Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus

1998 ◽  
Vol 111 (1) ◽  
pp. 45-60 ◽  
Author(s):  
S. Rottger ◽  
J. White ◽  
H.H. Wandall ◽  
J.C. Olivo ◽  
A. Stark ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Immunoelectron Microscopy ◽  
Hela Cells ◽  
Subcellular Fractionation ◽  
Transferase Activity ◽  
Medial Part ◽  
Membrane Structures ◽  
Golgi Stack ◽  
Vesicular Membrane

O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-transferases.

The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells

1996 ◽  
Vol 109 (7) ◽  
pp. 1975-1989 ◽  
Author(s):  
T. Nilsson ◽  
C. Rabouille ◽  
N. Hui ◽  
R. Watson ◽  
G. Warren
Keyword(s):  
Golgi Apparatus ◽  
Cell Lines ◽  
Hela Cells ◽  
Kin Recognition ◽  
Dramatic Effect ◽  
Golgi Stack ◽  
Stable Cell Lines ◽  
Membrane Spanning ◽  
Necessary And Sufficient

Using a series of chimeric and truncated N-acetylglucosaminyltransferase I (NAGT I) molecules we have shown that part of the lumenal stalk region is both necessary and sufficient for kin recognition of mannosidase II and retention in the Golgi stack. The membrane-spanning domain was not required for retention, but replacing part or all of this domain with leucine residues did have a dramatic effect on Golgi morphology. In stable cell lines, stacked cisternae were replaced by tubulo-vesicular clusters containing the mutated NAGT I. The loss of stacked cisternae was proportional to the number of leucines used to replace the membrane-spanning domain.

scholarly journals Okadaic Acid Induces Selective Arrest of Protein Transport in the Rough Endoplasmic Reticulum and Prevents Export into COPII-Coated Structures

1998 ◽  
Vol 18 (2) ◽  
pp. 1125-1135 ◽  
Author(s):  
James G. Pryde ◽  
Theodora Farmaki ◽  
John M. Lucocq
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Okadaic Acid ◽  
Protein Transport ◽  
Subcellular Fractionation ◽  
Cell Fractionation ◽  
Membrane Structures ◽  
Golgi Stack ◽  
Aluminium Fluoride ◽  
Quantitative Immunoelectron Microscopy

ABSTRACT Quantitative immunoelectron microscopy and subcellular fractionation established the site of endoplasmic reticulum (ER)-Golgi transport arrest induced by the phosphatase inhibitor okadaic acid (OA). OA induced the disappearance of transitional element tubules and accumulation of the anterograde-transported Chandipura (CHP) virus G protein only in the rough ER (RER) and not at more distal sites. The block was specific to the early part of the anterograde pathway, because CHP virus G protein that accumulated in the intermediate compartment (IC) at 15°C could gain access to Golgi stack enzymes. OA also induced RER accumulation of the IC protein p53/p58 via an IC-RER recycling pathway which was resistant to OA and inhibited by the G protein activator aluminium fluoride. The role of COPII coats in OA transport block was investigated by using immunofluorescence and cell fractionation. In untreated cells the COPII coat protein sec 13p colocalized with p53/p58 in Golgi-IC structures of the juxtanuclear region and peripheral cytoplasm. During OA treatment, p53/p58 accumulated in the RER but was excluded from sec 13p-containing membrane structures. Taken together our data indicate that OA induces an early defect in RER export which acts to prevent entry into COPII-coated structures of the IC region.

Intact Ca(2+)-binding sites are required for targeting of annexin 1 to endosomal membranes in living HeLa cells

2000 ◽  
Vol 113 (22) ◽  
pp. 3931-3938 ◽  
Author(s):  
U. Rescher ◽  
N. Zobiack ◽  
V. Gerke
Keyword(s):  
Hela Cells ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Membrane Binding ◽  
Growth Factor Receptor ◽  
Intracellular Distribution ◽  
Live Cells ◽  
Membrane Structures ◽  
Annexin 1

Annexin 1 is a Ca(2+)-regulated membrane binding protein and a major substrate of the epidermal growth factor receptor kinase. Because of its properties and intracellular distribution, the protein has been implicated in endocytic trafficking of the receptor, in particular in receptor sorting occurring in multivesicular endosomes. Up to now, however, the localization of annexin 1 to cellular membranes has been limited to subcellular fractionation and immunocytochemical analyses of fixed cells. To establish its localization in live cells, we followed the intracellular fate of annexin 1 molecules fused to the Green Fluorescent Protein (GFP). We show that annexin 1-GFP associates with distinct, transferrin receptor-positive membrane structures in living HeLa cells. A GFP chimera containing the Ca(2+)/phospholipid-binding protein core of annexin 1 also shows a punctate intracellular distribution, although the structures labeled here do not resemble early but, at least in part, late endosomes. In contrast, the cores of annexins 2 and 4 fused to GFP exhibit a cytoplasmic or a different punctate distribution, respectively, indicating that the highly homologous annexin core domains carry distinct membrane specificities within live cells. By inactivating the three high-affinity Ca(2+) binding sites in annexin 1 we also show that endosomal membrane binding of the protein in live HeLa cells depends on the integrity of these Ca(2+) binding sites. More detailed analysis identifies a single Ca(2+) site in the second annexin repeat that is crucially involved in establishing the membrane association. These results reveal for the first time that intracellular membrane binding of an annexin in living cells requires Ca(2+) and is mediated in part through an annexin core domain that is capable of establishing specific interactions.

scholarly journals Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step

1994 ◽  
Vol 124 (1) ◽  
pp. 55-70 ◽  
Author(s):  
J Krijnse-Locker ◽  
M Ericsson ◽  
PJ Rottier ◽  
G Griffiths
Keyword(s):  
Golgi Complex ◽  
Hepatitis Virus ◽  
Vesicular Transport ◽  
Helix Pomatia ◽  
M Protein ◽  
Membrane Structures ◽  
Golgi Stack ◽  
Permeabilized Cells ◽  
Intermediate Compartment ◽  
Rough Er

Mouse hepatitis coronavirus (MHV) buds into pleomorphic membrane structures with features expected of the intermediate compartment between the ER and the Golgi complex. Here, we characterize the MHV budding compartment in more detail in mouse L cells using streptolysin O (SLO) permeabilization which allowed us to better visualize the membrane structures at the ER-Golgi boundary. The MHV budding compartment shares membrane continuities with the rough ER as well as with cisternal elements on one side of the Golgi stack. It also labeled with p58 and rab2, two markers of the intermediate compartment, and with PDI, usually considered to be a marker of the rough ER. The membranes of the budding compartment, as well as the budding virions themselves, but not the rough ER, labeled with the N-acetyl-galactosamine (GalNAc)-specific lectin Helix pomatia. When the SLO-permeabilized cells were treated with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), the budding compartment accumulated a large number of beta-cop-containing buds and vesicular profiles. Complementary biochemical experiments were carried out to determine whether vesicular transport was required for the newly synthesized M protein, that contains only O-linked oligosaccharides, to acquire first, GalNAc and second, the Golgi modifications galactose and sialic acid. The results from both in vivo studies and from the use of SLO-permeabilized cells showed that, while GalNAc addition occurred under conditions which block vesicular transport, both cytosol and ATP were prerequisites for the M protein oligosaccharides to acquire Golgi modifications. Collectively, our data argue that transport from the rough ER to the Golgi complex requires only one vesicular transport step and that the intermediate compartment is a specialized domain of the endoplasmatic reticulum that extends to the first cisterna on the cis side of the Golgi stack.

Association of kinesin with the Golgi apparatus in rat hepatocytes

1994 ◽  
Vol 107 (9) ◽  
pp. 2417-2426 ◽  
Author(s):  
D.L. Marks ◽  
J.M. Larkin ◽  
M.A. McNiven
Keyword(s):  
Golgi Apparatus ◽  
Immunofluorescence Microscopy ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Immunoblot Analysis ◽  
Subcellular Fractionation ◽  
Rat Hepatocyte ◽  
Membranous Structure ◽  
Microtubule Disruption ◽  
Motor Enzymes

The Golgi apparatus is a dynamic membranous structure, which has been observed to alter its location and morphology during the cell cycle and after microtubule disruption. These dynamics are believed to be supported by a close structural interaction of the Golgi with the microtubule cytoskeleton and associated motor enzymes. One microtubule-dependent motor enzyme, kinesin, has been implicated in Golgi movement and function although direct evidence supporting this interaction is lacking. In this study, we utilized two well-characterized kinesin antibodies in conjunction with subcellular fractionation techniques, immunoblot analysis and immunofluorescence microscopy to conduct a detailed study on the association of kinesin with the Golgi and other membranous organelles in a polarized epithelial cell, the primary rat hepatocyte. We found that kinesin represents approximately 0.3% of total protein in rat liver homogenates, with approximately 30% membrane-associated and the remainder in the cytosol. Among membrane fractions, kinesin was concentrated markedly in Golgi-enriched fractions, which were prepared using two independent techniques. Kinesin was also abundant in fractions enriched in transcytotic carriers and secretory vesicles, with lower levels detected on fractions enriched in endosomes, endoplasmic reticulum, lysosomes and mitochondria. Immunofluorescence microscopy showed that kinesin is concentrated on Golgi-like structures in both primary cultured hepatocytes and rat hepatocyte-derived clone 9 cells. Double-label immunofluorescence demonstrated that kinesin staining colocalizes with the Golgi marker, alpha-mannosidase II, in both cell types. These results provide compelling evidence showing that kinesin is associated with the Golgi complex in cells and implicate this motor enzyme in Golgi structure, function and dynamics.

scholarly journals Fragmentation and partitioning of the Golgi apparatus during mitosis in HeLa cells.

1987 ◽  
Vol 6 (11) ◽  
pp. 3239-3246 ◽  
Author(s):  
J. M. Lucocq ◽  
G. Warren
Keyword(s):  
Golgi Apparatus ◽  
Hela Cells

scholarly journals Partitioning of the Golgi Apparatus during Mitosis in Living HeLa Cells

1997 ◽  
Vol 137 (6) ◽  
pp. 1211-1228 ◽  
Author(s):  
David T. Shima ◽  
Kasturi Haldar ◽  
Rainer Pepperkok ◽  
Rose Watson ◽  
Graham Warren
Keyword(s):  
Golgi Apparatus ◽  
Hela Cells ◽  
Direct Evidence ◽  
Fluorescent Protein ◽  
Immunofluorescence Microscopy ◽  
Critical Feature ◽  
Directed Movement ◽  
Late Telophase ◽  
Green Fluorescent ◽  
Retention Signal

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I–GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.

scholarly journals GABARAPL2 Is Critical for Growth Restriction of Toxoplasma gondii in HeLa Cells Treated with Gamma Interferon

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Zhaoxia Zhang ◽  
Haorong Gu ◽  
Qi Li ◽  
Jun Zheng ◽  
Shinuo Cao ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Hela Cells ◽  
Critical Role ◽  
Gamma Interferon ◽  
Growth Restriction ◽  
Membrane Structures ◽  
Content Type ◽  
Receptor Associated Protein ◽  
Ifn Γ ◽  
And Function

ABSTRACT Gamma interferon (IFN-γ)-induced innate immune responses play important roles in the inhibition of Toxoplasma gondii infection. It has been reported that IFN-γ stimulates non-acidification-dependent growth restriction of T. gondii in HeLa cells, but the mechanism remains unclear. Here, we found that γ-aminobutyric acid (GABA) receptor-associated protein-like 2 (GABARAPL2) plays a critical role in parasite restriction in IFN-γ-treated HeLa cells. GABARAPL2 is recruited to membrane structures surrounding parasitophorous vacuoles (PV). Autophagy adaptors are required for the proper localization and function of GABARAPL2 in the IFN-γ -induced immune response. These findings provide further understanding of a noncanonical autophagy pathway responsible for IFN-γ-dependent inhibition of T. gondii growth in human HeLa cells and demonstrate the critical role of GABARAPL2 in this response.

scholarly journals Subcellular distribution of selenium in deficient mouse liver

1989 ◽  
Vol 258 (2) ◽  
pp. 541-545 ◽  
Author(s):  
R Reiter ◽  
R Otter ◽  
A Wendel
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Trace Element ◽  
Fold Increase ◽  
Subcellular Fractionation ◽  
Fast Response ◽  
Cell Compartment ◽  
Deficient Mice

Selenium (Se)-deficient mice were labelled in vivo with single pulses of [75Se]selenite, and the intrahepatic distribution of the trace element was studied by subcellular fractionation. At 1 h after intraperitoneal injection of 3.3 or 10 micrograms of Se/kg body weight, 15% of the respective doses were found in the liver. Accumulation in the subcellular fractions followed the order: Golgi vesicular much greater than lysosomal greater than cytosolic = microsomal greater than mitochondrial, peroxisomal, nuclear and plasma-membrane fraction. At a dose of 3.3 micrograms/kg, more than 90% of the hepatic Se was protein-bound. When cross-contamination was accounted for, the following specific Se contents of the subcellular compartments were extrapolated: Golgi apparatus, 7.50 pmol/mg; cytosol, 0.90 pmol/mg; endoplasmic reticulum, 0.80 pmol/mg; mitochondria, 0.49 pmol/mg; nuclei, lysosomes, peroxisomes and plasma membrane, less than 0.4 pmol/mg. At 10 micrograms/kg, a roughly 2-3-fold increase in Se content of all fractions was found without major changes in the intrahepatic distribution pattern. An extraordinary rise in the cytosolic fraction was due to an apparently non-protein-bound Se pool. At 24 h after dosing, total hepatic Se had decreased to 6% of the initial dose and had become predominantly protein-bound. The 60% decrease in hepatic Se was reflected in a similar fall in the subcellular levels of the trace element. The Golgi apparatus still had the highest specific Se content, although accumulation was 5 times less than that after 1 h. The cytosolic pool accounted for 50% of the hepatic Se at both labelling times. After 1 h the Golgi apparatus was, with 19%, the second largest intrahepatic pool, followed by the endoplasmic reticulum with 16%. The high affinity and fast response of the Golgi apparatus to Se supplementation of deficient mice is interpreted in terms of a predominant function of this cell compartment in the processing and the export of Se-proteins from the liver.

scholarly journals Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level.

1987 ◽  
Vol 35 (3) ◽  
pp. 327-333 ◽  
Author(s):  
E van Tuinen ◽  
H Riezman
Keyword(s):  
Immunoelectron Microscopy ◽  
Ultrastructural Level ◽  
Subcellular Fractionation ◽  
Glycolytic Enzymes ◽  
Yeast Cells ◽  
Carboxypeptidase Y ◽  
Yeast Vacuole

We have developed a simple and effective method to embed whole yeast cells in Lowicryl resins with excellent ultrastructural and antigenic preservation. Using affinity-purified antibodies eluted from electrophoretically separated proteins transferred to nitrocellulose, we have shown by immunoelectron microscopy that two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and hexokinase, are present in the cytoplasm and the nucleus. Carboxypeptidase Y is localized in the yeast vacuole. These results agree with earlier localization studies based on subcellular fractionation.

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