Rat Liver Rough Endoplasmic Reticulum Research Articles

Characterization of the ATP transporter in the reconstituted rough endoplasmic reticulum proteoliposomes

Adenosine triphosphate (ATP) transporter from rat liver rough endoplasmic reticulum (RER) was solubilized and reconstituted into phosphatidylcholine liposomes. The RER proteoliposomes, resulting from optimizing some reconstitution parameters, had an apparent K m value of 1.5 μM and a V max of 286 pmol min −1 (mg protein) −1 and showed higher affinity for ATP and a lower V max value than intact RER ( K m of 6.5 μM and V max of 1 nmol). ATP transport was time- and temperature-dependent, inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, which is known as an inhibitor of anion transporters including ATP transporter, but was not affected by atractyloside, a specific inhibitor of mitochondrial ADP/ATP carrier. The internal and external effects of various nucleotides on the ATP transport were examined. ATP transport was cis-inhibited strongly by ADP and weakly by AMP. ADP-preloaded RER proteoliposomes showed a specific increase of ATP transport activity while AMP-preloaded RER proteoliposomes did not show the enhanced overshoot peak in the ATP uptake plot. These results demonstrate the ADP/ATP antiport mechanism of ATP transport in rat liver RER.

Identification of the ATP transporter of rat liver rough endoplasmic reticulum via photoaffinity labeling and partial purification.

In order to identify the ATP transporter in rat liver rough endoplasmic reticulum (RER), a photoreactive azido derivative of ATP, 3'-O-(p-azidobenzoyl)-ATP (AB-ATP), was synthesized by the reaction of ATP with N-hydroxysuccinimido 4-azidobenzoate (NHS-AB). The activity of the ATP transporter was determined by measuring the influx of [8-14C]ATP. The ATP transport had an apparent Km value of 6.5 microM and a Vmax of 1 nmol min-1 (mg of protein)-1. The transport of ATP was specifically inhibited by AB-ATP and 4, 4'-diisothiocyanatostilbene-2', 2'-disulfonic acid (DIDS). Under a dim light, AB-ATP was a competitive inhibitor of the ATP transport with Ki value of 0.19 microM, which indicates that AB-ATP has a high affinity for the ATP transporter, so it can be utilized as a photoaffinity probe for the identification of the ATP transporter in rat liver RER. An SDS--PAGE analysis of RER vesicles photolabeled with [gamma-32P]AB-ATP indicates the presence of a 56-kDa protein. The 56-kDa protein was completely protected from photoaffinity labeling by 10 microM ATP but not by 30 microM GTP. The specific labeling of the 56-kDa protein was sensitive to the anion transport inhibitor DIDS. In order to confirm whether the apparent uptake of ATP was due to the 56-kDa protein, the ATP transporter was partially purified through two successive ion-exchange chromatography steps (DEAE and Mono-S). The fraction showing the high activity of the ATP transporter also contained the 56-kDa protein photolabeled with [gamma-32P]AB-ATP. On the basis of the photoaffinity labeling and reconstitution experiment, we conclude that the 56-kDa protein represents the ATP transporter in rat liver RER.

Analysis of a novel double-barreled anion channel from rat liver rough endoplasmic reticulum

The presence of anionic channels in stripped rough endoplasmic reticulum membranes isolated from rat hepatocytes was investigated by fusing microsomes from these membranes to a planar lipid bilayer. Several types of anion-selective channels were observed including a voltage-gated Cl- channel, the activity of which appeared in bursts characterized by transitions among three distinct conductance levels of 0 pS (0 level), 160 pS (O1 level), and 320 pS (O2 level), respectively, in 450 mM (cis) 50 mM (trans) KCl conditions. A chi 2 analysis on current records where interburst silent periods were omitted showed that the relative probability of current levels 0 (baseline), O1, and O2 followed a binomial statistic. However, measurements of the conditional probabilities W(level 0 at tau/level O2 at 0) and W(level O2 at tau/level 0 at 0) provided clear evidence of direct transitions between the current levels 0 and O2 without any detectable transitions to the intermediate level O1. It was concluded on the basis of these results that the observed channel was controlled by at least two distinct gating processes, namely 1) a voltage-dependent activation mechanism in which the entire system behaves as two independent monomeric channels of 160 pS with each channel characterized by a simple Open-Closed kinetic, and 2) a slow voltage-dependent process that accounts for both the appearance of silent periods between bursts of channel activity and the transitions between the current levels 0 and O2. Finally, an analysis of the relative probability for the system to be in levels 0, O1, and O2 showed that our results are more compatible with a model in which all the states resulting from the superposition of the two independent monomeric channels have access at different rates to a common inactivated state than with a model where a simple Open-Closed main gate either occludes or exposes simultaneously two independent 160-pS monomers.

Changes in lipid distribution and dynamics in degranulated rat liver rough endoplasmic reticulum due to the membrane attachment of polyribosomes

Binding of rat liver polyribosomes to homologous degranulated rough endoplasmic reticulum (dRER) labeled with 10-(pyren-1-yl)decanoic acid (PDA) was studied. As a consequence of the membrane association of polysomes, the excimer/monomer fluorescence intensity ratios (Ie/Im) decreased, thus indicating alterations in the dynamics and organization of lipids. These fluorescence changes were complete within approximately 1 min, in accordance with the tight binding of ribosomes to RER. In order to characterize the changes in membrane lipid dynamics in more detail, polysomes were covalently labeled with trinitrobenzenesulfonic acid so as to allow their use as Förster-type resonance energy-transfer acceptors while utilizing PDA as a donor. Accordingly, assuming the binding of native and quencher-labeled ribosomes to the PDA-labeled membranes to be identical, we were able to discriminate fluorescence changes (a) in the proximity of the ribosome binding site from (b) those arising in the surrounding ribosome-free membrane and beyond the effective quenching radii of the TNP residues coupled to polysomes. Our data suggest that lipids in the polysome attachment site of dRER are less mobile than those in the remaining, ribosome-free membrane. In addition, there appears to be a relative enrichment of the PDA probe in the polyribosome membrane attachment sites.

59] Transport of sugar nucleotides into the lumen of vesicles derived from rat liver rough endoplasmic reticulum and golgi apparatus

Publisher Summary This chapter describes methods to determine whether an intact sugar nucleotide is transported into rat liver derived vesicles of the Golgi apparatus or the rough endoplasmic reticulum (RER). This assay also has the advantage that the total transport activity of a radiolabeled sugar nucleotide can be separated into two components: (1) those radiolabeled solutes which are within the lumen of the vesicles, and (2) those which have become transferred (covalently linked) to macromolecules. The chapter discusses the transport assays of the vesicles. To show that the intact sugar nucleotide, and not breakdown products, is being translocated across the membranes, vesicles are incubated with a mixture of the sugar nucleotide labeled with different radioisotopes in the sugar and the nucleotide. Following the incubation period, the double-radiolabeled ratio of the vesicle pellet is compared to the ratio of the incubation mixture at the beginning of the incubation period. If these two ratios are similar, it suggests that the intact sugar nucleotide was transported across the membrane.

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus.

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.

Characterization of an endopeptidase involved in pre-protein processing.

Proteolytic removal of the pre-segment from growing nascent chains of pre-human placental lactogen (hPL) occurred during in vitro translation of placental mRNA if crude membranes derived from ascites lysates, dog pancreas, or rat liver rough endoplasmic reticulum were added to the translation mixtures. The cotranslational proteolytic event was inhibited by the peptide protease inhibitor, chymostatin, but not by leupeptin, antipain, or elastatinal. The proteases involved in cleavage were solubilized with detergent and converted completed pre-hPL to hPL (post-translational processing). Direct assay of the solubilized membranes, with synthetic fluorogenic aminocoumarin peptide substrates, revealed no significant tryptic or elastase-like activity, but activity against a chymotrypsin substrate [(succinyl-Ala-Ala-Phe)-7-amino-4-methyl-coumarin] was found. This activity was dependent upon both an endopeptidase and an aminopeptidase. Although bestatin inhibited the aminopeptidase activity, it had no effect on the endopeptidase or on post-translational cleavage. Although this endopeptidase cleaved on the COOH side of an alanine residue, it was not inhibited by elastatinal. However, it was inhibited by high levels of chymostatin and by some serine protease inhibitors.

Characterization of the ribosomal binding site in rat liver rough microsomes: ribophorins I and II, two integral membrane proteins related to ribosome binding.

Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detergent Kyro EOB; iii) in intact rough microsomes ribophorins can be cross-linked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton-X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and "rough-inverted" vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosomes when these aggregate without detaching. Measurements of the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents suggest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them.

Antigenic properties of rat liver nuclear membrane

Antigenic properties of purified rat liver nuclear membrane were compared with those of other cellular membranes and nuclear components using the quantitative complement fixation assay. Rat liver nuclear membrane was prepared by heparin treatment and sucrose gradient centrifugation and found to be essentially free of plasma membrane, inner mitochondrial membrane and chromatin components as judged by biochemical analysis and electron microscopy. Antisera to rat liver nuclear membrane were produced in rabbits. These antisera fix complement not only in the presence of rat liver nuclear membrane but also in the presence of rat liver rough or smooth endoplasmic reticulum, plasma membrane or mitochondria or rat muscle sareoplasmic reticulum, indicating common antigenic sites in these membranes. Species specificity of these antigenic sites is suggested, since the antisera did not show complement fixation when rabbit skeletal muscle sarcoplasmic reticulum was tested. Rat liver chromatin or liver nonhistone protein. DNA complex also did not fix complement in the presence of antibodies to the nuclear membrane. When the anti-rat liver nuclear membrane sera were absorbed with rat liver rough endoplasmic reticulum or mitochondria, antibodies specific for the rat liver nuclear membrane were obtained. It is proposed that specific antigens can be used as a marker for the nuclear membrane. Furthermore it was found that sera from certain non-immunized rabbits contained antibodies which fixed complement in the presence of various membrane preparations.

The role of membrane proteins and phospholipids in the interaction of ribosomes with endoplasmic reticulum membranes.

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.

The comparison of rat liver rough endoplasmic reticulum membrane proteins before and after in vitro removal of its bound ribosomes.

The comparison of the proteins of rat liver rough membrane after stripping with EDTA or KCl-puromycin by two dimensional gel electrophoresis is described. By stripping the membrane with EDTA, most of the basic ribosomal proteins are still attached to the membrane; in contrast to the EDTA stripping method, treatment with KCl-puromycin removes most of the ribosomal proteins and does not remove any of the membranal proteins.

Increased protein synthetic capacity in vitro of rat liver rough endoplasmic reticulum following starvation

There is a dramatic rise in the protein synthetic activity of rough endoplasmic reticulum (RER) of liver from starved and starved-refed rats, compared to normal controls. This observation is similar to the doubling of protein synthetic activity of rat liver RER observed 20 hours after partial hepatectomy (1). Oxidised glutathione (GSSG), a potent inhibitor of protein synthesis by normal RER, was a much less effective inhibitor when assayed with RER from the livers of starved rats, this again is in parallel with our earlier observations in regenerating liver. This common pattern of response is discussed in terms of a cellular control system which responds to changes in the cytoplasmic sulphydryl concentration.

Effects of growth hormone and tri-iodothyronine on amino acid incorporation by microsomal subfractions from rat liver.

A two-step procedure without the use of detergents for the separation of smooth and rough endoplasmic reticulum of rat-liver and the fractionation of the latter into “light rough membranes”“heavy rough membranes” and “free polysomes” is described. The distribution of RNA, protein and phospholipid in these fractions as well as their amino acid incorporation characteristics are also described. The heavy rough membrane fraction incorporated 14C-labelled phenylalanine, isoleucine and lysine into protein at a rate 3–5 times greater than that exhibited by the light rough membranes and free polysomes. Stimulation of the incorporation of [14C]phenylalanine and [14C]-isoleucine by poly U and poly UA showed that, per mg ribosomal RNA, the heavy rough membrane fraction was also more active in its response to synthetic messenger RNAs than the other two fractions. Hypophysectomy caused a marked reduction in the amount of ribosomes and phospholipids, the amino acid incorporation capacity and the response to poly U and poly UA in all three rough microsomal subfractions. These effects were reversed in 3–4 days by a combined treatment of hypophysectomized rats with human growth hormone and 3,3′,5-tri-iodo-l-thyronine. Although the amino acid incorporation activity in the presence of endogenous and synthetic template RNA was affected in all the subfractions, the effect of hormone deprivation and replacement was most marked in the heavy rough membrane fraction. The relative stimulation of the incorporation of [14C]phenylalanine and [14C]isoleucine by poly U and poly UA, respectively, by free and membrane-bound polysomes was only slightly lowered in a few cases after hormonal treatment of hypophysectomized rats. However, per mg ribosomal RNA, hormone treatment led to a 2–4-fold enhancement of the incorporation of phenylalanine and isoleucine in response to the synthetic polyribonucleotides. Treatment of heavy rough membranes with sodium deoxycholate showed that much of the hormonal effect on amino acid incorporation was manifested in the ribosomes but that the maximal hormonal effect was detected when these were associated with the membranes. It is concluded that growth hormone and thyroid hormone together control both the amount and amino acid incorporation activity of free and membrane-associated polyribosomes of rat-liver.