scholarly journals The ER cholesterol sensor SCAP promotes CARTS biogenesis at ER-Golgi contact sites

2019 ◽  
Author(s):  
Yuichi Wakana ◽  
Kaito Hayashi ◽  
Takumi Nemoto ◽  
Chiaki Watanabe ◽  
Masato Taoka ◽  
...  
Keyword(s):  
Cholesterol Metabolism ◽  
Regulatory Protein ◽  
Cholesterol Transport ◽  
Lipid Transfer ◽  
Wild Type ◽  
Proteolytic Activation ◽  
Contact Sites ◽  
Transport Defect ◽  
Golgi Network

AbstractIn response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum (ER) to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here we show that in cholesterol-fed cells ER-localized SCAP interacts through Sac1 phosphoinositide 4-phosphate (PI4P) phosphatase with a VAP/OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER-Golgi contact sites. SCAP knockdown inhibited the turnover of PI4P perhaps due to a cholesterol transport defect and altered the subcellular distribution of the VAP/OSBP complex. As in the case of perturbation of lipid transfer complexes at ER-Golgi contact sites, SCAP knockdown inhibited the biogenesis of the trans-Golgi network-derived transport carriers CARTS, which was reversed by expression of wild-type SCAP but not cholesterol sensing-defective mutants. Altogether, our findings reveal a new role of SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis at ER-Golgi contact sites, depending on the ER cholesterol.SummarySCAP is the key regulatory protein in cholesterol metabolism. Wakana et al. describe a new role of SCAP in controlling Golgi PI4P turnover and the biogenesis of the Golgi-derived transport carries CARTS via cholesterol/PI4P exchange machinery at ER-Golgi contact sites.

scholarly journals The ER cholesterol sensor SCAP promotes CARTS biogenesis at ER–Golgi membrane contact sites

2020 ◽  
Vol 220 (1) ◽  
Author(s):  
Yuichi Wakana ◽  
Kaito Hayashi ◽  
Takumi Nemoto ◽  
Chiaki Watanabe ◽  
Masato Taoka ◽  
...  
Keyword(s):  
Lipid Transfer ◽  
Golgi Membrane ◽  
Proteolytic Activation ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Defective Mutant ◽  
Transport Defect ◽  
Membrane Contact ◽  
Phosphatidylinositol 4 ◽  
Golgi Network

In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here, we show that in cholesterol-fed cells, ER-localized SCAP interacts through Sac1 phosphatidylinositol 4-phosphate (PI4P) phosphatase with a VAP–OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER–Golgi membrane contact sites (MCSs). SCAP knockdown inhibited the turnover of PI4P, perhaps due to a cholesterol transport defect, and altered the subcellular distribution of the VAP–OSBP complex. As in the case of perturbation of lipid transfer complexes at ER–Golgi MCSs, SCAP knockdown inhibited the biogenesis of the trans-Golgi network–derived transport carriers CARTS, which was reversed by expression of wild-type SCAP or a Golgi transport–defective mutant, but not of cholesterol sensing–defective mutants. Altogether, our findings reveal a new role for SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis via ER–Golgi MCSs, depending on the ER cholesterol.

scholarly journals Role of the Sec22b–E-Syt complex in neurite growth and ramification

2020 ◽  
Vol 133 (18) ◽  
pp. jcs247148 ◽  
Author(s):  
Alessandra Gallo ◽  
Lydia Danglot ◽  
Francesca Giordano ◽  
Bailey Hewlett ◽  
Thomas Binz ◽  
...  
Keyword(s):  
Nervous System ◽  
Opposite Effect ◽  
Neurite Growth ◽  
Lipid Transfer ◽  
Wild Type ◽  
Lipid Transfer Proteins ◽  
Contact Sites ◽  
Developing Neurons ◽  
Clostridial Neurotoxin

ABSTRACTAxons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b–Stx1 SNARE complexes at endoplasmic reticulum (ER)–PM contacts. Here, we show that Sec22b interacts with members of the extended synaptotagmin (E-Syt) family of ER lipid transfer proteins (LTPs), and this interaction depends on the longin domain of Sec22b. Overexpression of E-Syts stabilizes Sec22b–Stx1 association, whereas silencing of E-Syts has the opposite effect. Overexpression of wild-type E-Syt2, but not mutants unable to transfer lipids or attach to the ER, increase the formation of axonal filopodia and ramification of neurites in developing neurons. This effect is inhibited by a clostridial neurotoxin cleaving Stx1, and expression of the Sec22b longin domain and a Sec22b mutant with an extended linker between the SNARE and transmembrane domains. We conclude that Sec22b–Stx1 ER–PM contact sites contribute to PM expansion by interacting with LTPs, such as E-Syts.This article has an associated First Person interview with the first author of the paper.

scholarly journals Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

2010 ◽  
Vol 84 (14) ◽  
pp. 7039-7052 ◽  
Author(s):  
Barbara Kropff ◽  
Yvonne Koedel ◽  
William Britt ◽  
Michael Mach
Keyword(s):  
Amino Acid ◽  
Human Cytomegalovirus ◽  
Intracellular Localization ◽  
Surface Expression ◽  
Wild Type Virus ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Carboxy Terminal ◽  
Golgi Network

ABSTRACT Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.

Role of the N-terminal region and of β-sheet residue Thr29 on the activity of the ω2 global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035

2005 ◽  
Vol 386 (9) ◽  
Author(s):  
Karin Welfle ◽  
Florencia Pratto ◽  
Rolf Misselwitz ◽  
Joachim Behlke ◽  
Juan C. Alonso ◽  
...  
Keyword(s):  
Host Range ◽  
Streptococcus Pyogenes ◽  
Regulatory Protein ◽  
Terminal Region ◽  
Broad Host Range ◽  
Wild Type ◽  
Global Regulator ◽  
Β Sheet

AbstractThe dimeric regulatory protein wild-type ω (wt ω

scholarly journals Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

2020 ◽  
Vol 61 (12) ◽  
pp. 1577-1588
Author(s):  
Ryunosuke Ohkawa ◽  
Hann Low ◽  
Nigora Mukhamedova ◽  
Ying Fu ◽  
Shao-Jui Lai ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Cholesterol Efflux ◽  
Cholesterol Metabolism ◽  
Plasma Cholesterol ◽  
Translocator Protein ◽  
Plasma Lipoproteins ◽  
Cholesterol Transport ◽  
Autologous Plasma

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1+/+ and Abca1−/− mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.

scholarly journals Role of Constitutive STAR in Leydig Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 2021 ◽  
Author(s):  
Melanie Galano ◽  
Yuchang Li ◽  
Lu Li ◽  
Chantal Sottas ◽  
Vassilios Papadopoulos
Keyword(s):  
Leydig Cells ◽  
Lipid Droplets ◽  
Regulatory Protein ◽  
Translocator Protein ◽  
Mouse Tumor ◽  
Liquid Chromatography Mass Spectrometry ◽  
Wild Type ◽  
Cell Functions ◽  
Steroidogenic Acute Regulatory

Leydig cells contain significant amounts of constitutively produced steroidogenic acute regulatory protein (STAR; STARD1). Hormone-induced STAR plays an essential role in inducing the transfer of cholesterol into the mitochondria for hormone-dependent steroidogenesis. STAR acts at the outer mitochondrial membrane, where it interacts with a protein complex, which includes the translocator protein (TSPO). Mutations in STAR cause lipoid congenital adrenal hyperplasia (lipoid CAH), a disorder characterized by severe defects in adrenal and gonadal steroid production; in Leydig cells, the defects are seen mainly after the onset of hormone-dependent androgen formation. The function of constitutive STAR in Leydig cells is unknown. We generated STAR knockout (KO) MA-10 mouse tumor Leydig cells and showed that STAR KO cells failed to form progesterone in response to dibutyryl-cAMP and to TSPO drug ligands, but not to 22(R)-hydroxycholesterol, which is a membrane-permeable intermediate of the CYP11A1 reaction. Electron microscopy of STAR KO cells revealed that the number and size of lipid droplets were similar to those in wild-type (WT) MA-10 cells. However, the density of lipid droplets in STAR KO cells was drastically different than that seen in WT cells. We isolated the lipid droplets and analyzed their content by liquid chromatography–mass spectrometry. There was a significant increase in cholesteryl ester and phosphatidylcholine content in STAR KO cell lipid droplets, but the most abundant increase was in the amount of diacylglycerol (DAG); DAG 38:1 was the predominantly affected species. Lastly, we identified genes involved in DAG signaling and lipid metabolism which were differentially expressed between WT MA-10 and STAR KO cells. These results suggest that constitutive STAR in Leydig cells is involved in DAG accumulation in lipid droplets, in addition to cholesterol transport. The former event may affect cell functions mediated by DAG signaling.

scholarly journals Nanoscale architecture of a VAP-A-OSBP tethering complex at membrane contact sites

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Eugenio de la Mora ◽  
Manuela Dezi ◽  
Aurélie Di Cicco ◽  
Joëlle Bigay ◽  
Romain Gautier ◽  
...  
Keyword(s):  
Lipid Transfer Protein ◽  
Structural Information ◽  
Transmembrane Protein ◽  
Lipid Transfer ◽  
Oxysterol Binding Protein ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Golgi Network

AbstractMembrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and helical T-shape region. We propose that the molecular flexibility of VAP-A enables the recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains between membranes.

scholarly journals Prohibitin-1 Transgenic Mice Revealed an Important Role of Prohibitin in Testicular Steroidogenesis

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A771-A771
Author(s):  
Geetika Bassi ◽  
Suresh Mishra
Keyword(s):  
Transgenic Mice ◽  
Regulatory Protein ◽  
Phenotypic Characterization ◽  
Seminiferous Tubules ◽  
Testis Size ◽  
Wild Type ◽  
Testosterone Levels ◽  
Testicular Steroidogenesis ◽  
Prohibitin 1

Abstract Testosterone, the male sex hormone, plays an important role in the sexual development and fertility. Consequently, its deficiency causes infertility, obesity, osteoporosis and cardiovascular diseases. Leydig cells (LCs) are the testicular interstitial cells responsible for the biosynthesis of testosterone in response to luteinizing hormone (LH) from the pituitary. Cholesterol is the essential substrate for steroidogenesis which is translocated from the cytosol to the mitochondria where it gets converted to pregnenolone (by P450 side chain cleavage enzyme). Subsequently, pregnenolone translocate to endoplasmic reticulum where action of various enzymes results in the biosynthesis of testosterone. Prohibitin-1 (PHB1) is an evolutionary conserved ubiquitously expressed protein with cell compartment and cell-type specific functions. Mitochondrial function of PHB1 has been widely studied but its role in testicular steroidogenesis is unexplored. Recently, we have reported two transgenic mice models of PHB1, PHB-Tg and mutant-PHB-Tg (mPHB-Tg), expressing PHB1 or Y114F (mutant PHB1) respectively under the control of Fabp-4 gene promoter. During phenotypic characterization of these mice models, we observed a drastic size/weight difference in the testis of PHB-Tg and mPHB-Tg when compared with wild type mice. The mPHB-Tg mice testis was significantly smaller than the PHB-Tg and wild type mice. Further analysis of mPHB-Tg testis revealed wider testicular interstitium with LC hyperplasia and elongated seminiferous tubules. Ultrastructure investigation revealed that LCs of mPHB-Tg mice have prominent nucleus with increased number of mitochondria and lipid droplets. In addition, electron microscopic images of mPHB-Tg mice LCs revealed a sign of lipophagy and mitophagy. This prompted us to measure testosterone levels in these mice; surprisingly mPHB-Tg mice showed significantly higher testosterone levels as compared to PHB-Tg and wild type mice. Furthermore, testicular lysates and primary LCs cell lysates from transgenic mice models revealed that overexpression of PHB/mPHB in LCs inversely effect expression levels of steroidogenic acute regulatory protein (StAR). Moreover, co-immunoprecipitation of PHB1 displayed an interaction with StAR, P450scc and LC3 further revealing a key role of PHB1 in cholesterol translocation, testicular steroidogenesis and autophagy. Taken together, this finding suggests that PHB1 plays a multifaceted role in testicular steroidogenesis from determining testis size to the translocation of cholesterol into the mitochondria, in maintaining lipid homeostasis and biosynthesis of testosterone. Implications of our findings are broad because cholesterol translocation to the mitochondria and its subsequent utilization for steroidogenesis is conserved in all steroidogenic tissues.

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