scholarly journals Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Rodney Elwood Infante ◽  
Arun Radhakrishnan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Reversible Inhibitor ◽  
Cholesterol Transport ◽  
Cellular Cholesterol ◽  
Cholesterol Uptake ◽  
Regulatory Domains ◽  
Cholesterol Levels ◽  
Anthrolysin O ◽  
Lysosomal Transport

Cells employ regulated transport mechanisms to ensure that their plasma membranes (PMs) are optimally supplied with cholesterol derived from uptake of low-density lipoproteins (LDL) and synthesis. To date, all inhibitors of cholesterol transport block steps in lysosomes, limiting our understanding of post-lysosomal transport steps. Here, we establish the cholesterol-binding domain 4 of anthrolysin O (ALOD4) as a reversible inhibitor of cholesterol transport from PM to endoplasmic reticulum (ER). Using ALOD4, we: (1) deplete ER cholesterol without altering PM or overall cellular cholesterol levels; (2) demonstrate that LDL-derived cholesterol travels from lysosomes first to PM to meet cholesterol needs, and subsequently from PM to regulatory domains of ER to suppress activation of SREBPs, halting cholesterol uptake and synthesis; and (3) determine that continuous PM-to-ER cholesterol transport allows ER to constantly monitor PM cholesterol levels, and respond rapidly to small declines in cellular cholesterol by activating SREBPs, increasing cholesterol uptake and synthesis.

scholarly journals Ostreolysin A and anthrolysin O use different mechanisms to control movement of cholesterol from the plasma membrane to the endoplasmic reticulum

2019 ◽  
Vol 294 (46) ◽  
pp. 17289-17300 ◽  
Author(s):  
Kristen A. Johnson ◽  
Shreya Endapally ◽  
Danya C. Vazquez ◽  
Rodney E. Infante ◽  
Arun Radhakrishnan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Lipid Binding ◽  
Cholesterol Homeostasis ◽  
Bacterial Toxin ◽  
Live Cells ◽  
Cholesterol Transport ◽  
Animal Cells ◽  
Anthrolysin O ◽  
Binding Mechanisms

Recent studies using two cholesterol-binding bacterial toxin proteins, perfringolysin O (PFO) and domain 4 of anthrolysin O (ALOD4), have shown that cholesterol in the plasma membranes (PMs) of animal cells resides in three distinct pools. The first pool comprises mobile cholesterol, accessible to both PFO and ALOD4, that is rapidly transported to the endoplasmic reticulum (ER) to signal cholesterol excess and maintain cholesterol homeostasis. The second is a sphingomyelin (SM)-sequestered pool inaccessible to PFO and ALOD4 but that becomes accessible by treatment with SM-degrading sphingomyelinase (SMase). The third is an essential pool also inaccessible to PFO and ALOD4 that cannot be liberated by SMase treatment. The accessible cholesterol pool can be trapped on PMs of live cells by nonlytic ALOD4, blocking its transport to the ER. However, studies of the two other pools have been hampered by a lack of available tools. Here, we used ostreolysin A (OlyA), which specifically binds SM/cholesterol complexes in membranes, to study the SM-sequestered cholesterol pool. Binding of nonlytic OlyA to SM/cholesterol complexes in PMs of live cells depleted the accessible PM cholesterol pool detectable by ALOD4. Consequently, transport of accessible cholesterol from PM to ER ceased, thereby activating SREBP transcription factors and increasing cholesterol synthesis. Thus, OlyA and ALOD4 both control movement of PM cholesterol, but through different lipid-binding mechanisms. We also found that PM-bound OlyA was rapidly internalized into cells, whereas PM-bound ALOD4 remained on the cell surface. Our findings establish OlyA and ALOD4 as complementary tools to investigate cellular cholesterol transport.

scholarly journals VAPs and ACBD5 tether peroxisomes to the ER for peroxisome maintenance and lipid homeostasis

2017 ◽  
Vol 216 (2) ◽  
pp. 367-377 ◽  
Author(s):  
Rong Hua ◽  
Derrick Cheng ◽  
Étienne Coyaud ◽  
Spencer Freeman ◽  
Erminia Di Pietro ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Lipid Homeostasis ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Cellular Cholesterol ◽  
Cholesterol Levels ◽  
Lipid Exchange ◽  
Associated Proteins ◽  
Mediated Contact

Lipid exchange between the endoplasmic reticulum (ER) and peroxisomes is necessary for the synthesis and catabolism of lipids, the trafficking of cholesterol, and peroxisome biogenesis in mammalian cells. However, how lipids are exchanged between these two organelles is not understood. In this study, we report that the ER-resident VAMP-associated proteins A and B (VAPA and VAPB) interact with the peroxisomal membrane protein acyl-CoA binding domain containing 5 (ACBD5) and that this interaction is required to tether the two organelles together, thereby facilitating the lipid exchange between them. Depletion of either ACBD5 or VAP expression results in increased peroxisome mobility, suggesting that VAP–ACBD5 complex acts as the primary ER–peroxisome tether. We also demonstrate that tethering of peroxisomes to the ER is necessary for peroxisome growth, the synthesis of plasmalogen phospholipids, and the maintenance of cellular cholesterol levels. Collectively, our data highlight the importance of VAP–ACBD5–mediated contact between the ER and peroxisomes for organelle maintenance and lipid homeostasis.

scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

Modulation of intestinal cholesterol absorption by high glucose levels: impact on cholesterol transporters, regulatory enzymes, and transcription factors

2008 ◽  
Vol 295 (5) ◽  
pp. G873-G885 ◽  
Author(s):  
Z. Ravid ◽  
M. Bendayan ◽  
E. Delvin ◽  
A. T. Sane ◽  
M. Elchebly ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Protein Expression ◽  
High Glucose ◽  
Cholesterol Absorption ◽  
Cholesterol Homeostasis ◽  
Cholesterol Transport ◽  
Intestinal Cholesterol Absorption ◽  
Cholesterol Uptake ◽  
Glucose Levels

Growing evidence suggests that the small intestine may contribute to excessive postprandial lipemia, which is highly prevalent in insulin-resistant/Type 2 diabetic individuals and substantially increases the risk of cardiovascular disease. The aim of the present study was to determine the role of high glucose levels on intestinal cholesterol absorption, cholesterol transporter expression, enzymes controlling cholesterol homeostasis, and the status of transcription factors. To this end, we employed highly differentiated and polarized cells (20 days of culture), plated on permeable polycarbonate filters. In the presence of [14C]cholesterol, glucose at 25 mM stimulated cholesterol uptake compared with Caco-2/15 cells supplemented with 5 mM glucose ( P < 0.04). Because combination of 5 mM glucose with 20 mM of the structurally related mannitol or sorbitol did not change cholesterol uptake, we conclude that extracellular glucose concentration is uniquely involved in the regulation of intestinal cholesterol transport. The high concentration of glucose enhanced the protein expression of the critical cholesterol transporter NPC1L1 and that of CD36 ( P < 0.02) and concomitantly decreased SR-BI protein mass ( P < 0.02). No significant changes were observed in the protein expression of ABCA1 and ABCG8, which act as efflux pumps favoring cholesterol export out of absorptive cells. At the same time, 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was decreased ( P < 0.007), whereas ACAT activity remained unchanged. Finally, increases were noted in the transcription factors LXR-α, LXR-β, PPAR-β, and PPAR-γ along with a drop in the protein expression of SREBP-2. Collectively, our data indicate that glucose at high concentrations may regulate intestinal cholesterol transport and metabolism in Caco-2/15 cells, thus suggesting a potential influence on the cholesterol absorption process in Type 2 diabetes.

Reverse cholesterol transport: relationship between free cholesterol uptake and HDL3 in normolipidaemic and hyperlipidaemic subjects

1993 ◽  
Vol 23 (2) ◽  
pp. 122-129 ◽  
Author(s):  
M. CASTRO CABEZAS ◽  
G. P. H. VAN HEUSDEN ◽  
T. W. A. DE BRUIN ◽  
J. R. C. M. VAN BECKHOVEN ◽  
L. A. W. KOCK ◽  
...  
Keyword(s):  
Reverse Cholesterol Transport ◽  
Free Cholesterol ◽  
Cholesterol Transport ◽  
Cholesterol Uptake

FINE STRUCTURE IN CELLS OF PEA AND WHEAT EMBRYOS

1959 ◽  
Vol 37 (1) ◽  
pp. 65-72 ◽  
Author(s):  
G. Setterfield ◽  
H. Stern ◽  
F. B. Johnston
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Osmic Acid ◽  
Dense Granules ◽  
Cytoplasmic Bodies ◽  
Wheat Embryos ◽  
Electron Microscopes ◽  
Cell Fractions ◽  
Isolated Cell

To provide a basis for relating biochemical findings on isolated cell fractions to cytological structure in situ, embryos of pea and wheat were fixed with osmic acid, sectioned, and observed in phase-contrast and electron microscopes. The nuclei of all cells were similar, showing nuclear membranes, chromosomes, and prominent nucleoli. The cytoplasm contained highly developed structure which presumably reflected the incipient growth condition of the cells. Several cytoplasmic components were common to both embryos: small dense granules, endoplasmic reticulum, mitochondria, presumed proplastids, amyloplasts, irregular bodies, plasma membranes, and plasmodesmata. The small dense granules, presumably ribonucleoprotein particles, occurred profusely, both free and in association with extensively developed endoplasmic reticulum. These particles are probably responsible for the microsomal fractions obtainable from embryos and seedlings. The mitochondria were usually relatively small (0.25−0.5 μ diameter) although groups of very long (5 μ) ones were occasionally found. Bodies resembling mitochondria in size and shape, but lacking cristae, were present and represent either immature mitochondria or proplastids. Reserve material occurred as starch in structurally complex amyloplasts and possibly as protein in the irregular bodies. In addition to these structures cells of the wheat embryos remote from the meristems contained prominent cytoplasmic bodies classified as "dense" and "thick-walled". The dense bodies probably represent stored lipids while the significance of the thick-walled bodies, which showed a variety of forms, is unknown.

scholarly journals A role for NPC1 and NPC2 in intestinal cholesterol absorption – the hypothesis gutted

2007 ◽  
Vol 408 (1) ◽  
Author(s):  
Laura Liscum
Keyword(s):  
Endoplasmic Reticulum ◽  
Dietary Cholesterol ◽  
Cholesterol Absorption ◽  
Cholesterol Transport ◽  
Intestinal Epithelial ◽  
Intestinal Cholesterol Absorption ◽  
Biochemical Journal ◽  
Good Target ◽  
Type C ◽  
Niemann Pick

Dietary and biliary cholesterol are taken up by intestinal epithelial cells and transported to the endoplasmic reticulum. At the endoplasmic reticulum, cholesterol is esterified, packaged into chylomicrons and secreted into the lymph for delivery to the bloodstream. NPC1L1 (Niemann–Pick C1-like 1) is a protein on the enterocyte brush-border membrane that facilitates cholesterol absorption. Cholesterol's itinerary as it moves to the endoplasmic reticulum is unknown, as is the identity of any cellular proteins that facilitate the movement. Two proteins that play an important role in intracellular cholesterol transport and could potentially influence NPC1L1-mediated cholesterol uptake are NPC1 and NPC2 (Niemann–Pick type C disease proteins 1 and 2). In this issue of the Biochemical Journal, Dixit and colleagues show that the absence or presence of NPC1 and NPC2 has no effect on intestinal cholesterol absorption in the mouse. Thus neither protein fills the gap in our knowledge of intra-enterocyte cholesterol transport. Furthermore, the NPC1/NPC2 pathway would not be a good target for limiting the uptake of dietary cholesterol.

scholarly journals Rapid isolation of plasma membranes in high yield from cultured fibroblasts

1977 ◽  
Vol 168 (2) ◽  
pp. 187-194 ◽  
Author(s):  
D Thom ◽  
A J Powell ◽  
C W Lloyd ◽  
D A Rees
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
High Yield ◽  
Chemical Compositions ◽  
Differential Centrifugation ◽  
Rapid Preparation ◽  
Cultured Fibroblasts ◽  
Good Agreement

1. A method was developed which allows the rapid preparation of pure plasma membranes in high yield from cultured fibroblasts. 2. Cells are lysed in hypo-osmotic borate/EDTA and, after differential centrifugation, the membranes collected by centrifugation on a sucrose barrier. 3. Electron microscopy of the isolated material shows large membrane vesicles essentially free from contaminating organelles. 4. There is no detectable activity of the endoplasmic-reticulum enzyme marker, NADH2—lipoamide oxidoreductase (EC 1.6.4.3), and that of succinate dehydrogenase (EC 1.3.99.1), a marker for mitochondria, is substantially decreased. Chemical compositions are in good agreement with previous observations. 5. This study confirms the usefulness of applied isotopic markers for isolating plasma membranes.

scholarly journals Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Nicholas L. Cianciola ◽  
Stacey Chung ◽  
Danny Manor ◽  
Cathleen R. Carlin
Keyword(s):  
Innate Immunity ◽  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Lipid Droplets ◽  
Cholesterol Transport ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Human Adenoviruses ◽  
Early Region ◽  
Early Region 3

ABSTRACT Human adenoviruses (Ads) generally cause mild self-limiting infections but can lead to serious disease and even be fatal in high-risk individuals, underscoring the importance of understanding how the virus counteracts host defense mechanisms. This study had two goals. First, we wished to determine the molecular basis of cholesterol homeostatic responses induced by the early region 3 membrane protein RIDα via its direct interaction with the sterol-binding protein ORP1L, a member of the evolutionarily conserved family of oxysterol-binding protein (OSBP)-related proteins (ORPs). Second, we wished to determine how this interaction regulates innate immunity to adenovirus. ORP1L is known to form highly dynamic contacts with endoplasmic reticulum-resident VAP proteins that regulate late endosome function under regulation of Rab7-GTP. Our studies have demonstrated that ORP1L-VAP complexes also support transport of LDL-derived cholesterol from endosomes to the endoplasmic reticulum, where it was converted to cholesteryl esters stored in lipid droplets when ORP1L was bound to RIDα. The virally induced mechanism counteracted defects in the predominant cholesterol transport pathway regulated by the late endosomal membrane protein Niemann-Pick disease type C protein 1 (NPC1) arising during early stages of viral infection. However, unlike NPC1, RIDα did not reconstitute transport to endoplasmic reticulum pools that regulate SREBP transcription factors. RIDα-induced lipid trafficking also attenuated proinflammatory signaling by Toll-like receptor 4, which has a central role in Ad pathogenesis and is known to be tightly regulated by cholesterol-rich “lipid rafts.” Collectively, these data show that RIDα utilizes ORP1L in a way that is distinct from its normal function in uninfected cells to fine-tune lipid raft cholesterol that regulates innate immunity to adenovirus in endosomes. IMPORTANCE Early region 3 proteins encoded by human adenoviruses that attenuate immune-mediated pathology have been a particularly rich source of information regarding intracellular protein trafficking. Our studies with the early region 3-encoded RIDα protein also provided fundamental new information regarding mechanisms of nonvesicular lipid transport and the flow of molecular information at membrane contacts between different organelles. We describe a new pathway that delivers cholesterol from endosomes to the endoplasmic reticulum, where it is esterified and stored in lipid droplets. Although lipid droplets are attracting renewed interest from the standpoint of normal physiology and human diseases, including those resulting from viral infections, experimental model systems for evaluating how and why they accumulate are still limited. Our studies also revealed an intriguing relationship between lipid droplets and innate immunity that may represent a new paradigm for viruses utilizing these organelles.

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