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 Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level.

1994 ◽  
Vol 42 (7) ◽  
pp. 917-929 ◽  
Author(s):  
E Spiess ◽  
A Brüning ◽  
S Gack ◽  
B Ulbricht ◽  
H Spring ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Tumor Cell ◽  
Enzymatic Activity ◽  
Cell Lines ◽  
Cathepsin B ◽  
Lung Tumor ◽  
Cell Types ◽  
Cellular Level ◽  
Tumor Cell Lines

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefins A and B, at the cellular level in human tumor cell lines HS-24, derived from a primary lung tumor (squamous cell), and SB-3, derived from a metastasis (lung adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and functionally related cathepsin L, but not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefin B was found in both cell types. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized and quantified. Thus, the cellular cathepsin B kinetics for the synthetic substrates Z-Arg-Arg-4M beta NA and Z-Val-Lys-Lys-Arg-4M beta NA, its pH dependence and inhibition by E64, stefins A and B, and cystatin C could be determined. From these measurements it appeared that the enzyme exhibited different cleavage rates for these substrates in the different cell types, showed considerable cleavage activity at neutral pH, which was stable under these conditions for extended time periods, and was highly sensitive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefins, particularly stefin A. By conventional light microscopy, confocal laser scanning microscopy, and electron microscopy the enzymatic activity was localized in lysosomes, as expected, but also in the endoplasmic reticulum, nuclear membrane, and plasma membrane. The endoplasmic reticulum is a site at which only pre-mature enzyme forms exist, which are usually not active. The appearance of enzymatic activity at the plasma membrane confirms earlier biochemical and immunofluorescence microscopic investigations. The different sites of localization within the cells make it likely that different forms of the enzyme are expressed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative processes.

scholarly journals Internalization of Monomeric Lipopolysaccharide Occurs after Transfer Out of Cell Surface Cd14

1999 ◽  
Vol 190 (4) ◽  
pp. 509-522 ◽  
Author(s):  
Thierry Vasselon ◽  
Eric Hailman ◽  
Rolf Thieringer ◽  
Patricia A. Detmers
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Soluble Cd14 ◽  
Stable Transfectants ◽  
Intracellular Vesicles ◽  
Green Fluorescent

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14–EGFP) was used to follow trafficking of mCD14 and BODIPY–LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex–like structure. mCD14–EGFP was functional in mediating binding of and responses to LPS. BODIPY–LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14–EGFP on the cell surface. However, within 5–10 min, the BODIPY–LPS distributed to intracellular vesicles that did not contain mCD14–EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

scholarly journals Syntaxin 5-Dependent Retrograde Transport to thetrans-Golgi Network Is Required for Adeno-Associated Virus Transduction

2014 ◽  
Vol 89 (3) ◽  
pp. 1673-1687 ◽  
Author(s):  
Mathieu E. Nonnenmacher ◽  
Jean-Christophe Cintrat ◽  
Daniel Gillet ◽  
Thomas Weber
Keyword(s):  
Gene Delivery ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Transport Pathway ◽  
Adeno Associated Virus ◽  
Viral Attachment ◽  
Golgi Network ◽  
Endosomal System

ABSTRACTIntracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward thetrans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment.IMPORTANCEGene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne muscular dystrophy, hemophilia, heart failure, Parkinson's disease, and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to thetrans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from that of known Golgi apparatus-targeted cargos, and we raise the possibility that this nonclassical pathway is shared by most AAV variants, regardless of their attachment receptors.

scholarly journals MIGRATION OF GLYCOPROTEIN FROM THE GOLGI APPARATUS TO THE SURFACE OF VARIOUS CELL TYPES AS SHOWN BY RADIOAUTOGRAPHY AFTER LABELED FUCOSE INJECTION INTO RATS

1974 ◽  
Vol 60 (1) ◽  
pp. 258-284 ◽  
Author(s):  
Gary Bennett ◽  
C. P. Leblond ◽  
Antonio Haddad
Keyword(s):  
Plasma Membrane ◽  
Electron Microscope ◽  
Golgi Apparatus ◽  
Early Time ◽  
Cell Types ◽  
Cell Type ◽  
Time Intervals ◽  
Cell Coat ◽  
Golgi Region ◽  
Silver Grains

A single intravenous injection of L-[3H]fucose, a specific glycoprotein precursor, was given to young 35–45 g rats which were sacrificed at times varying between 2 min and 30 h later. Radioautography of over 50 cell types, including renewing and nonrenewing cells, was carried out for light and electron microscope study. At early time intervals (2–10 min after injection), light microscope radioautography showed a reaction over nearly all cells investigated in the form of a discrete clump of silver grains over the Golgi region. This reaction varied in intensity and duration from cell type to cell type. Electron microscope radioautographs of duodenal villus columnar cells and kidney proximal and distal tubule cells at early time intervals revealed that the silver grains were restricted to Golgi saccules. These observations are interpreted to mean that glycoproteins undergoing synthesis incorporate fucose in the saccules of the Golgi apparatus. Since fucose occurs as a terminal residue in the carbohydrate side chains of glycoproteins, the Golgi saccules would be the site of completion of synthesis of these side chains. At later time intervals, light and electron microscope radioautography demonstrated a decrease in the reaction intensity of the Golgi region, while reactions appeared over other parts of the cells: lysosomes, secretory material, and plasma membrane. The intensity of the reactions observed over the plasma membrane varied considerably in various cell types; furthermore the reactions were restricted to the apical surface in some types, but extended to the whole surface in others. Since the plasma membrane is covered by a "cell coat" composed of the carbohydrate-rich portions of membrane glycoproteins, it is concluded that newly formed glycoproteins, after acquiring fucose in the Golgi apparatus, migrate to the cell surface to contribute to the cell coat. This contribution implies turnover of cell coat glycoproteins, at least in nonrenewing cell types, such as those of kidney tubules. In the young cells of renewing populations, e.g. those of gastro-intestinal epithelia, the new glycoproteins seem to contribute to the growth as well as the turnover of the cell coat. The differences in reactivity among different cell types and cell surfaces imply considerable differences in the turnover rates of the cell coats.

scholarly journals A unique combination of plasma membrane Ca2+-ATPase isoforms is expressed in islets of Langerhans and pancreatic β-cell lines

1996 ◽  
Vol 314 (2) ◽  
pp. 663-669 ◽  
Author(s):  
Anikó VÁRADI ◽  
Elek MOLNÁR ◽  
Stephen J. H. ASHCROFT
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Islets Of Langerhans ◽  
Cell Lines ◽  
Cell Types ◽  
Human Islets ◽  
Unique Combination ◽  
Β Cells ◽  
Β Cell ◽  
Calmodulin Binding

Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic β-cells. The existence of steep Ca2+ gradients within the β-cell requires the presence of specialized Ca2+ exclusion systems. In this study we have characterized the plasma membrane Ca2+-ATPases (PMCAs) which extrude Ca2+ from the cytoplasm. PMCA isoform- and subtype-specific mRNA expression was investigated in rodent pancreatic α- and β-cell lines, and in human and rat islets of Langerhans using reverse-transcription PCR with primers flanking the calmodulin-binding region of rat PMCA. The expression pattern of PMCA 1 and 2 was conserved in different species and islet-cell types since both rat and human islets of Langerhans and all cell lines tested contained the 1b and 2b forms. PMCA 4 isoform subtypes, however, were expressed in a cell-type-specific manner since β-cells expressed PMCA 4b only, whereas in islets of Langerhans, which contain α, β, δ and polypeptide-secreting cells, PMCA 4a and 4b were simultaneously present. No evidence was obtained for the expression of PMCA 3. Characterization of the β-cell Ca2+-pump protein showed that it shared several similarities with the erythrocyte PMCA. It is a P-type ATPase; its phosphorylated intermediate was stabilized by La3+; it reacted with a PMCA-specific antibody; and it was not N-glycosylated. However, the β-cell PMCA had a higher molecular mass than that of the erythrocyte; this difference could be explained by either predominant translation of the PMCA 2 form, which has a molecular mass 3–8 kDa higher than the erythrocyte PMCA 1 and 4 proteins, or by a possible sequence insertion. Thus a unique combination of functionally distinct PMCA isoforms (1b, 2b, 4b) participates in Ca2+ homoeostasis in the β-cell.

scholarly journals Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.

1984 ◽  
Vol 99 (3) ◽  
pp. 1101-1109 ◽  
Author(s):  
A A Rogalski ◽  
J E Bergmann ◽  
S J Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
G Protein ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Temperature Shift ◽  
Surface Expression ◽  
Microtubule Assembly ◽  
In Cells

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.

scholarly journals Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins.

1987 ◽  
Vol 105 (3) ◽  
pp. 1227-1240 ◽  
Author(s):  
S A Green ◽  
K P Zimmer ◽  
G Griffiths ◽  
I Mellman
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Rat Kidney ◽  
Fc Receptor ◽  
Lysosomal Membrane ◽  
Plasma Membrane Proteins ◽  
Kinetics Of

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.

scholarly journals Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy.

1989 ◽  
Vol 109 (5) ◽  
pp. 2067-2079 ◽  
Author(s):  
R E Pagano ◽  
M A Sepanski ◽  
O C Martin
Keyword(s):  
Golgi Apparatus ◽  
Light And Electron Microscopy ◽  
Cell Types ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Fluorescent Marker ◽  
Cellular Lipids ◽  
Endogenous Lipids ◽  
Fluorescent Lipid

We have previously shown that a fluorescent derivative of ceramide, N-(epsilon-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-eryth ro-sphingosin e (C6-NBD-Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6-NBD-Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6-NBD-Cer at 2 degrees C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24 degrees C removed excess C6-NBD-Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland, 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6-NBD-Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6-NBD-Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6-NBD-Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.

scholarly journals Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts.

1989 ◽  
Vol 108 (6) ◽  
pp. 2169-2181 ◽  
Author(s):  
M Koval ◽  
R E Pagano
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Chinese Hamster ◽  
Membrane Lipid ◽  
Cell Periphery ◽  
Perinuclear Region ◽  
Cultured Fibroblasts ◽  
Plasma Membrane Lipid ◽  
Intracellular Compartments ◽  
Fluorescent Lipid

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.

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.

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