The in vitro reassembly of rough endoplasmic reticulum: Ribosome binding capacity

A system for study and measurement of the attachment in vitro of exogenous polyribosomes to membranes has been presented. Its main features are use of low temperature, post-microsomal supernatant, pyrophosphate and citric acid to remove ribosomes from the surface of rough endoplasmic reticulum, and a method for quantitative separation of unattached from membrane-associated polyribosomes. The following were found. (1) Rough endoplasmic reticulum, from which ribosomes had been removed by treatment with pyrophosphate and citrate, bound over 50% of added polyribosomes, whereas the untreated (or control) rough and smooth endoplasmic reticulum and the smooth endoplasmic reticulum treated with pyrophosphate–citrate did not bind polyribosomes. (2) The polyribosome-binding capacity of rough endoplasmic reticulum stripped of its ribosomes decayed upon storage of the membranes at 0–4°C. The half-life of this decay was about 6 days whereas that of the polyribosome-binding capacity of hepatoma stripped rough endoplasmic reticulum was about 1.5 days. (3) Preparations of stripped rough endoplasmic reticulum after reassociation with polyribosomes in vitro were quite similar to preparations of native rough endoplasmic reticulum as viewed with the electron microscope. Evidence is presented to support the contention that association of polyribosomes with membranes was the result of polyribosomal reattachment to the membranes rather than trapping of the polyribosomes between vesicles of the membranes.

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RADIOAUTOGRAPHIC VISUALIZATION OF THE INCORPORATION OF GALACTOSE-3H AND MANNOSE-3H BY RAT THYROIDS IN VITRO IN RELATION TO THE STAGES OF THYROGLOBULIN SYNTHESIS

The Journal of Cell Biology ◽

10.1083/jcb.43.2.289 ◽

1969 ◽

Vol 43 (2) ◽

pp. 289-311 ◽

Cited By ~ 192

Author(s):

P. Whur ◽

Annette Herscovics ◽

C. P. Leblond

Keyword(s):

Endoplasmic Reticulum ◽

Electron Microscope ◽

Golgi Apparatus ◽

Rough Endoplasmic Reticulum ◽

Light Microscope ◽

Detectable Effect ◽

Apical Vesicles ◽

Rat Thyroid

Rat thyroid lobes incubated with mannose-3H, galactose-3H, or leucine-3H, were studied by radioautography. With leucine-3H and mannose-3H, the grain reaction observed in the light microscope is distributed diffusely over the cells at 5 min, with no reaction over the colloid. Later, the grains are concentrated towards the apex, and colloid reactions begin to appear by 2 hr. With galactose-3H, the reaction at 5 min is again restricted to the cells but it consists of clumped grains next to the nucleus. Soon after, grains are concentrated at the cell apex and colloid reactions appear in some follicles as early as 30 min. Puromycin almost totally inhibits incorporation of leucine-3H and mannose-3H, but has no detectable effect on galactose-3H incorporation during the 1st hr. Quantitation of electron microscope radioautographs shows that mannose-3H label localizes initially in the rough endoplasmic reticulum, and by 1–2 hr much of this reaction is transferred to the Golgi apparatus. At 3 hr and subsequently, significant reactions are present over apical vesicles and colloid, while the Golgi reaction declines. Label associated with galactose-3H localizes initially in the Golgi apparatus and rapidly transfers to the apical vesicles, and then to the colloid. These findings indicate that mannose incorporation into thyroglobulin precursors occurs within the rough endoplasmic reticulum; these precursors then migrate to the Golgi apparatus, where galactose incorporation takes place. The glycoprotein thus formed migrates via the apical vesicles to the colloid.

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The biosynthesis of cytochrome P450 by rough endoplasmic reticulum in vitro

FEBS Letters ◽

10.1016/0014-5793(79)80227-6 ◽

1979 ◽

Vol 98 (2) ◽

pp. 403-407 ◽

Cited By ~ 6

Author(s):

John A. Craft ◽

Michael B. Cooper ◽

Margaret R. Estall ◽

Brian R. Rabin

Keyword(s):

Endoplasmic Reticulum ◽

Cytochrome P450 ◽

Rough Endoplasmic Reticulum

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THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN

The Journal of Cell Biology ◽

10.1083/jcb.52.2.231 ◽

1972 ◽

Vol 52 (2) ◽

pp. 231-245 ◽

Cited By ~ 102

Author(s):

Colvin M. Redman ◽

M. George Cherian

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Rough Endoplasmic Reticulum ◽

Serum Proteins ◽

Rat Serum ◽

Secretory Pathways ◽

Specific Antisera ◽

Microsome Fraction

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the 14C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.

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DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES

The Journal of Cell Biology ◽

10.1083/jcb.49.2.288 ◽

1971 ◽

Vol 49 (2) ◽

pp. 288-302 ◽

Cited By ~ 92

Author(s):

A. Leskes ◽

P. Siekevitz ◽

G. E. Palade

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Enzyme Reaction ◽

Regional Differentiation ◽

Lead Phosphate ◽

Cytochemical Localization ◽

Actual Distribution ◽

A Cell ◽

Microsome Fraction

Electron microscope cytochemical localization of glucose-6-phosphatase in the developing hepatocytes of fetal and newborn rats indicates that the enzyme appears simultaneously in all the rough endoplasmic reticulum of a cell, although asynchronously within the hepatocyte population as a whole. To confirm that the pattern of cytochemical deposits reflects the actual distribution of enzyme sites, a method to subfractionate rough endoplasmic reticulum was developed. The procedure is based on the retention of the cytochemical reaction product (precipitated lead phosphate) within freshly prepared rough microsomes reacted in vitro with glucose-6-phosphate and lead ions. Lead phosphate increases the density of the microsomes which have glucose-6-phosphatase activity and thereby makes possible their separation from microsomes lacking the enzyme; separation is obtained by isopycnic centrifugation on a two-step density gradient. The procedure was applied to rough microsomes isolated from rats at several stages during hepatocyte differentiation and the results obtained agree with those given by cytochemical studies in situ. Before birth, when only some of the cells react positively for glucose-6-phosphatase, only a commensurate proportion of the rough microsome fraction can be rendered dense by the enzyme reaction. At the time of birth and in the adult, when all cells react positively, practically all microsomes acquire deposit and become dense after reaction. Thus, the results of the microsome subfractionation confirm the cytochemical findings; the enzyme is evenly distributed throughout all the endoplasmic reticulum of a cell and there is no regional differentiation within the rough endoplasmic reticulum with respect to glucose-6-phosphatase. These findings suggest that new components are inserted molecule-by-molecule into a pre-existing structural framework. The membranes are thus mosaics of old and new molecules and do not contain large regions of entirely "new" membrane in which all of the components are newly synthesized or newly assembled.

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The beads in the Golgi complex-endoplasmic reticulum region.

The Journal of Cell Biology ◽

10.1083/jcb.70.2.384 ◽

1976 ◽

Vol 70 (2) ◽

pp. 384-394 ◽

Cited By ~ 37

Author(s):

M Locke ◽

P Huie

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Golgi Complex ◽

Insect Cell ◽

Cell Types ◽

Feulgen Staining ◽

Clear Halo ◽

Bismuth Salts ◽

Transition Vesicles

The region between the rough endoplasmic reticulum (ER) and the Golgi complex has been studied in a variety of insect cell types in an attempt to find a marker for the exit gate or gates from the ER. We have found that the smooth surface of the rough endoplasmic reticulum near Golgi complex transitional elements has beadlike structures arranged in rings at the base of transition vesicles. They occur in all insect cell types and a variety of other organisms. The beads can be seen only after staining in bismuth salts. They are 10-12 nm in diameter and are separated from the membrane and one another by a clear halo giving them a center to center spacing of about 27 nm. The beads are not sensitive to nucleases under conditions which disrupt ribosomes or remove all Feulgen staining material from the nucleus. Under conditions similar to those used to stain tissue, bismuth does not react in vitro with nucleic acids. The component of the beads that stains preferentially with bismuth is therefore probably not nucleic acid.

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The biosynthesis of Trypanosoma brucei variant surface glycoproteins — in vitro processing of signal peptide and glycosylation using heterologous rough endoplasmic reticulum vesicles

Molecular and Biochemical Parasitology ◽

10.1016/0166-6851(82)90075-5 ◽

1982 ◽

Vol 6 (3) ◽

pp. 161-174 ◽

Cited By ~ 16

Author(s):

Josie McConnell ◽

John S. Cordingley ◽

Mervyn J. Turner

Keyword(s):

Endoplasmic Reticulum ◽

Trypanosoma Brucei ◽

Rough Endoplasmic Reticulum ◽

Signal Peptide ◽

In Vitro Processing ◽

Variant Surface Glycoproteins ◽

Surface Glycoproteins

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Ultrastructure of mononuclear phagocytes developing in liquid bone marrow cultures. A study on peroxidatic activity

Journal of Experimental Medicine ◽

10.1084/jem.149.1.17 ◽

1979 ◽

Vol 149 (1) ◽

pp. 17-26 ◽

Cited By ~ 46

Author(s):

JWM Van Der Meer ◽

RHJ Beelen ◽

DM Fluitsma ◽

R Van Furth

Keyword(s):

Bone Marrow ◽

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Nuclear Envelope ◽

Rough Endoplasmic Reticulum ◽

Precursor Cells ◽

Mononuclear Phagocytes ◽

Peroxidatic Activity

Monoblasts, promonocytes, and macrophages in in vitro cultures of murine bone marrow were studied ultrastructurally, with special attention to peroxidatic activity. Monoblasts show peroxidatic activity in the rough endoplasmic reticulum and nuclear envelope as well as in the granules. The presence of peroxidatic activity in the Golgi apparatus could not be determined. Promonocytes have peroxidase-positive rough endoplasmic reticulum, Golgi apparatus, nuclear envelope, and granules, as previously reported. During culture, cells are formed with peroxidatic activity similar to that of monocytes or exudate macrophages (positive granules; negative Golgi apparatus, RER, and nuclear envelope); we call these cells early macrophages. In addition, transitional macrophages with both positive granules and positive RER, nuclear envelope, negative Golgi apparatus (as in exudate- resident macrophages in vivo), and mature macrophages with peroxidatic activity only in the RER and nuclear envelope (as in resident macrophages in vivo) were found. A considerable number of cells without detectable peroxidatic activity were also encountered. Our finding that macrophages with the peroxidatic pattern of monocytes (early macrophages), exudate-resident macrophages (transitional macrophages), and resident macrophages (mature macrophages), develop in vitro from proliferating precursor cells deriving from the bone marrow, demonstrates once again that resident macrophages in tissues originate from precursor cells in the bone marrow. Therefore, this conclusion can no longer be challenged on the basis of a cytochemical difference between monocytes and exudate macrophages on the one hand and resident macrophages on the other.

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