scholarly journals The Arabidopsis AtGCD3 protein is a glucosylceramidase that preferentially hydrolyzes long-acyl-chain glucosylceramides

2019 ◽  
Vol 295 (3) ◽  
pp. 717-728 ◽  
Author(s):  
Guang-Yi Dai ◽  
Jian Yin ◽  
Kai-En Li ◽  
Ding-Kang Chen ◽  
Zhe Liu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Fusion Proteins ◽  
Biosynthetic Pathway ◽  
Acyl Chain ◽  
Specific Inhibitor ◽  
Endoplasmic Reticulum Membrane ◽  
Growth Defects ◽  
The Common

Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.

Biochemical approaches to the study of cytosolic calcium regulation in nerve endings

1981 ◽  
Vol 296 (1080) ◽  
pp. 115-122 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Steady State ◽  
Nerve Ending ◽  
Cytosolic Calcium ◽  
Endoplasmic Reticulum Membrane ◽  
Mitochondrial Inner Membrane ◽  
Voltage Dependent ◽  
Minor Significance

The nerve ending cytosol is bounded by the plasma membrane, the mitochondrial inner membrane and the endoplasmic reticulum membrane, transport across each of which is capable, in theory, of regulating the cytosolic free Ca 2+ concentration. By parallel monitoring of mitochondrial and plasma membrane potentials, ATP levels, Na + gradients and intrasynaptosomal Ca 2+ distribution in preparations of isolated synaptosomes, we conclude the following: ( a ) mitochondria in situ represent a major Ca 2+ pool, regulating the upper steady-state limit of the cytosolic free Ca 2+ concentration by sequestering Ca 2+ reversibly; ( b ) this limit is responsive to the cytosolic Na + concentration, but is below the concentration required for significant exocytosis; ( c ) plasma membrane Ca 2+ transport can be resolved into a constant slow influx, a voltage-dependent and verapamil-sensitive influx and an ATP-dependent efflux, while Ca 2+ efflux driven by the sodium electrochemical potential cannot be detected; ( d ) Ca 2+ regulation by intrasynaptosomal endoplasmic reticulum appears to be of minor significance in the present preparation.

scholarly journals Host Vesicle Fusion Proteins VAPB, Rab11b and Rab18 Contribute to HSV-1 Infectivity by Facilitating Egress through the Nuclear Membrane

2016 ◽  
Author(s):  
Natalia Saiz-Ros ◽  
Rafal Czapiewski ◽  
Andrew Stevenson ◽  
Ilaria Epifano ◽  
Selene K. Swanson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Life Cycle ◽  
Nuclear Envelope ◽  
Host Cell ◽  
Nuclear Membrane ◽  
Fusion Proteins ◽  
Vesicle Fusion ◽  
Endoplasmic Reticulum Membrane ◽  
Nuclear Egress ◽  
Hsv 1

AbstractThe herpesvirus process of primary envelopment and de-envelopment as viral particles exit the nucleus has been for many years one of the least understood steps in the virus life cycle. Though viral proteins such as pUL31, pUL34, pUS3 and others are clearly important, these are likely insufficient for efficient fusion with the nuclear membrane. We postulated that host nuclear membrane proteins involved in virus nuclear egress would move from the inner to outer nuclear membranes due to membrane fusion events in primary envelopment and de-envelopment and then diffuse into the endoplasmic reticulum. Membrane fractions were prepared enriched in the nuclear envelope or the endoplasmic reticulum with and without HSV-1 infection and analyzed by mass spectrometry, revealing several vesicle fusion proteins as candidates in the viral nuclear egress pathway. Knockdown of three of these, VAPB, Rab11b, and Rab18, significantly reduced titers of released virus while yielding nuclear accumulation of encapsidated particles. Antibody staining revealed that VAPB visually accumulates in the inner nuclear membrane during HSV-1 infection. VAPB also co-localizes at early time points with the viral pUL34 protein known to be involved in nuclear egress. Most strikingly, VAPB was also observed on HSV-1 virus particles by immunogold labelling electron microscopy. Thus, these data reveal several new host cell vesicle fusion proteins involved in viral nuclear egress.Author SummaryHuman herpesviruses are associated with common human diseases such as chicken pox, shingles and mononucleosis and infect a wide range of animals making them economically important pathogens for livestock. Herpes simplex virus 1 (HSV-1) is most commonly associated with cold sores, but is also the leading cause of blindness by infection in the Western world. All herpesviruses share many aspects of infection. As large nuclear replicating dsDNA viruses with capsid sizes too large to use the nuclear pores to exit the nucleus, they have evolved a complex mechanism for envelopment and de-envelopment of primary herpesvirus particles, but this critical step in the virus lifecycle remains poorly understood. We have identified several host cell vesicle fusion proteins, VAPB, Rab11b and Rab18 that appear to contribute to this step in the HSV-1 life cycle. VAPB accumulates at the nuclear envelope with the HSV-1 pUL34 protein important for viral nuclear egress. Knockdown of any of these vesicle fusion proteins reduces viral titers, further arguing that they are important for nuclear egress. As there appears to be a specific subset of vesicle fusion proteins involved in viral egress, they could possibly represent novel targets for therapeutic interventions.

Evidence for a Vacuolar-Type Proton ATPase in Entamoeba histolytica

1990 ◽  
Vol 45 (3-4) ◽  
pp. 229-232 ◽  
Author(s):  
Ursula Löhden-Bendinger ◽  
Tilly Bakker-Grunwald
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Entamoeba Histolytica ◽  
Specific Inhibitor ◽  
Eukaryotic Cells ◽  
Proton Atpase ◽  
Membrane Bound ◽  
Higher Eukaryotes ◽  
Type V ◽  
Pinocytic Vesicles

Abstract Entamoeba histolytica Entamoeba histolytica is a primitive eukaryote that lacks mitochondria, Golgi and a well-developed endoplasmic reticulum. Close to half of the cell volume is occupied by pinocytic vesicles, which are in continuous turnover with the plasma membrane and perform functions that in higher eukaryotic cells are taken over by lysosomes. Similar to the latter, the amebal vesicles are acidified. We report here that bafilomycin AI, a specific inhibitor of vacuolar-type (V-) ATPases, suppressed this acidification at submicromolar concentrations; concom itantly, it inhibited pinocytosis. These results strongly suggest the presence of a V-ATPase in pinocytic vesicles of E. histolytica, and thereby support the notion that the V-ATPases in the organelles of higher eukaryotes are derived from an archaic plasma membrane-bound form.

scholarly journals Subcellular localization of inositol lipids in blood platelets as deduced from the use of labelled precursors

1987 ◽  
Vol 244 (3) ◽  
pp. 757-761 ◽  
Author(s):  
G Mauco ◽  
P Dajeans ◽  
H Chap ◽  
L Douste-Blazy
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Blood Platelets ◽  
Blood Platelet ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Inositol Lipids ◽  
Hormone Sensitive ◽  
Reported Data ◽  
Phosphatidylinositol 4

1. By rapid fractionation of blood platelet lysates on Percoll density gradients at alkaline pH (9.6), a very pure plasma-membrane fraction was obtained, as well as discrimination between endoplasmic reticulum and lysosomes. 2. Labelling of intact platelets with [32P]Pi followed by subcellular fractionation showed an exclusive localization of all inositol lipids in the plasma membrane. 3. Preincubation of whole platelets with myo-[3H]inositol in a buffer containing 1 mM-MnCl2 allowed incorporation of the label into PtdIns (phosphatidylinositol) of both plasma and endoplasmic-reticulum membrane, whereas [3H]PtdIns4P (phosphatidylinositol 4-phosphate) and [3H]PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) were exclusively found on the plasma membrane. 4. It is concluded that PtdIns4P and PtdIns(4,5)P2 are exclusively localized in the plasma membrane, whereas PtdIns is present in both plasma and endoplasmic-reticulum membranes. This could provide an explanation for previously reported data on hormone-sensitive and -insensitive inositol lipid pools.

Protein translocation through the Sec61/SecY channel

2010 ◽  
Vol 30 (3) ◽  
pp. 201-207 ◽  
Author(s):  
Zhiliang Cheng
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Lipid Bilayer ◽  
Protein Translocation ◽  
Biological Membranes ◽  
Structural Data ◽  
Endoplasmic Reticulum Membrane ◽  
Primary Sequence ◽  
Membrane Channel ◽  
Final Destination

Special codes are embedded in the primary sequence of newly synthesized proteins to determine their final destination. Protein translocation across biological membranes requires co-operation between the targeting and translocation machineries. A conserved membrane channel, the Sec61/SecY complex, mediates protein translocation across or integration into the endoplasmic reticulum membrane in eukaryotes and the plasma membrane in prokaryotes. A combination of recent biochemical and structural data provides novel insights into the mechanism of how the channel allows polypeptide movement into the exoplasmic space and the lipid bilayer.

scholarly journals A comparative study of the molecular structures of the plasma membranes and the smooth and the rough endoplasmic-reticulum membranes from rat liver

1972 ◽  
Vol 129 (3) ◽  
pp. 781-788 ◽  
Author(s):  
F. Morin ◽  
S. Tay ◽  
H. Simpkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Molecular Structures ◽  
Endoplasmic Reticulum Membrane ◽  
Marker Enzyme ◽  
Membrane Phospholipids

Plasma-membrane as well as smooth-, rough- and degranulated-endoplasmic-reticulum-membrane fractions were isolated from the microsomal pellet of rat liver. The purity of these fractions, as determined by marker-enzyme activities, electron microscopy, cholesterol content and RNA content, was found to be adequate for a comparative structural study. Major differences in lipid and protein composition were found to exist between the plasma membrane and the endoplasmic reticulum, but not between the smooth and the rough fractions of the endoplasmic reticulum. Differences in the location of membrane protein thiol groups and the mobility of the membrane phospholipids were observed between the plasma membranes and the endoplasmic reticulum, and these could be explained by differences in protein and lipid composition. However, by employing fluorescence and spin-labelling techniques structural changes were also observed between the smooth and the rough endoplasmic-reticulum fractions. These results suggest that the structural heterogeneity existing between the two latter membrane fractions occurs near or on their membrane surfaces and is not due to the greater number of ribosomes bound to the rough endoplasmic-reticulum fraction.

scholarly journals The Endoplasmic Reticulum Membrane Is Permeable to Small Molecules

2004 ◽  
Vol 15 (2) ◽  
pp. 447-455 ◽  
Author(s):  
Sylvie Le Gall ◽  
Andrea Neuhof ◽  
Tom Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Low Temperature ◽  
Small Molecules ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Permeation ◽  
Permeabilized Cells ◽  
Golgi Membranes ◽  
Pass Through ◽  
Er Membrane

The lumen of the endoplasmic reticulum (ER) differs from the cytosol in its content of ions and other small molecules, but it is unclear whether the ER membrane is as impermeable as other membranes in the cell. Here, we have tested the permeability of the ER membrane to small, nonphysiological molecules. We report that isolated ER vesicles allow different chemical modification reagents to pass from the outside into the lumen with little hindrance. In permeabilized cells, the ER membrane allows the passage of a small, charged modification reagent that is unable to cross the plasma membrane or the lysosomal and trans-Golgi membranes. A larger polar reagent of ∼5 kDa is unable to pass through the ER membrane. Permeation of the small molecules is passive because it occurs at low temperature in the absence of energy. These data indicate that the ER membrane is significantly more leaky than other cellular membranes, a property that may be required for protein folding and other functions of the ER.

Sign in / Sign up

Export Citation Format

Share Document