scholarly journals Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

1985 ◽  
Vol 101 (5) ◽  
pp. 1733-1740 ◽  
Author(s):  
A Yamamoto ◽  
R Masaki ◽  
Y Tashiro
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Subcellular Fraction ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Cytochrome P 450 ◽  
Microscopic Techniques ◽  
Lysosomal Proteins

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P-450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.

scholarly journals Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes.

1978 ◽  
Vol 78 (2) ◽  
pp. 503-519 ◽  
Author(s):  
S Matsuura ◽  
Y Fujii-Kuriyama ◽  
Y Tashiro
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Control Level ◽  
Rat Hepatocytes ◽  
Present Observation ◽  
Membrane Structures ◽  
Rat Liver Cells ◽  
Microsomal Membranes ◽  
Almost All ◽  
Cytochrome P 450

Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.

Synthesis and Turnover of Membrane Proteins in Rat Liver: An Examination of the Membrane Flow Hypothesis

1971 ◽  
Vol 26 (10) ◽  
pp. 1031-1039 ◽  
Author(s):  
Werner W. Franke ◽  
D. James Morre ◽  
Barbara Deumling ◽  
Ronald D. Cheetham ◽  
Jürgen Kartenbeck ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Plasma Membranes ◽  
Degradation Kinetics ◽  
Specific Radioactivity ◽  
Membrane Flow ◽  
Rapid Turnover ◽  
Associated Proteins

The kinetics of synthesis and degradation of the protein constituents of nuclear membranes, endoplasmic reticulum membranes (rough-surfaced microsomes), Golgi apparatus membranes and plasma membranes were determined following a single administration of L- [guanido-14C] arginine by intraperitoneal injection. Membrane protein was determined as the fraction which resists sonication and sequential extrations with 1.5 M KCl, 0.1% deoxycholate and water to remove intravesicular, intracisternal (secretory), nucleo-, adsorbed and ribosome-associated proteins.The order of maximum labeling of membrane proteins was a) endoplasmic reticulum (nuclear membrane), b) Golgi apparatus, and c) plasma membrane. Rapid decreases in specific radioactivity followed maximal labeling of endoplasmic reticulum and Golgi apparatus membranes. These rapid turnover components of endoplasmic reticulum and Golgi apparatus were sufficient to account for labeling of plasma membranes via a flow mechanism.Incorporation of radioactivity into plasma membranes showed two distinct phases. The ultrastructural features underlying the biphasic pattern of incorporation into plasma membranes are discussed.Following initial incorporation and rapid turnover, membrane proteins were characterized by degradation kinetics approximating 1st order. Rates of degradation for Golgi apparatus and plasma membranes were faster than those for nuclear envelope and endoplasmic reticulum membranes.Assuming steady state conditions, an absolute synthetic rate of 7.1 mpg/min/avergage hepatocyte was calculated for membrane proteins of the plasma membrane.The results are compatible with intracellular movement and conversion of rough endoplasmic reticulum to plasma membrane via the membranes of the Golgi apparatus, i. e., membrane flow. Additionally, the kinetics indicate that membrane synthesis and transfer is restricted to specific parts of the endoplasmic reticulum and Golgi apparatus.

Immunoelectron-microscopic studies of endoplasmic reticulum-Golgi relationships in the intracellular transport process of lipoprotein particles in rat hepatocytes

1979 ◽  
Vol 39 (1) ◽  
pp. 273-290 ◽  
Author(s):  
S. Matsuura ◽  
Y. Tashiro
Keyword(s):  
Endoplasmic Reticulum ◽  
Transport Process ◽  
Intracellular Transport ◽  
Control Level ◽  
Rat Hepatocytes ◽  
Secretory Proteins ◽  
Marker Enzyme ◽  
Lipoprotein Particles ◽  
Antibody Conjugates ◽  
Cytochrome P 450

Endoplasmic reticulum (ER)-Golgi relationships in the intracellular transport process of secretory proteins in rat hepatocytes have been studied using lipoprotein particles as a marker for the secretory protein and cytochrome P-450 as a marker enzyme for the ER membranes. Ferritin immunoelectron-microscopic observation revealed that, while almost all the microsomal vesicles derived from ER membranes are heavily labelled with ferritin anti-cytochrome P-450 antibody conjugates, labelling of the small peripheral vesicles containing lipoprotein particles, the stacks of Golgi saccules, especially the outermost saccule which is sometimes fenestrated, condensing vacuoles in the trans-Golgi region and the secretion droplets of lipoprotein were scanty and at the control level. Such a characteristic pattern of labelling was especially evident when these structures were prepared from phenobarbital-treated rats. These findings indicate that the membranes of the small peripheral vesicles do not contain cytochrome P-450 and that the cytochrome is probably not transferred to Golgi saccules in the transport process of lipoprotein from ER to Golgi. It is suggested, therefore, that the small peripheral vesicles are formed by budding of the special regions of ER membrane where microsomal marker proteins such as cytochrome P-450 are excluded and the membrane proteins destined to the Golgi complexes are clustered. It is also shown that lysosomal membranes are not labelled with the anti P-450 antibody conjugates.

scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

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.

The Occurrence of Phenylalanine Ammonia-Lyase and Cinnamic Acid p-Hydroxylase on the Endoplasmic Reticulum of Cell Suspension Cultures of Glycine max

1978 ◽  
Vol 33 (1-2) ◽  
pp. 65-69 ◽  
Author(s):  
C. Postius ◽  
H. Kindi
Keyword(s):  
Endoplasmic Reticulum ◽  
Glycine Max ◽  
Golgi Apparatus ◽  
Cinnamic Acid ◽  
Time Course ◽  
Phenylalanine Ammonia Lyase ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Activity Increase ◽  
Membrane Bound

Abstract 1. The time course of activity of soluble and microsomal phenylalanine ammonia-lyase (PAL) was studied in dark grown cell cultures of soybean (Glycine max). A distinct activity increase of PAL in the soluble and microsomal fraction occurred prior to the stationary phase of the cell culture. Cinnamic acid p-hydroxylase and NADH : cytochrome c reductase, too, exhibited maximal activity in the log phase, 5 days after the transfer of soybean cells to fresh culture medium.2. Upon subfractionation of the once washed microsomal fraction by sedimentation velocity centrifugation on a sucrose gradient, membranes of the endoplasmic reticulum could be separated from fractions containing mainly membranes from the Golgi apparatus or plasma membranes, respectively. PAL and cinnamic acid p-hydroxylase were found in fractions of endoplasmic reticulum whereas no activity of either enzymes could be detected in fractions containing Golgi apparatus or plasma membranes.3. Repeated washing of microsomal fractions led to a residual membrane-bound PAL representing about 1% of the total PAL activity of the cells. This residual membrane-bound activity could be solubilized almost completely by Triton X-100 or digitonin at concentrations of 0.5 - 5%.

scholarly journals The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes

1986 ◽  
Vol 237 (1) ◽  
pp. 33-39 ◽  
Author(s):  
E Fries ◽  
I Lindström
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Low Temperatures ◽  
Intracellular Transport ◽  
Rat Hepatocytes ◽  
Subcellular Fractionation ◽  
Isolated Rat Hepatocytes ◽  
Different Temperatures ◽  
Lag Period ◽  
Time Courses

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.

scholarly journals Immunoisolaton of the Yeast Golgi Subcompartments and Characterization of a Novel Membrane Protein, Svp26, Discovered in the Sed5-Containing Compartments

2005 ◽  
Vol 25 (17) ◽  
pp. 7696-7710 ◽  
Author(s):  
Hironori Inadome ◽  
Yoichi Noda ◽  
Hiroyuki Adachi ◽  
Koji Yoda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Membrane Protein ◽  
Considerable Portion ◽  
Marker Proteins ◽  
Evolutionarily Conserved ◽  
Golgi Compartment

ABSTRACT The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.

scholarly journals CORRELATED MORPHOMETRIC AND BIOCHEMICAL STUDIES ON THE LIVER CELL

1969 ◽  
Vol 42 (1) ◽  
pp. 92-112 ◽  
Author(s):  
Willy Stäubli ◽  
Robert Hess ◽  
Ewald R. Weibel
Keyword(s):  
Endoplasmic Reticulum ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Rat Hepatocytes ◽  
Dense Bodies ◽  
Biochemical Studies ◽  
Ploidy Pattern ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Specific Number ◽  
Cytochrome P 450

The changes occurring in rat hepatocytes during a 5 day period of treatment with phenobarbital were determined by morphometric and biochemical methods, particular attention being paid to the endoplasmic reticulum. The hepatocytic cytoplasm played an overwhelming part in the liver hypertrophy, while the hepatocytic nuclei contributed to only a moderate extent. The endoplasmic reticulum accounted for more than half of the increase in cytoplasmic volume. The increase in the volume and number of hepatocytic nuclei in the course of phenobarbital treatment was associated with changes in the ploidy pattern. Until the 2nd day of treatment both the rough-surfaced endoplasmic reticulum (RER) and the smooth-surfaced endoplasmic reticulum (SER) participated in the increase in volume and surface of the whole endoplasmic reticulum (ER). Subsequently, the values for RER fell again to control levels, whereas those for SER continued to increase, with the result that by the 5th day of treatment the SER constituted the dominant cytoplasmic element. The specific volume of mitochondria and microbodies (peroxisomes) remained constant throughout the duration of the experiment, while that of the dense bodies increased. The specific number of mitochondria and microbodies displayed a significant increase, associated with a decrease in their mean volume. The phenobarbital-induced increase in the phospholipid and cytochrome P-450 content of the microsomes, as well as in the activities of microsomal reduced nicotinamide-adenine dinucleotide phosphate-cytochrome c reductase and N-demethylase, was correlated with the morphometric data on the endoplasmic reticulum.

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