Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver

To establish on a quantitative basis the subcellular distribution of the enzymes that glycosylate dolichyl phosphate in rat liver, preliminary kinetic studies on the transfer of mannose, glucose, and N-acetylglucosamine-1-phosphate from the respective (14)C- labeled nucleotide sugars to exogenous dolichyl phosphate were conducted in liver microsomes. Mannosyltransferase, glucosyltransferase, and, to a lesser extent, N- acetylglucosamine-phosphotransferase were found to be very unstable at 37 degrees C in the presence of Triton X-100, which was nevertheless required to disperse the membranes and the lipid acceptor in the aqueous reaction medium. The enzymes became fairly stable in the range of 10-17 degrees C and the reactions then proceeded at a constant velocity for at least 15 min. Conditions under which the reaction products are formed in amount proportional to that of microsomes added are described. For N- acetylglucosaminephosphotransferase it was necessary to supplement the incubation medium with microsomal lipids. Subsequently, liver homogenates were fractionated by differential centrifugation, and the microsome fraction, which contained the bulk of the enzymes glycosylating dolichyl phosphate, was analyzed by isopycnic centrifugation in a sucrose gradient without any previous treatment, or after addition of digitonin. The centrifugation behavior of these enzymes was compared to that of a number of reference enzymes for the endoplasmic reticulum, the golgi complex, the plasma membranes, and mitochondria. It was very simily to that of enzymes of the endoplasmic reticulum, especially glucose-6-phosphatase. Subcellular preparations enriched in golgi complex elements, plasma membranes, outer membranes of mitochondira, or mitoplasts showed for the transferases acting on dolichyl phosphate relative activities similar to that of glucose- 6-phosphatase. It is concluded that glycosylations of dolichyl phosphate into mannose, glucose, and N-acetylglucosamine-1-phosphate derivatives is restricted to the endoplasmic reticulum in liver cells, and that the enzymes involved are similarly active in the smooth and in the rough elements

Réticulum endoplasmique : anatomie d'une membrane biologique

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Subcellular topology of rat liver methionyl-, leucyl-, and arginyl-tRNA synthetases.

We have investigated the distribution of methionyl-, leucyl-, and arginyl- tRNA synthetases in primary liver fractions obtained by differential centrifugation of homogenates in isotonic sucrose: 78-93% of synthetase activities are recovered in the cytosolic fraction. Microsomes contain only 4.8%, 19.4%, and 6.4% of the methionyl-, leucyl-, and arginyl-tRNA synthetases activities, respectively. This proportion increases up to 11.3%, 26.1%, and 20.7%, respectively, when the homogenization medium is supplemented with 5 mM Mg2+ and 25 mM K+. The presence of protease inhibitors in the homogenization medium does not increase the proportion of synthetases recovered in microsomes. After subfractionation of microsomes by isopycnic centrifugation, the distributions of the 3 synthetases display a second peak overlapping that of at a density of 1.12. In addition, methionyl- and leucyl- tRNA synthetases display a second peak overlapping that of RNA. This suggests that a small proportion of these synthetases (0.7% and 5.71% of total activities, respectively) bind to the d domain of the ER. The Golgi complex, the plasma membranes, and the peroxisomes lack aminoacyl-tRNA synthetase activity. The 3 synthetases are readily detached from membranes when intact microsomes are washed with 250 mM sucrose alone or containing 5 mM PPi, or 320 mM KCl. The binding of methionyl-tRNA synthetases to microsomes was measured in vitro, at 4 degrees C, with a sample of the cytosolic fraction as a source of synthetase. Microsomes stripped of their bound polysomes display a binding capacity that is not significantly different from that of unstripped microsomes. Even in the presence of cations, the amount of synthetase bound to the membranes remained low by comparison with the cytosolic content

Translocation and proteolytic processing of nascent secretory polypeptide chains: two functions associated with the ribosomal domain of the endoplasmic reticulum.

Rat liver microsomes were subfractionated by isopycnic centrifugation in sucrose gradient. The subfractions were assayed for translocation and proteolytic processing of nascent polypeptides in a rabbit reticulocyte lysate programmed with total RNA from human term placenta. The distribution of the translocation and processing of prelactogen through the gradient correlated with that of the microsomal RNA (ribosomes). Microsomes became inactive upon incubation with elastase, but the proteolyzed membranes recovered their activity by recombination with the soluble and active fragment of the docking protein (SRP-receptor) from dog pancreas. When this fragment was combined with the gradient subfractions, or with the subfractions inactivated by incubation with elastase, the density profile of the translocation activity remained similar to that of RNA. Thus, its distribution cannot be accounted for merely by that of the docking protein; another membrane constituent, still unidentified, is both necessary for translocation of polypeptides and restricted to the rough portions of the endosplamic reticulum. Signal peptidase was assayed in the absence of protein synthesis, by use of preformed prelactogen and detergent-disrupted microsomes. Its density distribution was also similar to that of RNA. Several components of the endosplamic reticulum now appear to be segregated within restricted areas on either side of the membrane, and to make up a biochemically distinct domain. We propose to call it the ribosomal domain in consideration of its contribution to protein biosynthesis by bound ribosomes. This domain probably accounts for a greater part of the membrane area at the cytoplasmic than at the luminal surface, as postulated earlier to explain how enzymes of the cytoplasmic surface are relatively less abundant in the rough microsomes than those of the luminal surface [Amar-Costesec A. & Beaufay H. (1981) J. Theor. Biol. 89, 217-230]

A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum.

We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane-bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na-pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6-phosphatase with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also contains the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co- and post-translationally the signal peptide of pre-secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain