CDC50A is required for aminophospholipid transport and cell fusion in mouse C2C12 myoblasts

ABSTRACT Myoblast fusion is essential for the formation of multinucleated muscle fibers and is promoted by transient changes in the plasma membrane lipid distribution. However, little is known about the lipid transporters regulating these dynamic changes. Here, we show that proliferating myoblasts exhibit an aminophospholipid flippase activity that is downregulated during differentiation. Deletion of the P4-ATPase flippase subunit CDC50A (also known as TMEM30A) results in loss of the aminophospholipid flippase activity and compromises actin remodeling, RAC1 GTPase membrane targeting and cell fusion. In contrast, deletion of the P4-ATPase ATP11A affects aminophospholipid uptake without having a strong impact on cell fusion. Our results demonstrate that myoblast fusion depends on CDC50A and may involve multiple CDC50A-dependent P4-ATPases that help to regulate actin remodeling.

MMPL-Family Proteins in Bacteria, Protozoa, Fungi, Plants and Animals: A Bioinformatics and Structural Investigation

Lipid transporters play an important role in most if not all organisms, ranging from bacteria to humans. For example, in Mycobacterium tuberculosis, the trehalose monomycolate transporter MmpL3 is involved in cell wall biosynthesis, while in humans, cholesterol transporters are involved in normal cell function as well as in disease. Here, using structural and bioinformatics information, we propose that there are proteins that also contain “MmpL3-like” (MMPL) transmembrane (TM) domains in many protozoa, including Trypanosoma cruzi, as well as in the bacterium Staphylococcus aureus, where the fatty acid transporter FarE has the same set of “active-site” residues as those found in the mycobacterial MmpL3s, and in T. cruzi. We also show that there are strong sequence and predicted structural similarities between the TM proton-translocation domain seen in the X-ray structures of mycobacterial MmpL3s and several human as well as fungal lipid transporters, leading to the proposal that there are similar proteins in apicomplexan parasites, and in plants. The animal, fungal, apicomplexan and plant proteins have larger extra-membrane domains than are found in the bacterial MmpL3, but they have a similar TM domain architecture, with the introduction of a (catalytically essential) Phe>His residue change, and a Ser/Thr H-bond network, involved in H -transport. Overall, the results are of interest since they show that MMPL-family proteins are present in essentially all life-forms: archaea, bacteria, protozoa, fungi, plants and animals and, where known, they are involved in “lipid” (glycolipid, phospholipid, sphingolipid, fatty acid, cholesterol, ergosterol) transport, powered by transmembrane molecular pumps having similar structures.

Evidence for a trap-and-flip mechanism in a proton-dependent lipid transporter

Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters participate in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acids synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a trap-and-flip mechanism that might be shared among MFS lipid transporters.

Lipid Transporters Beam Signals from Cell Membranes

Lipid composition in cellular membranes plays an important role in maintaining the structural integrity of cells and in regulating cellular signaling that controls functions of both membrane-anchored and cytoplasmic proteins. ATP-dependent ABC and P4-ATPase lipid transporters, two integral membrane proteins, are known to contribute to lipid translocation across the lipid bilayers on the cellular membranes. In this review, we will highlight current knowledge about the role of cholesterol and phospholipids of cellular membranes in regulating cell signaling and how lipid transporters participate this process.

Ist2 recruits the lipid transporters Osh6/7 to ER–PM contacts to maintain phospholipid metabolism

ER-plasma membrane (PM) contacts are proposed to be held together by distinct families of tethering proteins, which in yeast include the VAP homologues Scs2/22, the extended-synaptotagmin homologues Tcb1/2/3, and the TMEM16 homologue Ist2. It is unclear whether these tethers act redundantly or whether individual tethers have specific functions at contacts. Here, we show that Ist2 directly recruits the phosphatidylserine (PS) transport proteins and ORP family members Osh6 and Osh7 to ER–PM contacts through a binding site located in Ist2’s disordered C-terminal tethering region. This interaction is required for phosphatidylethanolamine (PE) production by the PS decarboxylase Psd2, whereby PS transported from the ER to the PM by Osh6/7 is endocytosed to the site of Psd2 in endosomes/Golgi/vacuoles. This role for Ist2 and Osh6/7 in nonvesicular PS transport is specific, as other tethers/transport proteins do not compensate. Thus, we identify a molecular link between the ORP and TMEM16 families and a role for endocytosis of PS in PE synthesis.

Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate a rapid bi-directional movement of lipids without metabolic energy input. Here, we established a new fluorescence microscopy-based assay for detecting phospholipid scramblase activity of membrane proteins upon their reconstitution into giant unilamellar vesicles formed from proteoliposomes by electroformation. The assay is based on chemical bleaching of fluorescence of a photostable ATTO-dye labeled phospholipid with the membrane-impermeant reductant sodium dithionite. We demonstrate that this new methodology is suitable for the study of the scramblase activity of the yeast endoplasmic reticulum at single vesicle level.

Transporters Beam Signals from Cell Membranes

Lipid composition in the cellular membranes plays an important role in maintaining the struc-tural integrity of cells and in regulating cellular signaling that controls functions of both mem-brane-anchored and cytoplasmic proteins. ATP-dependent ABC and P4-ATPase lipid transport-ers, two integral membrane proteins, are known to contribute to lipid translocation across the li-pid bilayers on the cellular membranes. In this review, we will highlight current knowledge about the role of cholesterol and phospholipids of cellular membrane in regulating cell signaling and how lipid transporters participate this process.

Investigating the Role Intestinal-Specific FXR and SHP in Regulating Lipid Metabolism in Mice

Dysregulation of lipid metabolism is a causal factor that can lead to a variety of disorders, such as obesity and metabolic syndrome. Dietary fats are digested in the small intestine by the physiological detergents known as bile acids. They emulsify the fats and break them down into smaller molecules in order for the enterocytes to absorb the nutrients through simple diffusion or through the utilization of specific lipid transporters. Interestingly, the nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP) not only regulates bile acid synthesis and circulation, but also lipid metabolism. Although many studies have examined the role of FXR in hepatic and intestinal lipid metabolism, studies investigating the role of SHP in the intestine are still lacking. Although FXR and SHP cooperate to regulate many metabolic pathways, FXR or SHP knockout models exhibit different lipid phenotypes. These data indicate there are FXR-dependent and -independent pathways of SHP that controls lipid metabolism. To delineate these two interconnecting yet separate pathways, we will utilize intestine-specific Shp knockout (IShpKO) and intestine-specific Fxr knockout (IFxrKO) mice model and place them on high fat diet to investigate their intestinal intestinal absorption and transportation of lipids. We will also monitor the bile acid pool in the intestine, serum, and liver in these knockouts to evaluate the consequence of intestinal deletion of Fxr as well as Shp on bile acid homeostasis and how this may affect lipid absorption. These experiments will identify how FXR and/or SHP regulates intestinal fat digestion and absorption and if this is secondary to the alterations in bile acid concentration and lipid transporters. In addition, we will also investigate the intestinal Fxr-Shp double knockout (IDKO) mice model to determine their combined contribution in intestinal lipid metabolism. Overall, the results obtained from this research will elucidate if intestinal FXR and SHP cooperate or can independently regulate lipid metabolism and homeostasis.