irritants, such as allyl isothiocyanate (AITC), acrolein, and 4-hydroxynonena, and contributes to cellular mechanisms underlying inflammatory pain (6-9).TRPA1 channels show species-specific functional variation to suit their physiological roles. For example, snakes are unique among vertebrates in that their TRPA1 channels are heat-sensitive, which some species (rattlesnakes, boas, and pythons) have exploited to detect infrared radiation (10). Similarly, insect TRPA1 channels are heat-sensitive and contribute to thermal avoidance behaviors (11)(12)(13)(14). In these cases, however, thermosensitivity comes at the expense of chemosensitivity, such that AITC and other chemical irritants still activate these channels but with reduced potency compared with mammalian orthologs (10). Despite clear physiological differences between snake and mammalian TRPA1 channels, they share significant amino acid identity (56%), providing a unique opportunity to exploit sequence comparisons and domain swaps to pinpoint structural elements associated with stimulus detection and/or gating. Delineating elements that contribute to TRPA1 function will provide insights into the evolutionary process whereby structural changes lead t...

The [PSI(+)] prion is the aggregated self-propagating form of the Sup35 protein from the yeast Saccharomyces cerevisiae. Aggregates of Sup35 in [PSI(+)] cells exist in different heritable conformations, called "variants," and they are composed of detergent-resistant Sup35 polymers, which may be closely associated with themselves, other proteins, or both. Here, we report that disassembly of the aggregates into individual Sup35 polymers and non-Sup35 components increases their infectivity while retaining their variant specificity, showing that variant-specific [PSI(+)] infection can be transmitted by Sup35 polymers alone. Morphological analysis revealed that Sup35 isolated from [PSI(+)] yeast has the appearance of short barrels, and bundles, which seem to be composed of barrels. We show that the major components of two different variants of [PSI(+)] are interacting infectious Sup35 polymers and Ssa1/2. Using a candidate approach, we detected Hsp104, Ssb1/2, Sis1, Sse1, Ydj1, and Sla2 among minor components of the aggregates. We demonstrate that Ssa1/2 efficiently binds to the prion domain of Sup35 in [PSI(+)] cells, but that it interacts poorly with the nonaggregated Sup35 found in [psi(-)] cells. Hsp104, Sis1, and Sse1 interact preferentially with the prion versus nonprion form of Sup35, whereas Sla2 and Ssb1/2 interact with both forms of Sup35 with similar efficiency.