scholarly journals Sphingosine-1-Phosphate Phosphohydrolase Regulates Endoplasmic Reticulum-to-Golgi Trafficking of Ceramide

2006 ◽  
Vol 26 (13) ◽  
pp. 5055-5069 ◽  
Author(s):  
Paola Giussani ◽  
Michael Maceyka ◽  
Hervé Le Stunff ◽  
Aki Mikami ◽  
Sandrine Lépine ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Trafficking ◽  
Fluorescent Protein ◽  
Fumonisin B1 ◽  
Sphingolipid Metabolism ◽  
Ceramide Synthase ◽  
C Protein ◽  
Sphingosine 1 Phosphate ◽  
Green Fluorescent ◽  
Ceramide Accumulation

ABSTRACT Previous studies demonstrated that sphingosine-1-phosphate (S1P) phosphohydrolase 1 (SPP-1), which is located mainly in the endoplasmic reticulum (ER), regulates sphingolipid metabolism and apoptosis (H. Le Stunff et al., J. Cell Biol. 158:1039-1049, 2002). We show here that the treatment of SPP-1-overexpressing cells with S1P, but not with dihydro-S1P, increased all ceramide species, particularly the long-chain ceramides. This was not due to inhibition of ceramide metabolism to sphingomyelin or monohexosylceramides but rather to the inhibition of ER-to-Golgi trafficking, determined with the fluorescent ceramide analog N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (DMB-Cer). Fumonisin B1, an inhibitor of ceramide synthase, prevented S1P-induced elevation of all ceramide species and corrected the defect in ER transport of DMB-Cer, readily allowing its detection in the Golgi. In contrast, ceramide accumulation had no effect on either the trafficking or the metabolism of 6-([N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl)-sphingosine, which rapidly labels the Golgi even at 4°C. Protein trafficking from the ER to the Golgi, determined with vesicular stomatitis virus ts045 G protein fused to green fluorescent protein, was also inhibited in SPP-1-overexpressing cells in the presence of S1P but not in the presence of dihydro-S1P. Our results suggest that SPP-1 regulates ceramide levels in the ER and thus influences the anterograde membrane transport of both ceramide and proteins from the ER to the Golgi apparatus.

scholarly journals Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis

2002 ◽  
Vol 158 (6) ◽  
pp. 1039-1049 ◽  
Author(s):  
Hervé Le Stunff ◽  
Ismael Galve-Roperh ◽  
Courtney Peterson ◽  
Sheldon Milstien ◽  
Sarah Spiegel
Keyword(s):  
Cell Fate ◽  
De Novo ◽  
Fumonisin B1 ◽  
Sphingolipid Metabolism ◽  
Ceramide Synthase ◽  
Serine Palmitoyltransferase ◽  
Intracellular Membranes ◽  
Sphingosine 1 Phosphate ◽  
Ceramide Accumulation ◽  
Membrane Treatment

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein–coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

scholarly journals Identification of a Novel Saturable Endoplasmic Reticulum Localization Mechanism Mediated by the C-Terminus of aDictyostelium Protein Disulfide Isomerase

2000 ◽  
Vol 11 (10) ◽  
pp. 3469-3484 ◽  
Author(s):  
Jean Monnat ◽  
Eva M. Neuhaus ◽  
Marius S. Pop ◽  
David M. Ferrari ◽  
Barbara Kramer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Disulfide Isomerase ◽  
Fluorescent Protein ◽  
Null Mutation ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
C Terminus ◽  
Complementary Action ◽  
Green Fluorescent ◽  
Protein Disulfide

Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.

Increased Redox-Sensitive Green Fluorescent Protein Reduction Potential in the Endoplasmic Reticulum following Glutathione-Mediated Dimerization

Biochemistry ◽  
2013 ◽  
Vol 52 (19) ◽  
pp. 3332-3345 ◽  
Author(s):  
Deboleena Dipak Sarkar ◽  
Sarah K. Edwards ◽  
Justin A. Mauser ◽  
Allen M. Suarez ◽  
Maxwell A. Serowoky ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Reduction Potential ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
Protein Reduction

scholarly journals Resveratrol Targets Sphingolipid Metabolism to Induce Growth Inhibition in FLT3 ITD Acute Myeloid Leukemia

Proceedings ◽  
2019 ◽  
Vol 40 (1) ◽  
pp. 4
Author(s):  
Ersöz ◽  
Adan
Keyword(s):  
Growth Inhibition ◽  
Cell Fate ◽  
De Novo ◽  
Therapeutic Potential ◽  
Annexin V ◽  
Sphingolipid Metabolism ◽  
Ceramide Synthase ◽  
Cytotoxic Effects ◽  
Sphingosine 1 Phosphate ◽  
First Time

Sphingolipids are important signaling lipids which play crucial roles to determine the cell fate. Ceramide, apoptotic central molecule of sphingolipid metabolism, which is produced through de novo pathway by serine palmitoyl transferase (SPT) and can be converted to antiapoptotic sphingosine-1-phosphate (S1P) and glucosyl ceramide (GC) by sphingosine kinase (SK) and glucosyl ceramide synthase (GCS), respectively. It is aimed to investigate therapeutic potential of resveratrol on FLT3-ITD (Internal Tandem Duplication) AML cells and to identify potential mechanism behind resveratrol-mediated growth inhibition by targeting of ceramide metabolism. The cytotoxic effects of resveratrol, SPT inhibitor (myricoin), SK-1 inhibitor (SKI II), GCS inhibitor (PDMP), resveratrol: SPT inhibitor, resveratrol: SK-1 inhibitor and resveratrol: GCS inhibitor combinations on MOLM-13 and MV4-11 FLT3 ITD AML cells were investigated by cell proliferation assay. Apoptosis was evaluated by annexin V/PI double staining. There were synergistic cytotoxic effects of resveratrol with co-administration of SPT inhibitor, SK-1 inhibitor and GCS inhibitor and apoptosis was synergistically induced for resveratrol and its combinations. This preliminary data showed for the first time that resveratrol might inhibit the growth of FLT3 ITD AML cells through targeting ceramide metabolism.

scholarly journals Chaperone-mediated reflux of secretory proteins to the cytosol during endoplasmic reticulum stress

2019 ◽  
Vol 116 (23) ◽  
pp. 11291-11298 ◽  
Author(s):  
Aeid Igbaria ◽  
Philip I. Merksamer ◽  
Ala Trusina ◽  
Firehiwot Tilahun ◽  
Jeffrey R. Johnson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Secretory Pathway ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Control Process ◽  
Secretory Proteins ◽  
Quality Control Process ◽  
Folded Proteins ◽  
Green Fluorescent

Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such “ER stress,” we employed an ER-targeted, redox-responsive, green fluorescent protein—eroGFP—that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to “reflux” back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.

scholarly journals mCLCA4 ER processing and secretion requires luminal sorting motifs

2008 ◽  
Vol 295 (1) ◽  
pp. C279-C287 ◽  
Author(s):  
Chunlei Huan ◽  
Kai Su Greene ◽  
Bo Shui ◽  
Gwendolyn Spizz ◽  
Haitao Sun ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chloride Transport ◽  
Proteolytic Processing ◽  
Regulatory Sequences ◽  
Cellular Processing ◽  
Green Fluorescent

Ca+-activated Cl− channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90- and 40-kDa fragments, and the NH2- and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4.

Regulation of glycine transporters

2001 ◽  
Vol 29 (6) ◽  
pp. 742-745 ◽  
Author(s):  
B. López-Corcuera ◽  
C. Aragón ◽  
A. Geerlings
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Fluorescent Protein ◽  
Surface Expression ◽  
Immunogold Labelling ◽  
Glycine Release ◽  
Glycine Transporters ◽  
Protein Receptor ◽  
Large Dense Core Vesicles ◽  
Green Fluorescent

The regulation of neurotransmitter transporters is a central aspect of their physiology. Recent studies that focused on syntaxin-1 transporter interactions led to the postulation that syntaxin-1 is somehow implicated in protein trafficking. Because syntax – in-1 is involved in the exocytosis of neurotransmitters and it interacts with glycine transporter 2 (GLYT2), we stimulated exocytosis in synaptosomes and examined its effect on GLYT2 surface-expression and transport activity. We found that GLYT2 is rapidly trafficked first towards the plasma membrane and then internalized under conditions that stimulate vesicular glycine release. However, when syntaxin-1 was inactivated by pre-treatment of synaptosomes with the botulinum neurotoxin C, GLYT2 was unable to reach the plasma membrane but still was able to leave it. These results indicate the existence of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated regulatory mechanism that controls the surface expression of GLYT2. Syntaxin-1 is involved in the transport of GLYT2 to, but not its retrieval from, the plasma membrane. Immunogold-labelling on purified vesicular preparations from synaptosomes showed that GLYT2 is present in small synaptic-like vesicles. This may represent neurotransmitter transporter that is being trafficked. The subcellular distribution of the glycine transporters was further examined in PC12 cells that were stably transfected with the fusions of GLYT1 and GLYT2 with green fluorescent protein. There was a clear difference in their intracellular distribution, GLYT1 being present mainly on the plasma membrane and GLYT2 being localized mainly on large, dense-core vesicles. We are trying to find signal sequences responsible for this differential localization.

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