scholarly journals Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops.

1989 ◽  
Vol 108 (6) ◽  
pp. 2221-2231 ◽  
Author(s):  
E Baumgart ◽  
A Völkl ◽  
T Hashimoto ◽  
H D Fahimi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Normal Liver ◽  
Membrane System ◽  
Peroxisomal Membrane ◽  
Lowicryl K4m ◽  
Membrane Reservoir ◽  
Liver Peroxisomes

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

1991 ◽  
Vol 69 (8) ◽  
pp. 499-508 ◽  
Author(s):  
Andrea G. Bodnar ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunoblot Analysis ◽  
In Vitro Translation ◽  
Peroxisome Proliferator ◽  
Peroxisomal Membrane ◽  
Molecular Masses ◽  
Fold Induction ◽  
Membrane Bound ◽  
Liver Peroxisomes

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.

Peroxisomal membrane proteins are properly targeted to peroxisomes in the absence of COPI- and COPII-mediated vesicular transport

2001 ◽  
Vol 114 (11) ◽  
pp. 2199-2204 ◽  
Author(s):  
Tineke Voorn-Brouwer ◽  
Astrid Kragt ◽  
Henk F. Tabak ◽  
Ben Distel
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Human Fibroblasts ◽  
Vesicle Transport ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Classic Model ◽  
Early Secretory Pathway

The classic model for peroxisome biogenesis states that new peroxisomes arise by the fission of pre-existing ones and that peroxisomal matrix and membrane proteins are recruited directly from the cytosol. Recent studies challenge this model and suggest that some peroxisomal membrane proteins might traffic via the endoplasmic reticulum to peroxisomes. We have studied the trafficking in human fibroblasts of three peroxisomal membrane proteins, Pex2p, Pex3p and Pex16p, all of which have been suggested to transit the endoplasmic reticulum before arriving in peroxisomes. Here, we show that targeting of these peroxisomal membrane proteins is not affected by inhibitors of COPI and COPII that block vesicle transport in the early secretory pathway. Moreover, we have obtained no evidence for the presence of these peroxisomal membrane proteins in compartments other than peroxisomes and demonstrate that COPI and COPII inhibitors do not affect peroxisome morphology or integrity. Together, these data fail to provide any evidence for a role of the endoplasmic reticulum in peroxisome biogenesis.

scholarly journals Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver

1989 ◽  
Vol 259 (3) ◽  
pp. 659-663 ◽  
Author(s):  
F Vanstapel ◽  
L Hammaker ◽  
K Pua ◽  
N Blanckaert
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Membrane System ◽  
Permeability Barrier ◽  
Membrane Bound ◽  
Marker Studies ◽  
High Degree ◽  
Regulatory Properties

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.

scholarly journals The use of proteinases to determine the topological location of cytochrome P-450 in vesicles derived from smooth endoplasmic reticulum of rat liver

1981 ◽  
Vol 196 (2) ◽  
pp. 585-589 ◽  
Author(s):  
M B Cooper ◽  
M R Estall ◽  
B R Rabin
Keyword(s):  
Lipid Peroxidation ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Phosphatase Activity ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Transverse Plane ◽  
Phospholipid Bilayer ◽  
Cytochrome P 450

1. The phospholipid bilayer of intact vesicles from smooth endoplasmic reticulum is impermeable to macromolecules. Specific and non-specific proteinases were used to investigate the site of membrane proteins in the transverse plane of the bilayer. 2. When two proteinases were used in conjunction, denaturing effects additional to proteolysis were observed on cytochrome P-450 content and glucose 6-phosphatase activity which did not depend on the integrity of the phospholipid bilayer. 3. When lipid peroxidation was inhibited, these effects were not observed.

scholarly journals Mutants of the Yeast Yarrowia lipolyticaDefective in Protein Exit from the Endoplasmic Reticulum Are Also Defective in Peroxisome Biogenesis

1998 ◽  
Vol 18 (5) ◽  
pp. 2789-2803 ◽  
Author(s):  
Vladimir I. Titorenko ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Membrane Proteins ◽  
Matrix Proteins ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Mutant Cells

ABSTRACT Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A andsrp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A,srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that inY. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54,PEX1, and PEX6 genes.

scholarly journals Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

1975 ◽  
Vol 152 (2) ◽  
pp. 291-302 ◽  
Author(s):  
Richard Harwood ◽  
Michael E. Grant ◽  
David S. Jackson
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Subcellular Fractions ◽  
Anterior Lens Capsule ◽  
Cartilage Cells ◽  
Membrane Bound ◽  
And Cartilage

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [14C]lysine were assayed for hydroxy[14C]lysine and hydroxy[14C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[14C]lysine and glucosylgalactosylhydroxy[14C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [14C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. 14C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5′-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.

Les systèmes membranaires de Scopulariopsis brevicaulis. II. Étude comparée de leur composition lipidique

1987 ◽  
Vol 33 (1) ◽  
pp. 78-81 ◽  
Author(s):  
Jean-Louis Fonvieille ◽  
Jane-Marie Touze-Soulet ◽  
Charles Montant
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Percoll Gradient ◽  
Mitochondrial Membranes ◽  
Isolation Method ◽  
Discontinuous Sucrose Gradient ◽  
Phospholipid Ratio ◽  
Scopulariopsis Brevicaulis

Biochemical studies of Scopulariopsis brevicaulis membranes (except mitochondrial membranes), in particular their lipid composition, were undertaken using plasma membranes and fractions containing smooth endoplasmic reticulum and Golgi apparatus membranes. The results obtained by two separation techniques, a continuous Percoll gradient and a discontinuous sucrose gradient centrifugation, were similar. Depending on the isolation method used, the plasma membrane contained 30 or 33% protein and 23 or 30% lipid of which 16.5 or 18% was phospholipid. The sterol/phospholipid ratio was 1.98 or 2.28. The endoplasmic reticulum + Golgi fraction contained 21 or 26.7% protein and 43 or 50% lipid of which 7 or 10% was phospholipid. The sterol/phospholipid ratio was 0.85 or 0.81.

Organization of intracellular calcium regulatory proteins in the neuronal endoplasmic reticulum

1994 ◽  
Vol 52 ◽  
pp. 26-27
Author(s):  
Maryann E. Martone ◽  
Victoria M. Edelman ◽  
Saul A. Alba ◽  
Thomas J. Deerinck ◽  
Mark H. Ellisman
Keyword(s):  
Endoplasmic Reticulum ◽  
Intracellular Calcium ◽  
Dendritic Spines ◽  
Calcium Binding ◽  
Smooth Endoplasmic Reticulum ◽  
Membrane System ◽  
Regulatory Proteins ◽  
Purkinje Neurons ◽  
Storage Site ◽  
Ip3 Receptor

The smooth endoplasmic reticulum (SER) has been established as an intracellular calcium storage site in neurons. Although the SER appears to form a continuous membrane system within neurons, immunolocalization studies suggest that calcium regulatory proteins are not evenly distributed within the SER but are selectively concentrated or excluded from certain domains. The subcompartmenalization of the SER has been clearly demonstrated in Purkinje neurons where two proteins involved in the release of calcium from intracellular stores, the IP3 and ryanodine receptor, were differentially localized within dendrites. Both proteins were found associated with the SER in cell bodies and dendrites of chick Purkinje neurons but only labeling for the IP3 receptor was found within dendritic spines. A similar differential localization was described in Purkinje cell dendrites for the SER Ca++ATPase and calsequestrin, a lumenal calcium binding protein. The Ca++ATPase was found throughout dendrites and dendritic spines while calsequestrin was restricted to membranous profiles within the dendritic shaft.

scholarly journals STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN LIVERS OF PHENOBARBITAL-TREATED RATS

1972 ◽  
Vol 55 (2) ◽  
pp. 282-298 ◽  
Author(s):  
Joan A. Higgins ◽  
Russell J. Barrnett
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Normal Liver ◽  
Subsequent Treatment ◽  
Cell Fractionation ◽  
Acyltransferase Activity ◽  
Normal Rat

The localization of acyltransferases involved in acylation of α-glycerophosphate, during phenobarbital induced proliferation of smooth endoplasmic reticulum (ser) membranes, has been investigated using cytochemical and cell fractionation techniques. In cytochemical studies of normal rat liver, reaction product marking acyltransferase activity was associated to the greatest extent with the rough endoplasmic reticulum (rer) membranes and to a lesser extent with ser membranes. In liver from phenobarbital-treated rats, reaction product was largely restricted to ser membranes. The specific activity of the acyltransferases of rough microsomes from normal rat liver was higher than that of the smooth microsomes. On injection of phenobarbital, this fell rapidly after three injections to a low level, at which it remained during subsequent treatment. The specific activity of the smooth microsomes, on injection of phenobarbital, rose to a peak 12 hr after the first injection, after which it fell to a level at an activity above that of smooth microsomes of normal liver. A mechanism is postulated for the biogenesis of smooth membranes in which the phospholipid is synthesized in situ and the protein is synthesized in the rer and moves to the site of newly synthesized phospholipid, where it is inserted to produce a whole membrane.

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