scholarly journals Multiple functions of inositolphosphorylceramides in the formation and intracellular transport of glycosylphosphatidylinositol-anchored proteins in yeast

2007 ◽  
Vol 74 ◽  
pp. 199-209 ◽  
Author(s):  
Régine Bosson ◽  
Andreas Conzelmann
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Fatty Acid ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Gpi Anchor ◽  
New Class ◽  
Golgi Transport ◽  
Lipid Moiety ◽  
Gpi Proteins

The mature sphingolipids of yeast consist of IPCs (inositolphosphorylceramides) and glycosylated derivatives thereof. Beyond being an abundant membrane constituent in the organelles of the secretory pathway, IPCs are also used to constitute the lipid moiety of the majority of GPI (glycosylphosphatidylinositol) proteins, while a minority of GPI proteins contain PI (phosphatidylinositol). Thus all GPI anchor lipids (as well as free IPCs) typically contain C26 fatty acids. However, the primary GPI lipid that isadded to newly synthesized proteins in the endoplasmic reticulum consists of a PI with conventional C16 and C18 fatty acids. A new class of enzymes is required to replace the fatty acid in sn-2 by a C26 fatty acid. Cells lacking this activity make normal amounts of GPI proteins but accumulate GPI anchors containing lyso-PI. As a consequence, the endoplasmic reticulum to Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. The GPI anchor containing C26 in sn-2 can further be remodelled by the exchange of diacylglycerol for ceramide. This process is also dependent on the presence of specific phosphorylethanolamine side-chains on the GPI anchor.

scholarly journals The differential effects of dithiothreitol and 2-mercaptoethanol on the secretion of partially and completely assembled immunoglobulins suggest that thiol-mediated retention does not take place in or beyond the Golgi.

1994 ◽  
Vol 5 (12) ◽  
pp. 1311-1324 ◽  
Author(s):  
C Valetti ◽  
R Sitia
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Reducing Agents ◽  
Differential Effects ◽  
Golgi Transport ◽  
Disulphide Bonds ◽  
Golgi Compartment ◽  
Interchange Reactions

Dithiothreitol (DTT) blocks the endoplasmic reticulum (ER)-Golgi transport of newly synthesized immunoglobulin (Ig) molecules, whereas 2-mercaptoethanol (2ME) allows secretion of unpolymerized Igs otherwise retained intracellularly by disulphide interchange reactions. To understand this dichotomy, we have compared the effects of DTT and 2ME on the assembly, intracellular transport, and secretion of a panel of chimeric Igs that are either constitutively secreted or retained intracellularly. Our results demonstrate that DTT, but not 2ME, reduces some of the inter- and intrachain disulphide bonds and causes partial disassembly of H2L2 complexes and unfolding of individual chains in the ER. Upon DTT removal, heavy (H) and light (L) chains reform hapten-binding H2L2 molecules, which are later secreted. Reduction of the H2L2 interchain disulphide bonds can occur along the entire secretory pathway; however, in or beyond the Golgi this does not result in efficient H-L disassembly or unfolding. As a consequence, DTT does not block the exit from the Golgi. Moreover, unpolymerized Igs--normally retained in a pre-Golgi compartment--no longer require reducing agents to be secreted once they have reached the Golgi. Thus, little if any thiol-mediated retention seems to take place in or beyond the Golgi complex.

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport

1991 ◽  
Vol 11 (6) ◽  
pp. 2980-2993
Author(s):  
R Ossig ◽  
C Dascher ◽  
H H Trepte ◽  
H D Schmitt ◽  
D Gallwitz
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Single Copy ◽  
Integral Membrane Protein ◽  
Carboxypeptidase Y ◽  
Null Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Transport ◽  
Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

Inhibition of binding to fatty acid binding protein reduces the intracellular transport of fatty acids

1996 ◽  
Vol 271 (1) ◽  
pp. G113-G120 ◽  
Author(s):  
B. A. Luxon
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Intracellular Transport ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Female Rats ◽  
Dependent Manner ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Cytoplasmic Transport

Male livers, containing lesser amounts of fatty acid binding protein (FABP), utilize fatty acids more slowly than female livers. Conventional wisdom dictates that FABP stimulates fatty acid use by increasing cytoplasmic transport rates. Previously, we showed that the cytoplasmic diffusion of a fatty acid analogue [12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazol-amino stearate (NBD-stearate)] is faster in female hepatocytes, paralleling the larger amounts of FABP. Sex differences in other cytoplasmic factors could also lead to faster diffusion, independent of FABP levels. The aim of this study was to determine the effect of inhibition of fatty acid binding to FABP on the directly measured intracellular transport rate of NBD-stearate. The binding of NBD-stearate to FABP was reduced by incubating hepatocytes isolated from male and female rats with alpha-bromo-palmitate (0-1,500 microM), a modified long-chain fatty acid that binds to FABP. The inhibition by alpha-bromo-palmitate on NBD-stearate binding to FABP was measured with the use of centrifugation to separate cytosol from cytoplasmic membranes. Laser photobleaching (fluorescence recovery after photobleaching) was used to measure the cytoplasmic diffusion of NBD-stearate in hepatocytes. Alpha-Bromo-palmitate incubation reduced NBD-stearate binding to FABP in a dose-dependent manner. The measured diffusion rate was also reduced in proportion to the degree of binding inhibition. We conclude that cytoplasmic transport of NBD-stearate is modulated by binding to soluble proteins like FABP. FABP enhances diffusive transport by reducing binding to immobile cytosolic membranes.

scholarly journals The role of lipid components of the diet in the regulation of the fatty acid composition of the rat liver endoplasmic reticulum and lipid peroxidation

1978 ◽  
Vol 174 (2) ◽  
pp. 585-593 ◽  
Author(s):  
Catherine T. Hammer ◽  
Eric D. Wills
Keyword(s):  
Lipid Peroxidation ◽  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Polyunsaturated Fatty Acids ◽  
Acid Composition ◽  
Corn Oil ◽  
Lipid Peroxide

The fatty acid compositions of the lipids and the lipid peroxide concentrations and rates of lipid peroxidation were determined in suspensions of liver endoplasmic reticulum isolated from rats fed on synthetic diets in which the fatty acid composition had been varied but the remaining constituents (protein, carbohydrate, vitamins and minerals) kept constant. Stock diet and synthetic diets containing no fat, 10% corn oil, herring oil, coconut oil or lard were used. The fatty acid composition of the liver endoplasmic reticulum lipid was markedly dependent on the fatty acid composition of the dietary lipid. Feeding a herring-oil diet caused incorporation of 8.7% eicosapentaenoic acid (C20:5) and 17% docosahexaenoic acid (C22:6), but only 5.1% linoleic acid (C18:2) and 6.4% arachidonic acid (C20:4), feeding a corn-oil diet caused incorporation of 25.1% C18:2, 17.8% C20:4 and 2.5% C22:6 fatty acids, and feeding a lard diet caused incorporation of 10.3% C18:2, 13.5% C20:4 and 4.3% C22:6 fatty acids into the liver endoplasmic-reticulum lipids. Phenobarbitone injection (100mg/kg) decreased the incorporation of C20:4 and C22:6 fatty acids into the liver endoplasmic reticulum of rats fed on a lard, corn-oil or herring-oil diet. Microsomal lipid peroxide concentrations and rates of peroxidation in the presence of ascorbate depended on the nature and quantity of the polyunsaturated fatty acids in the diet. The lipid peroxide content was 1.82±0.30nmol of malonaldehyde/mg of protein and the rate of peroxidation was 0.60±0.08nmol of malonaldehyde/min per mg of protein after feeding a fat-free diet, and the values were increased to 20.80nmol of malonaldehyde/mg of protein and 3.73nmol of malonaldehyde/min per mg of protein after feeding a 10% herring-oil diet in which polyunsaturated fatty acids formed 24% of the total fatty acids. Addition of α-tocopherol to the diets (120mg/kg of diet) caused a very large decrease in the lipid peroxide concentration and rate of lipid peroxidation in the endoplasmic reticulum, but addition of the synthetic anti-oxidant 2,6-di-t-butyl-4-methylphenol to the diet (100mg/kg of diet) was ineffective. Treatment of the animals with phenobarbitone (1mg/ml of drinking water) caused a sharp fall in the rate of lipid peroxidation. It is concluded that the polyunsaturated fatty acid composition of the diet regulates the fatty acid composition of the liver endoplasmic reticulum, and this in turn is an important factor controlling the rate and extent of lipid peroxidation in vitro and possibly in vivo.

scholarly journals Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

1999 ◽  
Vol 10 (4) ◽  
pp. 1043-1059 ◽  
Author(s):  
Wolfgang P. Barz ◽  
Peter Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Protein Translocation ◽  
Sequence Similarity ◽  
Surface Proteins ◽  
Golgi Transport ◽  
Er Membrane

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes inSaccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting thatLAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δcells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

scholarly journals The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport.

1991 ◽  
Vol 11 (6) ◽  
pp. 2980-2993 ◽  
Author(s):  
R Ossig ◽  
C Dascher ◽  
H H Trepte ◽  
H D Schmitt ◽  
D Gallwitz
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Single Copy ◽  
Integral Membrane Protein ◽  
Carboxypeptidase Y ◽  
Null Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Golgi Transport ◽  
Gtp Binding

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.

scholarly journals Identification of Regulators for Ypt1 GTPase Nucleotide Cycling

1998 ◽  
Vol 9 (10) ◽  
pp. 2819-2837 ◽  
Author(s):  
Sara Jones ◽  
Celeste J. Richardson ◽  
Robert J. Litt ◽  
Nava Segev
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Rab Proteins ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Golgi Transport ◽  
Transport Assay

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7and GYP1, nor is it influenced by mutations inSEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.

scholarly journals Intracellular cleavage of glycosylphosphatidylinositol by phospholipase D induces activation of protein kinase Cα

1999 ◽  
Vol 342 (2) ◽  
pp. 449-455 ◽  
Author(s):  
Hiroshi TSUJIOKA ◽  
Noboru TAKAMI ◽  
Yoshio MISUMI ◽  
Yukio IKEHARA
Keyword(s):  
Signal Transduction ◽  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Secretory Pathway ◽  
Gradient Centrifugation ◽  
Endoplasmic Reticulum Membrane ◽  
Gpi Anchor ◽  
Protein Kinase Cα ◽  
In Cells

Many proteins are anchored to the cell membrane by glycosylphosphatidylinositol (GPI). One of the functions proposed for the GPI anchor is as a possible mediator in signal transduction through its hydrolysis. GPI-specific phospholipase D (GPI-PLD) is a secretory protein that is suggested to be involved in the release of GPI-anchored protein from the membrane. In the present study we examined how GPI-PLD is involved in signal transduction. When introduced exogenously and overexpressed in cells, GPI-PLD cleaved the GPI anchors in the early secretory pathway, possibly in the endoplasmic reticulum, resulting in an increased production of diacylglycerol. Experiments in vitro and in vivo showed that the association of protein kinase Cα (PKCα) with membranes was increased markedly by expression of GPI-PLD in cells. Furthermore, sucrose-density-gradient centrifugation and immunofluorescence microscopy demonstrated that PKCα was translocated to the endoplasmic reticulum membrane in cells expressing GPI-PLD, in contrast with its association with the plasma membrane in cells treated with PMA. We also confirmed that the phosphorylation of c-Fos as well as PKCα itself was greatly enhanced by the expression of GPI-PLD. Taken together, these results suggest that GPI-PLD is involved in intracellular cleavage of the GPI anchor, which is a new potential source of diacylglycerol production to activate PKCα.

scholarly journals The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

1998 ◽  
Vol 9 (5) ◽  
pp. 1149-1162 ◽  
Author(s):  
Gabriele Dürr ◽  
Jochen Strayle ◽  
Richard Plemper ◽  
Saskia Elbs ◽  
Saskia K. Klee ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Adenosine Triphosphatase ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Protein Glycosylation ◽  
Carboxypeptidase Y ◽  
Dual Function ◽  
Ion Pump ◽  
Endoplasmic Reticulum Protein ◽  
Sorting Receptor

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects ofpmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

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