Protein glycosylation in the endoplasmic reticulum and the Golgi apparatus and cell type-specificity of cell surface glycoconjugate expression: analysis by the protein A-gold and lectin-gold techniques

1996 ◽  
Vol 106 (1) ◽  
pp. 79-92 ◽  
Author(s):  
J. Roth
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Expression Analysis ◽  
Protein A ◽  
Protein Glycosylation ◽  
Cell Type ◽  
Cell Type Specificity

Altered labelling of the cell surface and intracellular organelles with [3H]mannose in enucleated amoebae

1984 ◽  
Vol 68 (1) ◽  
pp. 83-94
Author(s):  
C.J. Flickinger
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Cell Organelles ◽  
Intracellular Organelles ◽  
And Control ◽  
Kinetics Of ◽  
Heavy Labelling

The production, transport, and disposition of material labelled with [3H]mannose were studied in microsurgically enucleated and control amoebae. Cells were injected with the precursor and samples were prepared for electron-microscope radioautography at intervals, up to 24 h later. Control cells showed heavy labelling of the rough endoplasmic reticulum and the Golgi apparatus at early intervals after injection. Later, labelling of groups of small vesicles increased, and the percentage of grains over the cell surface peaked 12 h after administration of the precursor. Two major changes were detected in enucleate amoebae. First, the kinetics of labelling of cell organelles with [3H]mannose were altered in the absence of the nucleus. The Golgi apparatus and cell surface both displayed maximal labelling at later intervals in enucleates, and the percentage of grains over the rough endoplasmic reticulum varied less with time in enucleated than in control cells. Second, the distribution of radioactivity was altered. A greater percentage of grains was associated with lysosomes in enucleates than in control cells. The change in the kinetics of labelling of the endoplasmic reticulum, Golgi apparatus and cell surface indicates that intracellular transport of surface material was slower in the absence of the nucleus. It is suggested that this is related to the decreased motility of enucleate cells.

scholarly journals Multiple Domains in Caveolin-1 Control Its Intracellular Traffic

2000 ◽  
Vol 148 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Thomas Machleidt ◽  
Wei-Ping Li ◽  
Pingsheng Liu ◽  
Richard G.W. Anderson
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Integral Membrane Protein ◽  
Intracellular Traffic ◽  
Caveolin 1 ◽  
Multiple Domains ◽  
Control Traffic ◽  
Multiple Signaling

Caveolin-1 is an integral membrane protein of caveolae that is thought to play an important role in both the traffic of cholesterol to caveolae and modulating the activity of multiple signaling molecules at this site. The molecule is synthesized in the endoplasmic reticulum, transported to the cell surface, and undergoes a poorly understood recycling itinerary. We have used mutagenesis to determine the parts of the molecule that control traffic of caveolin-1 from its site of synthesis to the cell surface. We identified four regions of the molecule that appear to influence caveolin-1 traffic. A region between amino acids 66 and 70, which is in the most conserved region of the molecule, is necessary for exit from the endoplasmic reticulum. The region between amino acids 71 and 80 controls incorporation of caveolin-1 oligomers into detergent-resistant regions of the Golgi apparatus. Amino acids 91–100 and 134–154 both control oligomerization and exit from the Golgi apparatus. Removal of other portions of the molecule has no effect on targeting of newly synthesized caveolin-1 to caveolae. The results suggest that movement of caveolin-1 among various endomembrane compartments is controlled at multiple steps.

scholarly journals Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products.

1984 ◽  
Vol 99 (6) ◽  
pp. 2011-2023 ◽  
Author(s):  
J W Wills ◽  
R V Srinivas ◽  
E Hunter
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Nuclear Membrane ◽  
Rous Sarcoma Virus ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Rous Sarcoma ◽  
Sarcoma Virus

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.

scholarly journals The amounts and rates of export of polysaccharides found within the membrane system of maize root cells

1974 ◽  
Vol 142 (1) ◽  
pp. 139-144 ◽  
Author(s):  
Dianna J. Bowles ◽  
D. H. Northcote
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Rough Endoplasmic Reticulum ◽  
Membrane System ◽  
Maize Root ◽  
Wall Material ◽  
Cell Wall Material ◽  
Root Cells

1. Maize seedling roots were incubated in vivo with d-[U-14C]glucose for 2, 5, 10, 15, 30 and 45min. The total incorporation of radioactivity into polysaccharide components in isolated fractions was investigated, and the pattern of incorporation into different polysaccharide components within the rough endoplasmic reticulum, Golgi apparatus and exported material was analysed. 2. The membrane compartments reached a saturation value of radioactivity in polysaccharide components by 30min incubation. Radioactivity in exported polysaccharide continued to increase after that time. The latter was formed and maintained by a steady-state turnover of polysaccharide synthesis and transport from the membrane system. 3. If the only access of the slime polysaccharide to the cell surface is via dictyosome-derived vesicles, the amount of slime components in the Golgi apparatus would have to be displaced every 0.3min in order to maintain the observed rates of increase in slime. This is in contrast with a displacement time of about 2.5min that is necessary for polysaccharide components in the Golgi apparatus to produce the observed increase in cell-wall material. The activity of the membrane system in the production of maize root slime is 8 times as great as that of the membrane system involved in cell-wall synthesis. 4. If the amount of polysaccharide material in the Golgi apparatus is maintained only by inflow of polymeric material from the rough endoplasmic reticulum the total amount of slime components in the rough endoplasmic reticulum would have to be displaced every 7min to maintain a constant amount in the Golgi apparatus. If the endoplasmic reticulum contributed directly to the cell surface in the synthesis of cell-wall material, displacement times necessary to maintain the observed rate of polymer production would be very slow.

scholarly journals Human Antiviral Protein MxA Forms Novel Metastable Membraneless Cytoplasmic Condensates Exhibiting Rapid Reversible Tonicity-Driven Phase Transitions

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Deodate Davis ◽  
Huijuan Yuan ◽  
Feng-Xia Liang ◽  
Yang-Ming Yang ◽  
Jenna Westley ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Viral Infection ◽  
Protein A ◽  
Cell Cytoplasm ◽  
Intact Cells ◽  
N Protein ◽  
P Bodies ◽  
Huh7 Cells ◽  
Resistance Protein

ABSTRACT Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g., stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membraneless metastable (shape-changing) condensates in the cytoplasm. In our studies, we used the same cell lines and methods as those used by previous investigators but concluded that wild-type MxA formed variably sized spherical or irregular bodies, filaments, and even a reticulum distinct from that of ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of green fluorescent protein (GFP)-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24 ± 0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase, respectively, in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-α-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature and provide evidence for membraneless MxA biomolecular condensates in the uninfected cell cytoplasm. IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation and further report that MxA forms membraneless metastable (shape-changing) condensates in the cytoplasm consisting of variably sized spherical or irregular bodies, filaments, and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1 to 3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.

Redistribution of material labelled with [3H]mannose in amoebae induced to undergo pinocytosis

1982 ◽  
Vol 58 (1) ◽  
pp. 79-93
Author(s):  
C.J. Flickinger
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Rough Endoplasmic Reticulum ◽  
Lysosomal Enzymes ◽  
Surface Material ◽  
Albumin Solution ◽  
Electron Microscopic ◽  
Normal Medium ◽  
Electron Microscopic Radioautography

The synthesis, transport, and disposition of material labelled with [3H]mannose were studied by electron microscopic radioautography in normal amoebae and in cells that had internalized cell surface as a result of being induced to undergo pinocytosis. Control amoebae were injected with the precursor and placed in normal medium. The Golgi apparatus and rough endoplasmic reticulum were heavily labelled at the earliest intervals, while radioactivity of the cell surface peaked 12 h after injection of precursor. The experimental cells were injected, placed in bovine serum albumin solution from 15 to 60 min after injection, and then removed to normal medium until fixation. Incorporation of the precursor into the rough endoplasmic reticulum was near normal, but the proportions of grains associated with the Golgi apparatus and the cell surface were greatly reduced. The percentage of grains overlying vacuoles increased 12 h after injection, notably in the case of polymorphous vacuoles and dense vacuoles, both of which were identified as lysosomes with the acid phosphatase reaction. The results suggest that addition to the surface of components labelled with [3H]mannose was diminished following induction of pinocytosis. Incorporation of the precursor appeared to be shifted from cell surface material to lysosomal contents, possibly lysosomal enzymes. It is thought that this shift occurred in response to the need for the cell to digest unusually large amounts of endocytosed protein. Recycling of cell surface under these conditions is considered possible.

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