Effect of caffeine on intracellular transport of Semliki Forest virus membrane glycoproteins

1992 ◽  
Vol 102 (3) ◽  
pp. 505-513 ◽  
Author(s):  
E. Kuismanen ◽  
J. Jantti ◽  
V. Makiranta ◽  
M. Sariola
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Semliki Forest Virus ◽  
Precursor Protein ◽  
Baby Hamster Kidney ◽  
Intracellular Membrane ◽  
Membrane Glycoproteins ◽  
Exocytic Pathway

The effect of caffeine on the intracellular transport of Semliki Forest virus (SFV) membrane glycoproteins was studied in baby hamster kidney (BHK) cells. The movement of the proteins was affected at two steps in the exocytic pathway. The exit of the proteins from the endoplasmic reticulum (ER) was inhibited by 10 mM caffeine at 20 degrees C, a temperature that normally allows transport to the Golgi complex. At higher temperatures (28 degrees C and 37 degrees C) in the presence of 10 mM caffeine exit from the ER occurred, but the proteins accumulated at intracellular membrane elements. Immunofluorescence localization, endoglycosidase-H analysis, and analysis of the proteolytical cleavage of the p62 precursor protein suggested that transport in the presence of 10 mM caffeine was arrested at the membranes between the trans-Golgi and the plasma membrane.

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.

scholarly journals Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

1980 ◽  
Vol 87 (3) ◽  
pp. 783-791 ◽  
Author(s):  
L Kaariainen ◽  
K Hashimoto ◽  
J Saraste ◽  
I Virtanen ◽  
K Penttinen
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Semliki Forest Virus ◽  
Marginal Effect ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Membrane Glycoproteins ◽  
Infected Cells

Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 μg/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 μg/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of approximately 10 min. These results suggest a similar intracellular pathway in the maturation of both plasma membrane and secretory glycoproteins.

scholarly journals Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells.

1983 ◽  
Vol 97 (6) ◽  
pp. 1777-1787 ◽  
Author(s):  
J E Bergmann ◽  
S J Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
G Protein ◽  
Rough Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Vesicular Stomatitis

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.

The role of subcompartments of the Golgi complex in protein intracellular transport

1982 ◽  
Vol 300 (1099) ◽  
pp. 173-184 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Concanavalin A ◽  
Intracellular Transport ◽  
Subcellular Fractionation ◽  
Membrane Glycoproteins ◽  
Myeloma Cells ◽  
Golgi Stack ◽  
Wheatgerm Agglutinin

The functioning of the Golgi complex in protein intracellular transport is most simply understood in terms of its being composed of a sequence of functionally distinct subcompartments. For example, the influence of perturbation of cellular Na + -K + balance on the transport of secretory and membrane glycoproteins is to greatly slow their passage from relatively proximal to relatively distal subcompartments. To further the understanding of the nature of these subcompartments a rat IgM myeloma has been subjected to analytical subcellular fractionation. Fractions selectively enriched in distinct Golgi-associated activities have been prepared and their membrane proteins compared with those of rough microsomal fractions. The subfractionation is extensive and suggests the possibility of obtaining well resolved Golgi subfractions. Myeloma cells stained intracellularly with Goncanavalin A - and wheatgerm agglutinin-peroxidase conjugates show distinct labelling patterns. Concanavalin A stains the entirety of the rough endoplasmic reticulum as well as the proximal face of the Golgi stack. Wheatgerm agglutinin stains the distal face of the stack of Golgi cisternae. The staining patterns are not due to immunoglobulin as they are also observed in myeloma variants that fail to synthesize immunoglobulin.

scholarly journals Density of newly synthesized plasma membrane proteins in intracellular membranes. I. Stereological studies.

1984 ◽  
Vol 98 (6) ◽  
pp. 2133-2141 ◽  
Author(s):  
G Griffiths ◽  
G Warren ◽  
P Quinn ◽  
O Mathieu-Costello ◽  
H Hoppeler
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Semliki Forest Virus ◽  
Transport Pathway ◽  
Mean Cell Volume ◽  
Surface Areas ◽  
Sorting Process ◽  
Endogenous Proteins ◽  
Spike Proteins

As the spike proteins of Semliki Forest virus (SFV) pass from their site of synthesis in the endoplasmic reticulum (ER) to the cell surface, they must be concentrated and freed from endogenous proteins. To determine the magnitude of this sorting process we have measured the density of spike proteins in membranes of the intracellular transport pathway. In this first paper, using stereological procedures, we have estimated the surface areas of the ER, Golgi complex, and plasma membrane of infected and mock-infected baby hamster kidney cells. First, we estimated the mean cell volume in absolute units. This was done using a novel in situ method which is described in detail. Infection by SFV was found to have no effect on any of the parameters measured. In the accompanying paper ( Quinn , P., G. Griffiths, and G. Warren, 1984, J. Cell Biol., 2142-2147) these stereological estimates were combined with biochemical estimates of the amount of spike proteins in ER, Golgi complex, and plasma membrane to determine the density in the membranes of these compartments.

scholarly journals Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

1985 ◽  
Vol 232 (1) ◽  
pp. 71-78 ◽  
Author(s):  
J A Hedo ◽  
I A Simpson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Insulin Receptor ◽  
Golgi Complex ◽  
Microsomal Fraction ◽  
Primary Cultures ◽  
Sodium Dodecyl ◽  
High Density ◽  
Low Density ◽  
Adipose Cells

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.

scholarly journals RAB6 and microtubules restrict protein secretion to focal adhesions

2019 ◽  
Vol 218 (7) ◽  
pp. 2215-2231 ◽  
Author(s):  
Lou Fourriere ◽  
Amal Kasri ◽  
Nelly Gareil ◽  
Sabine Bardin ◽  
Hugo Bousquet ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Protein Secretion ◽  
Golgi Complex ◽  
Essential Role ◽  
Focal Adhesions ◽  
Secreted Proteins ◽  
Cell Compartments ◽  
Secretion Assay

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.

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 Compartmentalization of intracellular membrane glycocomponents is revealed by fracture-label.

1984 ◽  
Vol 98 (1) ◽  
pp. 29-34 ◽  
Author(s):  
M R Torrisi ◽  
P Pinto da Silva
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Secretory Granules ◽  
The Other ◽  
Rat Tissues ◽  
Intracellular Membrane ◽  
Definition Of

We used thin-section fracture-label to determine the distribution of wheat-germ agglutinin binding sites in intracellular membranes of secretory and nonsecretory rat tissues as well as in human leukocytes. In all cases, analysis of the distribution of wheat germ agglutinin led to the definition of two endomembrane compartments: one, characterized by absence of the label, includes the membranes of mitochondria and peroxisomes as well as those of the endoplasmic reticulum and nuclear envelope; the other, strongly labeled, comprises the membrane of lysosomes, phagocytic vacuoles, and secretory granules, as well as the plasma membrane. The Golgi apparatus was weakly labeled in all studied tissues.

scholarly journals The dynamic nature of the Golgi complex.

1989 ◽  
Vol 108 (2) ◽  
pp. 277-297 ◽  
Author(s):  
G Griffiths ◽  
S D Fuller ◽  
R Back ◽  
M Hollinshead ◽  
S Pfeiffer ◽  
...  
Keyword(s):  
Surface Area ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Semliki Forest Virus ◽  
Total Surface Area ◽  
Morphological Evidence ◽  
Golgi Stack ◽  
Golgi Compartment ◽  
Vesicular Stomatitis ◽  
Golgi Network

The intracellular transport of newly synthesized G protein of vesicular stomatitis virus is blocked at 20 degrees C and this spanning membrane glycoprotein accumulates in the last Golgi compartment, the trans Golgi-network (TGN). Previous morphological evidence suggested that the TGN enlarged significantly under this condition. In the present study we have used stereological procedures to estimate the volume and surface area of the Golgi stack and the TGN of baby hamster kidney cells under different conditions. The results indicate that the increase in the size of the TGN at 20 degrees C is accompanied by a significant decrease in the surface area and volume of the preceding Golgi compartments. A similar effect is also seen in uninfected cells at 20 degrees C, as well as during normal (37 degrees C) infection with Semliki Forest virus. In the latter case, however, the decrease in the size of the Golgi stack and the increase in that of the TGN is not accompanied by inhibition of transport from the Golgi complex to the cell surface. The results indicate that the Golgi stack and the TGN are dynamic and interrelated structures that are capable of rapid alteration in total surface area in response to changes in the rates of membrane transport.

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