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.

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 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.

scholarly journals Density of newly synthesized plasma membrane proteins in intracellular membranes II. Biochemical studies.

1984 ◽  
Vol 98 (6) ◽  
pp. 2142-2147 ◽  
Author(s):  
P Quinn ◽  
G Griffiths ◽  
G Warren
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Semliki Forest Virus ◽  
Companion Paper ◽  
Intracellular Membranes ◽  
Surface Areas ◽  
Quantitative Immunoblotting ◽  
Total Membrane

Using two independent methods, incorporation of radioactive amino-acid and quantitative immunoblotting, we have determined that the rate of synthesis of each of the Semliki Forest virus (SFV) proteins in infected baby hamster kidney (BHK) cells is 1.2 X 10(5) copies/cell/min. Given the absolute surface areas of the endoplasmic reticulum and Golgi complex presented in the companion paper (Griffiths, G., G. Warren, P. Quinn , O. Mathieu - Costello , and A. Hoppeler , 1984, J. Cell Biol. 98:2133-2141), and the approximate time spent in these organelles during their passage to the plasma membrane (Green J., G. Griffiths, D. Louvard , P. Quinn , and G. Warren 1981, J. Mol. Biol. 152:663-698), the mean density of each viral protein in these organelles can be calculated to be 90 and 750 molecules/micron 2 membrane, respectively. In contrast, we have determined that the density of total endogenous integral membrane proteins in these organelles is approximately 30,000 molecules/micron 2 so that the spike proteins constitute only 0.28 and 2.3% of total membrane protein in the endoplasmic reticulum and Golgi, respectively. Quantitative immunoblotting was used to give direct estimates of the concentrations of one of the viral membrane protein precursors (E1) in subcellular fractions; these agreed closely with the calculated values. The data are discussed with respect to the sorting of transported proteins from those endogenous to the intracellular membranes.

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 Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts.

1989 ◽  
Vol 108 (3) ◽  
pp. 821-832 ◽  
Author(s):  
J E Skibbens ◽  
M G Roth ◽  
K S Matlin
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Disulfide Bond ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Transport Pathway ◽  
Influenza Virus Hemagglutinin ◽  
Triton X

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.

Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells

1994 ◽  
Vol 107 (12) ◽  
pp. 3623-3633 ◽  
Author(s):  
J. Jantti ◽  
S. Keranen ◽  
J. Toikkanen ◽  
E. Kuismanen ◽  
C. Ehnholm ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Signal Sequence ◽  
Insertion Site ◽  
Membrane Insertion ◽  
Membrane Anchor

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

scholarly journals Free diffusion to and from the inner nuclear membrane of newly synthesized plasma membrane glycoproteins.

1987 ◽  
Vol 104 (3) ◽  
pp. 733-737 ◽  
Author(s):  
M R Torrisi ◽  
L V Lotti ◽  
A Pavan ◽  
G Migliaccio ◽  
S Bonatti
Keyword(s):  
Plasma Membrane ◽  
Outer Membrane ◽  
Nuclear Membrane ◽  
Sindbis Virus ◽  
Intracellular Transport ◽  
Plasma Membranes ◽  
Protein A ◽  
Membrane Glycoproteins ◽  
Transport Pathway ◽  
Inner Nuclear Membrane

Sindbis virus-infected baby hamster kidney (BHK) cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies was used in conjunction with colloidal gold-conjugated protein A. As we previously reported (Torrisi, M. R., and S. Bonatti, 1985, J. Cell Biol., 101:1300-1306), Sindbis transmembrane glycoproteins are present in the inner nuclear membrane as well as in the outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes. Viral glycoproteins located on the inner nuclear membrane resemble those present on the outer membrane in terms of amount, distribution, and preferential partition after fracture. We show in this paper that Sindbis glycoproteins after treatment with cycloheximide are removed from the inner nuclear membrane with the same kinetics as their counterparts present on the outer membrane. This finding strongly suggests that newly synthesized transmembrane glycoproteins may freely diffuse to and from the inner nuclear membrane before entering into the intracellular transport pathway to the plasma membrane.

scholarly journals Differences in intracellular transport of a fluorescent phosphatidylcholine analog in established cell lines

1989 ◽  
Vol 93 (2) ◽  
pp. 363-374 ◽  
Author(s):  
R.G. Sleight ◽  
M.N. Abanto
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Lines ◽  
Intracellular Transport ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Transport Pathway ◽  
Intracellular Lipid ◽  
Intracellular Vesicles ◽  
Fluorescent Lipid

The transport and metabolism of a fluorescent phosphatidylcholine analog, 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)- aminocaproyl-phosphatidylcholine [palmitoyl, C6-NBD)-PC), in BHK, CHO-K1, CHO-15B, MDCK, VA-2, Vero, V79 and WI-38 cells has been investigated. When liposomes containing (palmitoyl, C6-NBD)-PC were incubated with cells at 2 degrees C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells' plasma membranes occurred. Most of the lipid transferred to the cells could be removed by incubating the cells in the presence of nonfluorescent liposomes or media containing 10% serum, suggesting that the fluorescent probe resided exclusively in the outer leaflet of the plasma membrane at 2 degrees C. After insertion into the plasma membrane, internalization of (palmitoyl, C6-NBD)-PC occurred when the cells were warmed to 37 degrees C. This resulted in four different labeling patterns: (1) little or no internalization of (palmitoyl, C6-NBD)-PC into punctate vesicles was observed in Vero cells. (2) Transport of (palmitoyl, C6-NBD)-PC to the region of the Golgi apparatus and to a small number of intracellular vesicles was observed in both V79 and CHO-K1 cell lines. (3) A large number of fluorescently labeled intracellular vesicles with little or no labeling in the region of the Golgi apparatus appeared after the internalization of (palmitoyl, C6-NBD)-PC in BHK, CHO-15B, MDCK and WI-38 cell lines. (4) Accumulation of (palmitoyl, C6-NBD)-PC in small vesicles, mitochondria and the nuclear envelope was observed in VA-2 cells. In addition, cells having a defect in glycoprotein processing and those transformed with simian virus 40 (SV40) internalized the fluorescent lipid probe differently compared with parental lines. Neither differences in rates of endocytosis nor rates of (palmitoyl, C6-NBD)-PC degradation between cell types appears to cause the observed dissimilarities in intracellular lipid transport. We suggest that these different cell types may have dissimilar pathways of intracellular lipid trafficking or differential regulation of a common transport pathway, and that the predominant pathway of lipid translocation can be altered in cells by changing the composition of their glycoproteins or by viral transformation.

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.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

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