Fluorescent conjugates of brefeldin A selectively stain the endoplasmic reticulum and Golgi complex of living cells.

The fungal metabolite brefeldin A (BFA) interferes with vesicular trafficking in most animal cells. To gain insight into the mechanism of BFA action, we esterified it to the fluorophore, boron dipyromethene difluoride (BODIPY). BODIPY-BEA localized predominantly in the endoplasmic reticulum (ER) and Golgi complex of viable cells and was extracted by detergent treatment, suggesting it interacts primarily with lipid bilayers. The localization of the conjugate is conferred by BFA, since free BODIPY or BODIPY esterified to cyclopentanol did not specifically localize to internal membranes. BODIPY-BFA exhibited a similar biological activity to BFA, but only when used at higher concentrations and after a delay. HPLC analysis revealed that over this period, cells converted BODIPY-BFA to species co-eluting with free BODIPY and BFA. Therefore, BODIPY-BFA is probably inactive until BFA is released by cellular esterases. The specific localization of BODIPY-BFA to the ER and Golgi complex suggests that BFA might exert its effects on vesicular trafficking by perturbing the lipid bilayer of its target organelles. Because BODIPY-BFA intensely stains the ER at concentrations that have no discernible effects on intracellular transport or other cellular functions, it should be useful for visualizing the ER in living cells.

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Influenza virus hemagglutinin trimers and monomers maintain distinct biochemical modifications and intracellular distribution in brefeldin A-treated cells.

Cell Regulation ◽

10.1091/mbc.2.7.549 ◽

1991 ◽

Vol 2 (7) ◽

pp. 549-563 ◽

Cited By ~ 16

Author(s):

G Russ ◽

J R Bennink ◽

T Bächi ◽

J W Yewdell

Keyword(s):

Endoplasmic Reticulum ◽

Influenza Virus ◽

Monoclonal Antibodies ◽

Golgi Complex ◽

Brefeldin A ◽

Retrograde Transport ◽

Cell Fractionation ◽

Influenza Virus Hemagglutinin ◽

Infected Cells ◽

Vesicular Compartment

Brefeldin A (BFA) induces the retrograde transport of proteins from the Golgi complex (GC) to the endoplasmic reticulum (ER). It is uncertain, however, whether the drug completely merges the ER with post-ER compartments, or whether some of their elements remain physically and functionally distinct. We investigated this question by the use of monoclonal antibodies specific for monomers and trimers of the influenza virus hemagglutinin (HA). In untreated influenza virus-infected cells, monomers and trimers almost exclusively partition into the ER and GC, respectively. In BFA-treated cells, both monomers and trimers are detected in the ER by immunofluorescence. Cell fractionation experiments indicate, however, that whereas HA monomers synthesized in the presence of BFA reside predominantly in vesicles with a characteristic density of the ER, HA trimers are primarily located in lighter vesicles characteristic of post-ER compartments. Biochemical experiments confirm that in BFA-treated cells, trimers are more extensively modified than monomers by GC-associated enzymes. Additional immunofluorescence experiments reveal that in BFA-treated cells, HA monomers can exist in an ER subcompartment less accessible to trimers and, conversely, that trimers are present in a vesicular compartment less accessible to monomers. These findings favor the existence of a post-ER compartment for which communication with the ER is maintained in the presence of BFA and suggest that trimers cycle between this compartment and the ER, but have access to only a portion of the ER.

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The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes

Biochemical Journal ◽

10.1042/bj2370033 ◽

1986 ◽

Vol 237 (1) ◽

pp. 33-39 ◽

Cited By ~ 31

Author(s):

E Fries ◽

I Lindström

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Low Temperatures ◽

Intracellular Transport ◽

Rat Hepatocytes ◽

Subcellular Fractionation ◽

Isolated Rat Hepatocytes ◽

Different Temperatures ◽

Lag Period ◽

Time Courses

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.

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PROTEIN SYNTHESIS, STORAGE, AND DISCHARGE IN THE PANCREATIC EXOCRINE CELL

The Journal of Cell Biology ◽

10.1083/jcb.20.3.473 ◽

1964 ◽

Vol 20 (3) ◽

pp. 473-495 ◽

Cited By ~ 529

Author(s):

Lucien G. Caro ◽

George E. Palade

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Intracellular Transport ◽

Secretory Proteins ◽

Zymogen Granules ◽

Intravenous Injections ◽

Pancreatic Exocrine ◽

Exocrine Cell ◽

Secretory Products ◽

Electron Microscopical

The synthesis, intracellular transport, storage, and discharge of secretory proteins in and from the pancreatic exocrine cell of the guinea pig were studied by light- and electron microscopical autoradiography using DL-leucine-4,5-H3 as label. Control experiments were carried out to determine: (a) the length of the label pulse in the blood and tissue after intravenous injections of leucine-H3; (b) the amount and nature of label lost during tissue fixation, dehydration, and embedding. The results indicate that leucine-H3 can be used as a label for newly synthesized secretory proteins and as a tracer for their intracellular movements. The autoradiographic observations show that, at ∼5 minutes after injection, the label is localized mostly in cell regions occupied by rough surfaced elements of the endoplasmic reticulum; at ∼20 minutes, it appears in elements of the Golgi complex; and after 1 hour, in zymogen granules. The evidence conclusively shows that the zymogen granules are formed in the Golgi region by a progressive concentration of secretory products within large condensing vacuoles. The findings are compatible with an early transfer of label from the rough surfaced endoplasmic reticulum to the Golgi complex, and suggest the existence of two distinct steps in the transit of secretory proteins through the latter. The first is connected with small, smooth surfaced vesicles situated at the periphery of the complex, and the second with centrally located condensing vacuoles.

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Potassium depletion inhibits the intracellular transport of secretory proteins between the endoplasmic reticulum and the Golgi complex

Journal of Cell Science ◽

10.1242/jcs.92.2.173 ◽

1989 ◽

Vol 92 (2) ◽

pp. 173-185

Author(s):

J.D. Judah ◽

K.E. Howell ◽

J.A. Taylor ◽

P.S. Quinn

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Intracellular Transport ◽

Secretory Pathway ◽

Subcellular Fractionation ◽

Secretory Proteins ◽

Mature Form ◽

Processing Enzymes ◽

Microscopic Studies ◽

K Depletion

In this paper we show that hepatocytes that have been depleted of K+ secrete albumin, alpha-1-anti-trypsin and transferrin at a slower rate than cells to which K+ has been returned. K+ depletion has no effect on the intracellular nucleotide pools, and we provide evidence that the inhibitions of secretion caused by depletion of K+ and depletion of ATP are independent. Studies of the processing of alpha-1-anti-trypsin show that K+ depletion inhibits the formation of the mature form of the protein, but that immature forms are never secreted. In cells to which K+ was returned, secretion of the mature form was restored. This implies that transport is blocked at a point before the proteins reach the processing enzymes. Proteins delayed by K+ depletion are not removed from the secretory pathway, but are free to mix with protein synthesized subsequently. These data are supported by subcellular fractionation experiments, which show that the secretory proteins are delayed before reaching the Golgi complex, and by immunoelectron microscopic studies. These show that in K+-deficient cells the morphology of both the endoplasmic reticulum and the Golgi complex is normal. The secretory proteins are trapped in smooth vesicles that contain reaction product when incubated for glucose-6-phosphatase, a marker for the endoplasmic reticulum.

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Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

The Journal of Cell Biology ◽

10.1083/jcb.103.4.1179 ◽

1986 ◽

Vol 103 (4) ◽

pp. 1179-1191 ◽

Cited By ~ 243

Author(s):

C S Copeland ◽

R W Doms ◽

E M Bolzau ◽

R G Webster ◽

A Helenius

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Golgi Complex ◽

Intracellular Transport ◽

Secretory Pathway ◽

Quaternary Structure ◽

Subunit Interactions ◽

Oligomer Formation ◽

Influenza Hemagglutinin ◽

Triton X

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.

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Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

Molecular Biology of the Cell ◽

10.1091/mbc.4.4.413 ◽

1993 ◽

Vol 4 (4) ◽

pp. 413-424 ◽

Cited By ~ 24

Author(s):

L C Hendricks ◽

M McCaffery ◽

G E Palade ◽

M G Farquhar

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Brefeldin A ◽

Acinar Cells ◽

Atp Depletion ◽

Pancreatic Acinar Cells ◽

Coat Proteins ◽

Golgi Membranes ◽

Golgi Transport ◽

Transport Vesicles

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.

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Analysis of De Novo Golgi Complex Formation after Enzyme-based Inactivation

Molecular Biology of the Cell ◽

10.1091/mbc.e07-08-0799 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4637-4647 ◽

Cited By ~ 12

Author(s):

Florence Jollivet ◽

Graça Raposo ◽

Ariane Dimitrov ◽

Rachid Sougrat ◽

Bruno Goud ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

De Novo ◽

Living Cells ◽

Integrative Model ◽

Interphase Cells ◽

Golgi Structure ◽

Dynamics And Function ◽

And Function ◽

Drug Free

The Golgi complex is characterized by its unique morphology of closely apposed flattened cisternae that persists despite the large quantity of lipids and proteins that transit bidirectionally. Whether such a structure is maintained through endoplasmic reticulum (ER)-based recycling and auto-organization or whether it depends on a permanent Golgi structure is strongly debated. To further study Golgi maintenance in interphase cells, we developed a method allowing for a drug-free inactivation of Golgi dynamics and function in living cells. After Golgi inactivation, a new Golgi-like structure, containing only certain Golgi markers and newly synthesized cargos, was produced. However, this structure did not acquire a normal Golgi architecture and was unable to ensure a normal trafficking activity. This suggests an integrative model for Golgi maintenance in interphase where the ER is able to autonomously produce Golgi-like structures that need pre-existing Golgi complexes to be organized as morphologically normal and active Golgi elements.

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Dynein Supports Motility of Endoplasmic Reticulum in the FungusUstilago maydis

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0475 ◽

2002 ◽

Vol 13 (3) ◽

pp. 965-977 ◽

Cited By ~ 79

Author(s):

Roland Wedlich-Söldner ◽

Irene Schulz ◽

Anne Straube ◽

Gero Steinberg

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Intracellular Transport ◽

Fluorescent Protein ◽

Brefeldin A ◽

Cytoplasmic Dynein ◽

Organelle Transport ◽

Minor Importance ◽

Dependent Transport

The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER inU. maydis.

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Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum

The Journal of Cell Biology ◽

10.1083/jcb.137.2.319 ◽

1997 ◽

Vol 137 (2) ◽

pp. 319-333 ◽

Cited By ~ 60

Author(s):

Jochen Scheel ◽

Rainer Pepperkok ◽

Martin Lowe ◽

Gareth Griffiths ◽

Thomas E. Kreis

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Transport ◽

Golgi Complex ◽

Mammalian Cells ◽

Brefeldin A ◽

Retrograde Transport ◽

Marker Proteins ◽

Kdel Receptor

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the “EAGE”-peptide of β-COP, inhibit BFA-induced redistribution of β-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing β-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only ∼50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTPγS. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

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