scholarly journals Biosynthesis and transport of cathepsin D in cultured human fibroblasts.

1983 ◽  
Vol 97 (1) ◽  
pp. 1-5 ◽  
Author(s):  
V Gieselmann ◽  
R Pohlmann ◽  
A Hasilik ◽  
K Von Figura
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cathepsin D ◽  
Human Fibroblasts ◽  
Total Cell ◽  
Molecular Forms ◽  
Cell Extracts ◽  
Cultured Human Fibroblasts ◽  
Immune Precipitation ◽  
High Mannose

For study of the time order of glycosylation, formation of complex oligosaccharides and proteolytic maturation as well as the site of proteolytic maturation of cathepsin D, fibroblasts were subjected to pulse-chase labeling, and cathepsin D was isolated from either total cell extracts or subcellular fractions by immune precipitation and analyzed for its molecular forms and sensitivity to endo-beta-N-acetylglucosaminidase H. After a 10-min pulse, cathepsin D was detected in its glycosylated precursor form, indicating an early, probably a cotranslational, N-glycosylation of cathepsin D. Conversion of the high-mannose oligosaccharide side chains into forms resistant to endo-beta-N-acetylglucosaminidase H started after approximately 40 min, indicating that transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi apparatus requires approximately 40 min. Processing of the 53-kdalton precursor polypeptide of cathepsin D to a 47-kdalton intermediate followed about 20 min after the formation of complex oligosaccharides, and, another 30 min later, 31-kdalton mature forms of cathepsin D were detected. Processing of cathepsin D was first observed in light membranes as a partial conversion of the 53-kdalton precursor into the 47-kdalton intermediate. Both the precursor and the intermediate are transferred into the high density-class lysosomes. After 8 h, the processing to the mature 31-kdalton form of cathepsin D is mostly completed.

scholarly journals Inhibition by cyanate of the processing of lysosomal enzymes

1983 ◽  
Vol 210 (3) ◽  
pp. 795-802 ◽  
Author(s):  
A Hasilik ◽  
R Pohlmann ◽  
K von Figura
Keyword(s):  
Golgi Apparatus ◽  
Lysosomal Enzyme ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Human Fibroblasts ◽  
Cultured Human Fibroblasts ◽  
High Mannose ◽  
In Cells

In cultured human fibroblasts, maturation of the lysosomal enzymes beta-hexosaminidase and cathepsin D is inhibited by 10 mM-potassium cyanate. In cells treated with cyanate the two enzymes accumulate in precursor forms. The location of the accumulated precursor is probably non-lysosomal; in fractionation experiments the precursors separate from the bulk of the beta-hexosaminidase activity. The secretion of the precursor of cathepsin D, but not that of beta-hexosaminidase precursor, is enhanced in the presence of cyanate. The secreted cathepsin D, as well as that remaining within the cells, contains mostly high-mannose oligosaccharides cleavable with endo-beta-N-acetylglucosaminidase H. After removal of cyanate, the accumulated precursor forms of the lysosomal enzymes are largely released from the pretreated cells. It is concluded that cyanate interferes with the maturation of lysosomal-enzyme precursors by perturbing their intracellular transport. Most probably cyanate affects certain functions of the Golgi apparatus.

scholarly journals Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymes

1984 ◽  
Vol 217 (3) ◽  
pp. 649-658 ◽  
Author(s):  
R Pohlmann ◽  
S Krüger ◽  
A Hasilik ◽  
K von Figura
Keyword(s):  
Golgi Apparatus ◽  
Lysosomal Enzyme ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Human Fibroblasts ◽  
Free Medium ◽  
Intracellular Enzyme ◽  
Cultured Human Fibroblasts ◽  
Monensin A

In cultured human fibroblasts we observed that monensin, a Na+/H+-exchanging ionophore, (i) inhibits mannose 6-phosphate-sensitive endocytosis of a lysosomal enzyme, (ii) enhances secretion of the precursor of cathepsin D, while inhibiting secretion of the precursors of beta-hexosaminidase, (iii) induces secretion of mature beta-hexosaminidase and mature cathepsin D, and (iv) inhibits carbohydrate processing in and proteolytic maturation of the precursors remaining within the cells; this last effect appears to be secondary to an inhibition of the transport of the precursors. If the treated cells are transferred to a monensin-free medium, about half of the accumulated precursors are secreted, and the intracellular enzyme is converted into the mature form. Monensin blocks formation of complex oligosaccharides in lysosomal enzymes. In the presence of monensin, total phosphorylation of glycoproteins is partially inhibited, whereas the secreted glycoproteins are enriched in the phosphorylated species. The suggested inhibition by monensin of the transport within the Golgi apparatus [Tartakoff (1980) Int. Rev. Exp. Pathol. 22, 227-250] may be the cause of some of the effects observed in the present study (iv). Other effects (i, ii) are rather explained by interference by monensin with the acidification in the lysosomal and prelysosomal compartments, which appears to be necessary for the transport of endocytosed and of newly synthesized lysosomal enzymes.

Alloxan action on glucose metabolism in cultured fibroblasts. II. Effects on pentose-monophosphate shunt and tricarboxylic acid pathways

1981 ◽  
Vol 240 (6) ◽  
pp. E645-E648
Author(s):  
F. Ishibashi ◽  
P. H. Bennett ◽  
B. V. Howard
Keyword(s):  
Glucose Metabolism ◽  
Human Fibroblasts ◽  
Lactate Production ◽  
Tricarboxylic Acid ◽  
Cultured Fibroblasts ◽  
Cell Monolayers ◽  
Cell Extracts ◽  
Cultured Human Fibroblasts ◽  
Glucose Incorporation ◽  
14Co2 Production

The site of action of alloxan on glucose metabolism has been investigated using cultured human fibroblasts. Analysis of cell extracts after cell monolayers were exposed to D-[U-14C]glucose indicated that the initial stimulation of glucose incorporation by alloxan was observed primarily in the nucleotide fraction (ribose) with inhibition of lactate production. The subsequent inhibition of glucose incorporation was observed in the nucleotide fraction. Assay of 14CO2 production indicated that alloxan enhanced 14CO2 formation from D-[U-14C]glucose for approximately 10 min, followed by inhibition. To probe the site of alloxan action, rates of 14CO2 formation from 1- and 6-position labeled [14C]glucose, and [U-14C]pyruvate were compared. The initial stimulation was observed mainly in D-[1–14C]glucose oxidation, whereas inhibition was measurable with the 6-position tracer and [14C]pyruvate. The results suggest that alloxan initially stimulates the pentose-monophosphate shunt and then subsequently inhibits both the pentose-monophosphate shunt and tricarboxylic acid pathways.

scholarly journals The distribution of lysosomal cathepsin D in cardiac myocytes.

1980 ◽  
Vol 28 (3) ◽  
pp. 231-237 ◽  
Author(s):  
R S Decker ◽  
M L Decker ◽  
A R Poole
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Golgi Apparatus ◽  
Cardiac Myocytes ◽  
Cathepsin D ◽  
Immunohistochemical Method ◽  
Acid Proteinase ◽  
Reproducible Method ◽  
Secondary Lysosomes ◽  
Subcellular Level

Lysosomal cathepsin D has been localized with the electron microscope employing an indirect immunohistochemical method using peroxidase labeled, monospecific antibody Fab' subunits. The acid proteinase has been demonstrated within secondary lysosomes of cardiac myocytes and interstitial cells, but not in components of the Golgi apparatus or endoplasmic reticulum. Incubations with a variety of peroxidatic inhibitors suggests that the staining that is observed in secondary lysosomes is attributable to the peroxidase-labeled antibody and not to endogenous oxidation of DAB. The protocol outlined here provides a reproducible method to localize the major lysosomal acid proteinase of the heart at the subcellular level.

TERT enhances the survival rate of human fibroblasts under endoplasmic reticulum, Golgi apparatus, and lysosomal stresses

2018 ◽  
Vol 40 (6) ◽  
pp. 915-922 ◽  
Author(s):  
Amer Ali Abd El-Hafeez ◽  
Toru Hosoi ◽  
Kanako Nakatsu ◽  
Mina Thon ◽  
Akira Shimamoto ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Survival Rate ◽  
Golgi Apparatus ◽  
Human Fibroblasts

scholarly journals mTOR controls endoplasmic reticulum–Golgi apparatus trafficking of VSVg in specific cell types

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Alicja Koscielny ◽  
Ewa Liszewska ◽  
Katarzyna Machnicka ◽  
Michalina Wezyk ◽  
Katarzyna Kotulska ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Specific Cell ◽  
Energy Status ◽  
Mtor Inhibition ◽  
Cargo Transport ◽  
Image Movement

Abstract Background Mammalian/mechanistic target of rapamycin (mTOR) complexes are essential for cell proliferation, growth, differentiation, and survival. mTORC1 hyperactivation occurs in the tuberous sclerosis complex (TSC). mTORC1 localizes to the surface of lysosomes, where Rheb activates it. However, mTOR was also found on the endoplasmic reticulum (ER) and Golgi apparatus (GA). Recent studies showed that the same inputs regulate ER-to-GA cargo transport and mTORC1 (e.g., the level of amino acids or energy status of the cell). Nonetheless, it remains unknown whether mTOR contributes to the regulation of cargo passage through the secretory pathway. Methods The retention using selective hooks (RUSH) approach was used to image movement of model cargo (VSVg) between the ER and GA in various cell lines in which mTOR complexes were inhibited. We also investigated VSVg trafficking in TSC patient fibroblasts. Results We found that mTOR inhibition led to the overall enhancement of VSVg transport through the secretory pathway in PC12 cells and primary human fibroblasts. Also, in TSC1-deficient cells, VSVg transport was enhanced. Conclusions Altogether, these data indicate the involvement of mTOR in the regulation of ER-to-GA cargo transport and suggest that impairments in exocytosis may be an additional cellular process that is disturbed in TSC.

scholarly journals Recognition and receptor-mediated uptake of phosphorylated high mannose-type oligosaccharides by cultured human fibroblasts.

1983 ◽  
Vol 96 (3) ◽  
pp. 915-919 ◽  
Author(s):  
M Natowicz ◽  
D W Hallett ◽  
C Frier ◽  
M Chi ◽  
P H Schlesinger ◽  
...  
Keyword(s):  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Specific Activity ◽  
Human Fibroblasts ◽  
Lysosomal Hydrolases ◽  
Minimal Structure ◽  
Cultured Human Fibroblasts ◽  
Specific Uptake ◽  
High Mannose ◽  
Mannose 6 Phosphate

The intracellular transport of newly synthesized lysosomal hydrolases to lysosomes requires the presence of one or more phosphorylated high mannose-type oligosaccharides per enzyme. A receptor that mediates mannose-6-PO4-specific uptake of lysosomal enzymes is expressed on the surface of fibroblasts and presumably accounts for the intracellular transport of newly synthesized enzymes to the lysosome. In this study, we examined the internalization of lysosomal enzyme-derived phosphorylated oligosaccharides by cultured human fibroblasts. Oligosaccharides of known specific activity bearing a single phosphate in monoester linkage were internalized with Kuptake of 3.2 X 10(-7) M, whereas oligosaccharides bearing two phosphates in monoester linkage were internalized with a Kuptake of 3.9 X 10(-8) M. Thus, phosphorylated high mannose-type oligosaccharides appear to be the minimal structure required for recognition and uptake by the fibroblast receptor. The finding that the Kuptake for monophosphorylated oligosaccharides is 100-fold less than the reported Ki for mannose-6-phosphate indicates that the fibroblast phosphomannosyl receptor contains a binding site that recognizes features of the oligosaccharide in addition to mannose-6-phosphate.

scholarly journals Cell type-specific post-Golgi apparatus localization of a "resident" endoplasmic reticulum glycoprotein, glucosidase II.

1990 ◽  
Vol 110 (2) ◽  
pp. 309-318 ◽  
Author(s):  
D Brada ◽  
D Kerjaschki ◽  
J Roth
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Light And Electron Microscopy ◽  
Cell Types ◽  
Cell Type ◽  
Tubular Cells ◽  
Glucosidase Ii ◽  
Oligosaccharide Processing ◽  
Cell Type Specific ◽  
High Mannose

Glucosidase II, an asparagine-linked oligosaccharide processing enzyme, is a resident glycoprotein of the endoplasmic reticulum. In kidney tubular cells, in contrast to previous findings on hepatocytes, we found by light and electron microscopy immunoreactivity for glucosidase II predominantly in post-Golgi apparatus structures. The majority of immunolabel was in endocytotic structures beneath the plasma membrane. Immunoprecipitation confirmed presence of the glucosidase II subunit in purified brush border preparations. Kidney glucosidase II contained species carrying endo H-sensitive, high mannose as well as endo H-resistant oligosaccharide chains. Some species of glucosidase II contained sialic acid. The sialylated species were enzymatically active. This study demonstrates than an enzyme presumed to be a resident of the endoplasmic reticulum may show alternative localizations in some cell types.

scholarly journals Interaction of Coatomer with Aminoglycoside Antibiotics: Evidence That Coatomer Has at Least Two Dilysine Binding Sites

1997 ◽  
Vol 8 (10) ◽  
pp. 1901-1910 ◽  
Author(s):  
Robert Tod Hudson ◽  
Rockford K. Draper
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Golgi Apparatus ◽  
Binding Sites ◽  
Aminoglycoside Antibiotics ◽  
Carboxyl Terminus ◽  
Amino Groups ◽  
Cell Extracts ◽  
Golgi Membranes ◽  
Lysine Residues

Coatomer is the soluble precursor of the COPI coat (coat protein I) involved in traffic among membranes of the endoplasmic reticulum and the Golgi apparatus. We report herein that neomycin precipitates coatomer from cell extracts and from purified coatomer preparations. Precipitation first increased and then decreased as the neomycin concentration increased, analogous to the precipitation of a polyvalent antigen by divalent antibodies. This suggested that neomycin cross-linked coatomer into large aggregates and implies that coatomer has two or more binding sites for neomycin. A variety of other aminoglycoside antibiotics precipitated coatomer, or if they did not precipitate, they interfered with the ability of neomycin to precipitate. Coatomer is known to interact with a motif (KKXX) containing adjacent lysine residues at the carboxyl terminus of the cytoplasmic domains of some membrane proteins resident in the endoplasmic reticulum. All of the antibiotics that interacted with coatomer contain at least two close amino groups, suggesting that the antibiotics might be interacting with the di-lysine binding site of coatomer. Consistent with this idea, di-lysine itself blocked the interaction of antibiotics with coatomer. Moreover, di-lysine and antibiotics each blocked the coating of Golgi membranes by coatomer. These data suggest that certain aminoglycoside antibiotics interact with di-lysine binding sites on coatomer and that coatomer contains at least two of these di-lysine binding sites.

Sign in / Sign up

Export Citation Format

Share Document