Rate of Retrograde Transport of Cholera Toxin from the Plasma Membrane to the Golgi Apparatus and Endoplasmic Reticulum Decreases During Neuronal Development

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20–30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2–3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.

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Functional Protein Prenylation Is Required for the Brefeldin A-dependent Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum

Journal of Biological Chemistry ◽

10.1074/jbc.272.33.20828 ◽

1997 ◽

Vol 272 (33) ◽

pp. 20828-20834 ◽

Cited By ~ 13

Author(s):

N. Erwin Ivessa ◽

Diego Gravotta ◽

Carmen De Lemos-Chiarandini ◽

Gert Kreibich

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Brefeldin A ◽

Retrograde Transport ◽

Protein Prenylation ◽

Functional Protein

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Subcellular distribution of selenium in deficient mouse liver

Biochemical Journal ◽

10.1042/bj2580541 ◽

1989 ◽

Vol 258 (2) ◽

pp. 541-545 ◽

Cited By ~ 4

Author(s):

R Reiter ◽

R Otter ◽

A Wendel

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Golgi Apparatus ◽

Trace Element ◽

Fold Increase ◽

Subcellular Fractionation ◽

Fast Response ◽

Cell Compartment ◽

Deficient Mice

Selenium (Se)-deficient mice were labelled in vivo with single pulses of [75Se]selenite, and the intrahepatic distribution of the trace element was studied by subcellular fractionation. At 1 h after intraperitoneal injection of 3.3 or 10 micrograms of Se/kg body weight, 15% of the respective doses were found in the liver. Accumulation in the subcellular fractions followed the order: Golgi vesicular much greater than lysosomal greater than cytosolic = microsomal greater than mitochondrial, peroxisomal, nuclear and plasma-membrane fraction. At a dose of 3.3 micrograms/kg, more than 90% of the hepatic Se was protein-bound. When cross-contamination was accounted for, the following specific Se contents of the subcellular compartments were extrapolated: Golgi apparatus, 7.50 pmol/mg; cytosol, 0.90 pmol/mg; endoplasmic reticulum, 0.80 pmol/mg; mitochondria, 0.49 pmol/mg; nuclei, lysosomes, peroxisomes and plasma membrane, less than 0.4 pmol/mg. At 10 micrograms/kg, a roughly 2-3-fold increase in Se content of all fractions was found without major changes in the intrahepatic distribution pattern. An extraordinary rise in the cytosolic fraction was due to an apparently non-protein-bound Se pool. At 24 h after dosing, total hepatic Se had decreased to 6% of the initial dose and had become predominantly protein-bound. The 60% decrease in hepatic Se was reflected in a similar fall in the subcellular levels of the trace element. The Golgi apparatus still had the highest specific Se content, although accumulation was 5 times less than that after 1 h. The cytosolic pool accounted for 50% of the hepatic Se at both labelling times. After 1 h the Golgi apparatus was, with 19%, the second largest intrahepatic pool, followed by the endoplasmic reticulum with 16%. The high affinity and fast response of the Golgi apparatus to Se supplementation of deficient mice is interpreted in terms of a predominant function of this cell compartment in the processing and the export of Se-proteins from the liver.

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Cytochemical demonstration of extraperoxisomal catalase. I. Sheep liver.

Journal of Histochemistry & Cytochemistry ◽

10.1177/24.6.950458 ◽

1976 ◽

Vol 24 (6) ◽

pp. 713-724 ◽

Cited By ~ 39

Author(s):

F Roels

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Golgi Apparatus ◽

Rough Endoplasmic Reticulum ◽

Catalase Activity ◽

Nonparenchymal Cells ◽

Sheep Liver ◽

Heat Stable ◽

Cytochemical Demonstration ◽

Electron Microscopes

In sheep hepatocytes catalase activity was demonstrated both within peroxisomes and within the cytosol. In the cytosol the catalase reaction product is contiguous to the plasma membrane and surrounds the nuclei, rough endoplasmic reticulum, cisternae, mitochondria and Golgi apparatus. This is the first cytochemical demonstration of guine extraperoxisomal catalase. No catalase reaction product was seen in the cytosol of nonparenchymal cells. To demonstrate catalase, both glutaraldehyde and formaldehyde fixation were used, followed by a diaminobenzidine technique modified from Novikoff and Goldfischer. Control reactions were performed to distinguish catalase reaction product from adsorption of oxidized diaminobenzidine and from precipitate due to oxidase-, peroxidase- or heat-stable peroxidatic activities. The results were evaluated in the light and electron microscopes.

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The small molecule dispergo tubulates the endoplasmic reticulum and inhibits export

Molecular Biology of the Cell ◽

10.1091/mbc.e12-08-0575 ◽

2013 ◽

Vol 24 (7) ◽

pp. 1020-1029 ◽

Cited By ~ 6

Author(s):

Lei Lu ◽

Rami N. Hannoush ◽

Brian C. Goess ◽

Shankar Varadarajan ◽

Matthew D. Shair ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Small Molecule ◽

Continuous Flow ◽

Retrograde Transport ◽

Complex Processes ◽

Phenotypic Screen ◽

Reversible Loss

The mammalian endoplasmic reticulum (ER) is an organelle that maintains a complex, compartmentalized organization of interconnected cisternae and tubules while supporting a continuous flow of newly synthesized proteins and lipids to the Golgi apparatus. Using a phenotypic screen, we identify a small molecule, dispergo, that induces reversible loss of the ER cisternae and extensive ER tubulation, including formation of ER patches comprising densely packed tubules. Dispergo also prevents export from the ER to the Golgi apparatus, and this traffic block results in breakdown of the Golgi apparatus, primarily due to maintenance of the constitutive retrograde transport of its components to the ER. The effects of dispergo are reversible, since its removal allows recovery of the ER cisternae at the expense of the densely packed tubular ER patches. This recovery occurs together with reactivation of ER-to-Golgi traffic and regeneration of a functional Golgi with correct morphology. Because dispergo is the first small molecule that reversibly tubulates the ER and inhibits its export function, it will be useful in studying these complex processes.

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Development of the lateral palatal brush in larvae of Aedes aegypti (L.) (Diptera: Culicidae)

Canadian Journal of Zoology ◽

10.1139/z90-216 ◽

1990 ◽

Vol 68 (7) ◽

pp. 1454-1467 ◽

Cited By ~ 3

Author(s):

K. M. Fry ◽

S. B. McIver

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Golgi Apparatus ◽

Aedes Aegypti ◽

Rough Endoplasmic Reticulum ◽

Light And Electron Microscopy ◽

Fourth Instar ◽

Muscle Fibres ◽

Final Length

Light and electron microscopy were used to observe development of the lateral palatal brush in Aedes aegypti (L.) larvae. Development was sampled at 4-h intervals from second- to third-instar ecdyses. Immediately after second-instar ecdysis, the epidermis apolyses from newly deposited cuticle in the lateral palatal pennicular area to form an extensive extracellular cavity into which the fourth-instar lateral palatal brush filaments grow as cytoplasmic extensions. On reaching their final length, the filaments deposit cuticulin, inner epicuticle, and procuticle sequentially on their outer surfaces. The lateral palatal crossbars, on which the lateral palatal brush filaments insert, form after filament development is complete. At the beginning of development, the organelles involved in plasma membrane and cuticle production are located at the base and middle of the cells. As the filament rudiments grow, most rough endoplasmic reticulum, mitochondria, and Golgi apparatus move to the apex of the epidermal cells and into the filament rudiments. After formation of the lateral palatal brush filaments and lateral palatal crossbars, extensive organelle breakdown occurs. Lateral palatal brush formation is unusual in that no digestion and resorption of old endocuticle occurs prior to deposition of new cuticle. No mucopolysaccharide secretion by the lateral palatal brush epidermis was observed, nor were muscle fibres observed to attach to the lateral palatal crossbars, as has been suggested by other workers.

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The proteasomal deubiquitinating enzyme PSMD14 regulates macroautophagy by controlling Golgi-to-ER retrograde transport

10.1101/2020.01.29.925503 ◽

2020 ◽

Author(s):

HA Bustamante ◽

K Cereceda ◽

AE González ◽

GE Valenzuela ◽

Y Cheuquemilla ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Membrane Trafficking ◽

Retrograde Transport ◽

Deubiquitinating Enzyme ◽

20S Proteasome ◽

Biological Processes ◽

Pharmacological Inhibition ◽

Protein Membrane ◽

A Subunit

ABSTRACTUbiquitination regulates several biological processes. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1,187 genes of the human “ubiquitinome” using Amyloid Precursor Protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel key regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 caused a robust and rapid inhibition of Golgi-to-ER retrograde transport which leads to a potent blockage of macroautophagy by a mechanism associated with the retention of Atg9A and Rab1A at the Golgi apparatus. Because pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and their K-63-Ub chains, act as a crucial regulator factor for macroautophagy by controlling Golgi-to-ER retrograde transport.

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Gangliosides That Associate with Lipid Rafts Mediate Transport of Cholera and Related Toxins from the Plasma Membrane to Endoplasmic Reticulm

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0354 ◽

2003 ◽

Vol 14 (12) ◽

pp. 4783-4793 ◽

Cited By ~ 162

Author(s):

Yukako Fujinaga ◽

Anne A. Wolf ◽

Chiara Rodighiero ◽

Heidi Wheeler ◽

Billy Tsai ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Cholera Toxin ◽

Lipid Rafts ◽

Intestinal Cells ◽

B Subunit ◽

High Affinity ◽

A Subunit ◽

Human Intestinal Cells

Cholera toxin (CT) travels from the plasma membrane of intestinal cells to the endoplasmic reticulum (ER) where a portion of the A-subunit, the A1 chain, crosses the membrane into the cytosol to cause disease. A related toxin, LTIIb, binds to intestinal cells but does not cause toxicity. Here, we show that the B-subunit of CT serves as a carrier for the A-subunit to the ER where disassembly occurs. The B-subunit binds to gangliosides in lipid rafts and travels with the ganglioside to the ER. In many cells, LTIIb follows a similar pathway, but in human intestinal cells it binds to a ganglioside that fails to associate with lipid rafts and it is sorted away from the retrograde pathway to the ER. Our results explain why LTIIb does not cause disease in humans and suggest that gangliosides with high affinity for lipid rafts may provide a general vehicle for the transport of toxins to the ER.

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