Fractionation of rat liver proteins involved in translocation of newly synthesized proteins at the endoplasmic reticulum membrane (original) (raw)
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The Journal of cell biology, 1984
A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation sys...
Free and membrane-bound ribosomes in rat liver
The Journal of biological chemistry, 1967
Ribosomes in rat liver are present either free or attached to endoplasmic reticulum and in both states exist largely as polyribosomal aggregates. The present communication presents a quantitative study of the distribution of ribosomes between these two states and examines a number of properties of these two groups of ribosomes. It is shown that the base composition and the rate of synthesis of the ribosomal ribonucleic acid of free and membrane-bound ribosomes are the same, and these tindings, along with the results of previous studies of other properties, are consistent with the possibility that there are no intrinsic differences between the ribosomes found in these two states. Since certain conditions are known to produce alterations in the distribution of free and membrane-bound ribosomes which are rapid with respect to the turnover time of ribosomal RNA, it is further suggested that a given ribosome can exist in either state according to the needs of the cell. Although the administration of hydrocortisone in uivo induces acute rises in the levels of certain specific liver enzymes within a few hours, it has no marked effect upon the ratio of free to membrane-bound ribosomes during this period. The ribosomal aggregates obtained by the methods used here are shown to be sufficiently pure to permit the use of ultraviolet absorption as a direct measure of their concentration without applying a correction for ferritin absorption. Homogenization and zone centrifugation do not produce significant artifacts in the determination of the proportions of free and membrane-bound ribosomes. In a previous communication it was shown that the great majority of ribosomes in rat liver are attached to large membranous structures (1). Data were presented which showed that ribosomes in all fractions of liver homogenates are active in protein synthesis in tivo and in vitro, and it was further shown
Molecular Biology Reports, 1975
The separation of rat liver endoplasmic reticulum membrane proteins by two dimensional polyacrylamide gel electrophoresis is described. By this method, the proteins of the rough membrane ribosomes could be separated from the other rough membrane proteins. Both rough and smooth membrane fractions contain at least 30 defined membranal proteins. The electrophoretic patterns of rough and smooth membrane proteins are clearly different. I. iNTRODUCTION Electron microscopic pictures of liver cells clearly reveal the existence of an interlacing network of membranes, some of them studded with dense ribosomal particles (the rough membrane), and some lacking ribosomes (the smooth membrane) [ 1, 2]. Smooth and rough membranes can be separated in vitro by discontinuous sucrose gradient centrifugation, and then studied separately [3, 4]. The relationship between the rough and the smooth membrane fractions has been investigated but not completely clarified [5, 6]. Erikson studied the chemical composition and the enzymatic activities of the smooth and rough membrane fractions, and clearly showed that they differ both in their chemical composition and pattern of enzymatic activities [7]. The presence of structural proteins in the microsomal fraction was claimed by Criddle et al. [8], and Ward and Pollack [9]. The protein composition of rough and smooth membranes was investigated by SDS-polyacrylamide gel electrophoresis by Schnaitman, who found that the two microsomal membrane fractions contained a number of similar proteins, however similar proteins were not present in the same amounts in both microsomal membrane fractions, and some proteins were observed that were unique to each fraction [ 10]. Burka and Bulova reported on the difference in the ribosomal proteins of membrane-bound and free ribosomes [ 11 ], difference which could have been due to contamination of the bound ribosomes by membrane proteins [ 12]. Hanna et al. reported that free and membrane-bound ribosomes released by deoxycholate treatment have identical protein composition [ 13]. We decided to compare the protein composition of polyribosomes, smooth and rough membrane fractions by two dimensional polyacrylamide gel electrophoresis, in order to gain more information on the protein pattern of these different fractions.
Compartmentation of the rough endoplasmic reticulum
Molecular and Cellular Biochemistry, 1986
It has become evident during recent years that a wide variety of proteins are synthesized on membranebound polysomes, very many of which are not ultimately secreted from the cell. The majority of proteins appear to go through some form of post-translational modification before the final appearance of an 'active' product, and in some cases the polypeptide chain may be modified before the completed protein molecule is released from the ribosome. This then raises the question concerning the possibility of the organization of the rough endoplasmic reticulum into individual domains, or compartments, each of which may have the responsibility of performing definite and well defined functions. During recent years the behaviour of two subfractions of the rough endoplasmic reticulum in a variety of cell types and under a variety of conditions has been studied in order to gain insight into a possible compartmentation of this organelle.
Characterization of Ribosomal Aggregates Isolated From Liver
Proceedings of the National Academy of Sciences, 1964
Polyribosomal aggregates have been described in mammalian cells, bacteria, and plants.1-6 These aggregates are thought to consist of a linear array of ribosomes attached to a strand of messenger RNA and appear to be the units responsible for protein synthesis.
Journal of Lipid Research, 1999
We sought a rapid and non-ultracentrifugal method of recovering large amounts of highly pure rough endoplasmic reticulum (RER) membranes from livers. By substantially modifying a 20-year-old calcium precipitation technique, we obtained a RER fraction from rat liver and established its high degree of purity by quantitating classic membrane markers for different subcellular organelles. This RER fraction is highly enriched in four known proteins (or enzyme activities) required for lipoprotein assembly: apolipoprotein B, microsomal triglyceride transfer protein, acyl CoA:diacylglycerol acyltransferase, and acyl CoA:cholesterol acyltransferase, when compared to two classical RER markers, RNA and glucose-6-phosphatase. From one 10-12 g rat liver, we recover ten to twelve RER pellets of 1.5-1.6 cm in diameter containing ϳ 110-125 mg of total protein, about half of which is sodium carbonate-releasable. By electron microscopy, these large RER pellets from rat livers are homogeneously comprised largely of non-vesiculated short strips of ribosome-rich membranes. This novel technique for isolating RER membranes from liver may provide a useful tool for future studies on the assembly of apolipoprotein B-containing lipoproteins as well as for research focused on mechanisms of secretory and membrane protein translation, translocation, and folding.