Isolation of plasma membrane, Golgi apparatus, and endoplasmic reticulum fractions from single homogenates of mouse liver

Croze, Ed; Morré, D. James

doi:10.1002/jcp.1041190109

Cited by 92 publications

(54 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Differential subcellular fractionation was performed according to the procedure of Croze and Morre (22) as modified by Vance (23), yielding fractions corresponding to ER, nucleus, plasma membrane, mitochondria, MAM, and Golgi apparatus. Protein concentrations of individual fractions were measured by the Bradford method, using albumin as standard.…”

Section: Methodsmentioning

confidence: 99%

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Shields¹,

Lehner²,

Agellon³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

In liver, phosphatidylethanolamine is converted to phosphatidylcholine through a series of three sequential methylation reactions. Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes each transmethylation reaction, and S-adenosylmethionine is the methyl group donor. Biochemical analysis of human liver revealed that the methyltransferase activity is primarily localized to the endoplasmic reticulum and mitochondria-associated membranes. Bioinformatic analysis of the predicted amino acid sequence suggested that the enzyme adopts a polytopic conformation in those membranes. To elucidate the precise membrane topography of PEMT and thereby provide the basis for in-depth functional characterization of the enzyme, we performed endoproteinase-protection analysis of epitopetagged, recombinant protein. Our data suggest a topographical model of PEMT in which four transmembrane regions span the membrane such that both the N and C termini of the enzyme are localized external to the ER. Two hydrophilic connecting loops protrude into the luminal space of the microsomes whereas a corresponding loop on the cytosolic side remains proximate to the membrane. Further support for this model was obtained following endoproteinase-protection analysis of mutant recombinant PEMT derivatives in which specific protease cleavage sites had been genetically engineered or ablated.

show abstract

Section: Methodsmentioning

confidence: 99%

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Shields¹,

Lehner²,

Agellon³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The pellet was resuspended in 10 mmol/L TrisHCl, 0.15 mmol/L MgCl 2 , and 0.25 mol/L sucrose (pH 6.7). Mitochondria from livers of Pemt +/+ and Pemt 2/2 mice were isolated as previously described (11 (12). Fifteen minutes after injection, blood was collected by cardiac puncture into EDTA-containing tubes, plasma was prepared by centrifugation, and organs were harvested and snap-frozen in liquid nitrogen.…”

Section: Isolation Of Mitochondriamentioning

confidence: 99%

The Concentration of Phosphatidylethanolamine in Mitochondria Can Modulate ATP Production and Glucose Metabolism in Mice

et al. 2014

View full text Add to dashboard Cite

Phosphatidylethanolamine (PE) N-methyltransferase (PEMT) catalyzes the synthesis of phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected against diet-induced obesity and insulin resistance. We investigated the role of PEMT in hepatic carbohydrate metabolism in chow-fed mice. A pyruvate tolerance test revealed that PEMT deficiency greatly attenuated gluconeogenesis. The reduction in glucose production was specific for pyruvate; glucose production from glycerol was unaffected. Mitochondrial PC levels were lower and PE levels were higher in livers from Pemt 2/2 compared with Pemt +/+ mice, resulting in a 33% reduction of the PC-to-PE ratio. Mitochondria from Pemt 2/2 mice were also smaller and more elongated. Activities of cytochrome c oxidase and succinate reductase were increased in mitochondria of Pemt 2/2 mice. Accordingly, ATP levels in hepatocytes from Pemt 2/2 mice were double that in Pemt +/+ hepatocytes. We observed a strong correlation between mitochondrial PC-to-PE ratio and cellular ATP levels in hepatoma cells that expressed various amounts of PEMT. Moreover, mitochondrial respiration was increased in cells lacking PEMT. In the absence of PEMT, changes in mitochondrial phospholipids caused a shift of pyruvate toward decarboxylation and energy production away from the carboxylation pathway that leads to glucose production.

show abstract

“…COXIV, a mitochondrial-specific protein, was detected at a high level in the mitochondria. Calnexin and peroxisome proliferator-activated receptor ␦ were used as specific markers for the ER and nucleus, respectively (22) (Fig. 6A).…”

Section: Fsp27 Is Stabilized By ␤-Agonistmentioning

confidence: 99%

Fat-specific Protein 27 Undergoes Ubiquitin-dependent Degradation Regulated by Triacylglycerol Synthesis and Lipid Droplet Formation

Nian

Sun

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

The fat-specific protein 27 (Fsp27), a protein localized to lipid droplets (LDs), plays an important role in controlling lipid storage and mitochondrial activity in adipocytes. Fsp27-null mice display increased energy expenditure and are resistant to high fat diet-induced obesity and diabetes. However, little is known about how the Fsp27 protein is regulated. Here, we show that Fsp27 stability is controlled by the ubiquitin-dependent proteasomal degradation pathway in adipocytes. The ubiquitination of Fsp27 is regulated by three lysine residues located in the C-terminal region. Substitution of these lysine residues with alanines greatly increased Fsp27 stability and enhanced lipid storage in adipocytes. Furthermore, Fsp27 was stabilized and rapidly accumulated following treatment with ␤-agonists that induce lipolysis and fatty acid re-esterification in adipocytes. More importantly, Fsp27 stabilization was dependent on triacylglycerol synthesis and LD formation, because knockdown of diacylglycerol acyltransferase in adipocytes significantly reduced Fsp27 accumulation in adipocytes. Finally, we observed that increased Fsp27 during ␤-agonist treatment preferentially associated with LDs. Taken together, our data revealed that Fsp27 can be stabilized by free fatty acid availability, triacylglycerol synthesis, and LD formation. The stabilization of Fsp27 when free fatty acids are abundant further enhances lipid storage, providing positive feedback to regulate lipid storage in adipocytes.Cell death-inducing DNA fragmentation factor-45-like effector (Cide) proteins, including Cidea, Cideb, and fat-specific protein 27 (Fsp27, 3 also known as Cidec in humans), are a family of proteins shown to play critical roles in controlling metabolism homeostasis (1). Our previous work demonstrated that mice with a deficiency in Cidea or Cideb have higher energy expenditure and enhanced insulin sensitivity and are resistant to high fat diet-induced obesity and diabetes (2, 3). Fsp27 is enriched in adipocytes, in both white adipose tissue and brown adipose tissue (2, 4). The Fsp27 protein is detected in the lipid droplet (LD)-enriched fraction (5), and its overexpression can promote triacylglycerol (TAG) storage (6, 7). Interestingly, Fsp27 and Cidea mRNAs have also been detected in fatty livers, where an excess amount of lipids accumulate and large LDs form (8 -10). More recently, Fsp27 was demonstrated to be a direct mediator of peroxisome proliferator-activated receptor ␥-dependent hepatic steatosis (10). In accordance with a role for Fsp27 in LD formation, Fsp27 deficiency results in dramatically reduced white adipose tissue deposits and the acquisition of a brown fat-like morphology in these white adipose tissues, which is characterized by the appearance of smaller LDs and increased mitochondrial size and activity (11, 12). Furthermore, both Fsp27-deficient and Fsp27/leptin double-deficient mice display improved insulin sensitivity and lean phenotype (12). Except for one study showing that Cidea is degraded through the ubiquitin-m...

show abstract

Isolation of plasma membrane, Golgi apparatus, and endoplasmic reticulum fractions from single homogenates of mouse liver

Cited by 92 publications

References 31 publications

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

The Concentration of Phosphatidylethanolamine in Mitochondria Can Modulate ATP Production and Glucose Metabolism in Mice

Fat-specific Protein 27 Undergoes Ubiquitin-dependent Degradation Regulated by Triacylglycerol Synthesis and Lipid Droplet Formation

Contact Info

Product

Resources

About