scholarly journals Bead rings at the endoplasmic reticulum-golgi complex boundary: morphological changes accompanying inihibition of intracellular transport of secretory proteins in arthropod fat body tissue

1981 ◽  
Vol 90 (1) ◽  
pp. 92-100 ◽  
Author(s):  
DA Brodie
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Oxidative Phosphorylation ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Ring Structure ◽  
Prolonged Treatment ◽  
Antimycin A ◽  
Secretory Material ◽  
Rough Er

Golgi complex beads are 10-nm particles arranged in rings on the smooth surface of rough endoplasmic reticulum (ER) makind the forming face of the Golgi complex (GC). In arthropod cells they stain specifically with bismuth. Their morphology has been studied after treatment with reagents known to interfere with GC function. Inhibitors of oxidative phosphorylation (antimycin A, cyanide, and anoxia), but not an inhibitor of glycolysis (iodoacetate), both cause the bead rings to collapse and the GC saccules to round up, and inhibit transition vesicle (TV) formation. Cycloheximide blocks protein synthesis on ribosomes but does not stop TV formation or disrupt bead rings, even after prolonged treatment (6 h) to allow emptying of the rough ER cisternae. Thus the collapse of bead rings is not attributable to inhibition of protein synthesis, and the ring structure of beads does not require continued protein synthesis and secretion for its maintenance. Valinomycin has effects on the GC similar to those of antimycin A, but A23187, monensin, and lasalocid do not affect bead ring structure or TV formation. These results are consistent with valinomycin's secondarily uncoupling mitochondria, which collapses bead rings and prevents TV formation. Thus inhibitors of oxidative phosphorylation do not influence the beads through cation movement. Because mononsin and lasalocid block secretion at the level of the condensing vacuoles, bead rings are not influenced by blocks in secretion distal to them or by the backup of secretory material. These experiments are consistent with inhibitors of oxidative phosphorylation collapsing bead rings by decreasing intracellular ATP. The concomitant block to TV formation and the collapse of bead rings suggests that integrity of the bead rings is essential for the transport of secretory material from the rough ER to the GC.

Synthesis and Secretion of Growth Hormone in the Rat Anterior Pituitary

1973 ◽  
Vol 12 (1) ◽  
pp. 1-21
Author(s):  
S. L. HOWELL ◽  
MARGARET WHITFIELD
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Time Course ◽  
Transport Processes ◽  
Pulse Labelling ◽  
Electron Microscopic ◽  
Storage Granules ◽  
And Storage

The intracellular processes involved in synthesis, transport and storage of newly synthesized proteins in the rat somatotroph, together with their time course and metabolic requirements, have been investigated in a quantitative electron-microscopic radioautography study of the tissue following pulse labelling with tritium-labelled amino acids and chase incubations in various conditions. Proteins are synthesized initially on the rough-surfaced elements of the endoplasmic reticulum and are transported within 10 min after their synthesis to transitional areas between the rough-surfaced endoplasmic reticulum and Golgi complex. Transfer to the Golgi lamellae is achieved, probably via transfer vesicles, within about 60 min after synthesis, while formation of mature storage granules occurs within 2 h following protein synthesis. Further experiments utilizing cycloheximide or ouabain during the chase incubations showed that the intracellular transport of newly synthesized protein and its time course are not significantly affected by inhibitors of protein synthesis, or by inhibition of sodium-potassium dependent ATPase by ouabain. Inhibitors of oxidative phosphorylation (250 µM 2,4-dinitrophenol) or of respiration (10 µM antimycin A) markedly reduced intracellular ATP levels and inhibited the intracellular transport processes. The requirement for ATP appeared to be operative at 2 stages: in the movement of transfer vesicles to the Golgi complex and in the formation of storage granules; possible roles of ATP in these processes are discussed.

scholarly journals INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL

1968 ◽  
Vol 39 (3) ◽  
pp. 589-603 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Oxidative Phosphorylation ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Antimycin A ◽  
Cell Fractionation ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Cell Synthesis

Since in the pancreatic exocrine cell synthesis and intracellular transport of secretory proteins can be uncoupled (1), it is possible to examine separately the metabolic requirements of the latter process. To this intent, guinea pig pancreatic slices were pulse labeled with leucine-3H for 3 min and incubated post-pulse for 37 min in chase medium containing 5 x 10-4 M cycloheximide and inhibitors of glycolysis, respiration, or oxidative phosphorylation. In each case, the effect on transport was assessed by measuring the amount of labeled secretory proteins found in zymogen granule fractions isolated from the corresponding slices. This assay is actually a measure of the efficiency of transport of secretory proteins from the cisternae of the rough endoplasmic reticulum (RER) to the condensing vacuoles of the Golgi complex which are recovered in the zymogen granule fraction (16). The results indicate that transport is insensitive to glycolytic inhibitors (fluoride, iodoacetate) but is blocked by respiratory inhibitors (N2, cyanide, Antimycin A) and by inhibitors of oxidative phosphorylation (dinitrophenol, oligomycin). Except for Antimycin A, the effect is reversible. Parallel radioautographic studies and cell fractionation procedures applied to microsomal subfractions have indicated that the energy-dependent step is located between the transitional elements of the RER and the small, smooth-surfaced vesicles at the periphery of the Golgi complex. Radiorespirometric data indicate that the substrates oxidized to support transport are endogenous long-chain fatty acids.

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

1986 ◽  
Vol 237 (1) ◽  
pp. 33-39 ◽  
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.

scholarly journals SYNTHESIS, INTRACELLULAR TRANSPORT, AND DISCHARGE OF SECRETORY PROTEINS IN STIMULATED PANCREATIC EXOCRINE CELLS

1971 ◽  
Vol 50 (1) ◽  
pp. 135-158 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Protein Synthesis ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Cell Fractionation ◽  
Secretory Product ◽  
Zymogen Granules ◽  
Pancreatic Exocrine ◽  
Storage Granules

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-3H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.

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

1964 ◽  
Vol 20 (3) ◽  
pp. 473-495 ◽  
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.

scholarly journals Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver.

1975 ◽  
Vol 64 (2) ◽  
pp. 356-377 ◽  
Author(s):  
H Glaumann ◽  
A Bergstrand ◽  
J L Ericsson
Keyword(s):  
Chemical Composition ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
High Rate ◽  
Very Low Density Lipoproteins ◽  
Lipoprotein Particles ◽  
Parallel Change ◽  
Rough Er

Lipoprotein particles (d less than 1.03 g/ml) were isolated from rough and smooth microsomes and from the Golgi apparatus of rat liver, and were characterized chemically and morphologically. The rough endoplasmic reticulum (ER) particles were rich in protein (50%) and contained phospholipids (PLP) and triglycerides (TG) in smaller amounts, whereas the lipoprotein particles emanating from the smooth ER, and especially the Golgi apparatus, were rich in TG and PLP, resembling very low density lipoproteins (VLDL) of serum. The difference in chemical composition among the particles was associated with change in size both in situ and in isolated lipoprotein fractions. The rough ER particles were 200-800 A in diameter (mean similar to 420 A); the smooth er particles 200-900 A (mean similar to 520 A); the Golgi particles 350-950 A (mean similar to 580A); and serum VLDL 300-800 A (mean similar to 450 A). Generally, lipoprotein particles were rare in the rough ER, frequent but diffusely dispersed in smooth ER, and occurring mainly in clusters in "secretory vesicles" of the Golgi complex. They were seldom observed in the cisternal compartments of the Golgi complex. At short intervals (less than 15 min), intravenously injected radioactive glycerol was preferentially channelled into TG, whereas at later time points the majority of the isotope was recovered in the PLP. Three TG pools were distinguished: (a) a cytoplasmic pool with a slow turnover rate; (b) a membrane-associated TG pool; and (c) a pool corresponding to the TG moiety of lipoprotein particles, which showed the highest initial rate of labeling and fastest turnover. When, after pulse labeling, the appearance of incorporation of radioactive glycerol into TG or PLP of isolated lipoproteins was followed from one subcellular fraction to the other, a sequence of labeling was noted. During the first interval, TG from both rough and smooth microsomal lipoproteins displayed a high rate of labeling with peak value at 6 min, followed by a quick fall-off, while the Golgi lipoproteins reached maximal level at 10-20 min after administration. There was an interval of 10-15 min before the appearance of labeled VLDL in serum. It is concluded that the assembly of the apoproteins and lipid moieties into lipoprotein particles-presumed to be precursors of liver VLDL-begins in the rough ER and continues in the smooth ER. Also, there is a parallel change in chemical composition and size of the lipoprotein particles as they make their way through the ER and the Golgi apparatus. Some remodeling of the particles may take place in the Golgi apparatus before discharge into the circulation.

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

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.

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

1995 ◽  
Vol 43 (9) ◽  
pp. 907-915 ◽  
Author(s):  
Y Deng ◽  
J R Bennink ◽  
H C Kang ◽  
R P Haugland ◽  
J W Yewdell
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Brefeldin A ◽  
Living Cells ◽  
Vesicular Trafficking ◽  
Cellular Functions ◽  
Animal Cells ◽  
Specific Localization

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.

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

1986 ◽  
Vol 103 (4) ◽  
pp. 1179-1191 ◽  
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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