Biology subjects

Schneiter, R.

Publications and source records attributed to Schneiter, R..

6 recordsLinked to original sources

bioRxiv2024

The C. elegans LON-1 protein requires its CAP domain for function in regulating body size and BMP signaling

The CAP (Cysteine-rich secretory proteins, Antigen-5, Pathogenesis-Related) proteins are widely expressed and have been implicated to play diverse roles ranging from mammalian reproduction to plant immune response. Increasing evidence supports a role of CAP proteins in lipid binding. The C. elegans CAP protein LON-1 is known to regulate body size and Bone Morphogenetic Protein (BMP) signaling. LON-1 is a secreted protein with a conserved CAP domain and a C-terminal unstructured domain with no homology to other proteins. In this study, we report that the C-Terminal Domain (CTD) of LON-1 is dispensable for its function. Instead, key conserved residues located in the CAP domain are critical for LON-1 function in vivo. We further showed that LON-1 is capable of binding sterol, but not fatty acid, in vitro, and that certain key residues implicated in LON-1 function in vivo are also important for LON-1 sterol binding in vitro. These findings suggest a role of LON-1 in regulating body size and BMP signaling via sterol binding. ARTICLE SUMMARYThe C. elegans LON-1 protein is known to regulate body size and Bone Morphogenetic Protein (BMP) signaling. However, its molecular mode of action remains elusive. This study shows that LON-1 can bind sterol, but not fatty acid, in vitro. Furthermore, key conserved residues in the CAP domain of LON-1 are required for LON-1 function in vivo. These findings suggest a role of LON-1 in regulating body size and BMP signaling via sterol binding.

genetics↗

bioRxiv2022

Perilipin 3 promotes the formation of membrane domains enriched in diacylglycerol and lipid droplet biogenesis proteins

Lipid droplets (LDs) serve as intracellular stores of energy-rich neutral lipids. LDs form at discrete sites in the endoplasmic reticulum (ER) and they remain closely associated with the ER during lipogenic growth and lipolytic consumption. Their hydrophobic neutral lipid core is covered by a monolayer of phospholipids, which harbors a specific set of proteins. This LD surface is coated and stabilized by perilipins, a family of soluble proteins that specifically target LDs from the cytosol. We have previously used chimeric fusion proteins between perilipins and integral ER membrane proteins to test whether proteins that are anchored to the ER bilayer could be dragged onto the LD monolayer. Expression of these chimeric proteins induces repositioning of the ER membrane around LDs. Here, we test the properties of membrane-anchored perilipins in cells that lack LDs. Unexpectedly, membrane-anchored perilipins induce the formation of crescent-shaped ER membrane domains that have LD-like properties. They are stained by lipophilic dyes and harbor LD marker proteins. In addition, these ER domains are enriched in diacylglycerol (DAG) and in ER proteins that are important for early steps of LD biogenesis, including seipin and Pex30. These ER membrane domains dissolve upon induction of neutral lipid synthesis and the formation of nascent LDs. Formation of these membrane domains in vivo requires DAG, and we show that perilipin 3 (PLIN3) binds to liposomes containing DAG in vitro. Taken together, these observations indicate that perilipin not only serve to stabilize the surface of mature LDs but that they are likely to exert a more active role in early steps of LD biogenesis at ER subdomains enriched in DAG, seipin, and neutral lipid biosynthetic enzymes.

cell biology↗

bioRxiv2020

Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2) selectively binds an ascaroside (ascr#3)

During their infective stages, hookworms release excretory-secretory (E-S) products, including small molecules and proteins, to help evade and suppress the hosts immune system. Small molecules found in E-S products of mammalian hookworms include nematode derived metabolites like ascarosides, which are composed of the sugar ascarylose linked to a fatty acid side chain. Ascarosides play vital roles in signaling, development, reproduction, and survival. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). ASP belongs to the SCP/TAPS (sperm-coating protein / Tpx / antigen 5 / pathogenesis related-1 / Sc7) superfamily of proteins, members of which have previously been shown to bind to eicosanoids and fatty acids. These molecules are structurally similar to the fatty acid moieties of ascarosides. The objective of this study was to determine if the hookworm ASP; N. americanus Ancylostoma secreted protein 2 (Na-ASP-2) binds to the ascarosides or their fatty acid moieties. We describe investigations of our hypothesis that there is a functional relationship between the major secreted proteins and signaling small molecules found in hookworm E-S products. To accomplish this, several ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using a ligand competition assay and microscale thermophoresis. Our results reveal that the fatty acid moieties of the ascarosides, bind specifically to the palmitic acid binding cavity of Na-ASP-2. Additionally, ascr#3, an ascaroside that is present in mammalian hookworm E-S products binds to the palmitic acid binding cavity of Na-ASP-2, whereas oscr#10 which is not found in hookworm E-S products does not bind. Future studies are required to determine the structural basis of ascaroside binding by Na-ASP-2 and to understand the physiological significance of these observations.

biochemistry↗

bioRxiv2020

Targeting of integral membrane proteins to the surface of lipid droplets

Lipid droplets (LDs) are globular subcellular structures that mainly serve to store energy in form of neutral lipids, particularly triacylglycerols and steryl esters. LDs are closely associated with the membrane of the endoplasmic reticulum (ER), and are limited by a monolayer membrane of phospholipids harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here we address the question whether integral membrane spanning proteins could localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER resident proteins, such as Wbp1 (a N-glycosyl transferase complex subunit), Sec61 (a translocon subunit), and Pmt1 (a protein O-mannosyltransferase). The resulting fusion proteins localize to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, is due to redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners in cells coexpressing the membrane-anchored perilipin. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of OM14, an outer mitochondrial membrane protein. Expression of OM14-PLIN3 resulted in close apposition of mitochondria and LDs. Taken together, these data indicate that the LD surface constitutes a barrier for ER-localized integral membrane spanning proteins.

cell biology↗

bioRxiv2020

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER). Relatively little is known about how sites of LD formation are selected, and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)-synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER-LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG synthases and additional LD biogeneiss factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG. We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.

cell biology↗

bioRxiv2019

Lipid droplet biogenesis is a liquid phase separation spatially regulated by seipin and membrane curvature.

Cells store energy in the form of neutral lipids packaged into micrometer-sized organelles named lipid droplets (LD). These structures emerge from the endoplasmic reticulum (ER), but their biogenesis remains poorly understood. Using molecular simulations, we found that fat accumulation and LD formation are described by a liquid-liquid phase separation (LLPS) process. Within this framework, we could identify how ER membrane properties modulate LD formation, and we could directly test our computational predictions by combining yeast genetics with fluorescence microscopy. Our data suggest that the specific lipid composition of the ER together with its peculiar physical properties, such as low membrane tension and membrane curvature, promote the packaging of neutral lipids into LD, preventing their accumulation in the ER membrane. Our results provide a new conceptual understanding of LD biogenesis in the context of ER homeostasis and function.

cell biology↗