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.     

Biosynthesis of sphingomyelin in rat liver endoplasmic reticulum.

Rat liver microsomal sphingomyelin synthetase (CDPcholine: N-acylspingosine choline phosphotransferase (EC 2.7.8.3)) has been shown to be markedly stimulated by ATP and pantothenic acid derivatives such as CoA, pantethine, pantetheine and 4'-phosphopantetheine.     

Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.     

Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.     

Isolation, characterization, and expression of cDNA encoding a rat liver endoplasmic reticulum alpha-mannosidase

We have isolated a cDNA encoding an endoplasmic reticulum alpha-mannosidase, an asparagine-linked oligosaccharide processing enzyme, from a rat liver lambda gt11 library. Two degenerate oligonucleotides, based on amino acid sequence data from the purified enzyme, were used as primers in the polymerase chain reaction with liver cDNA as a template to generate an unambiguous cDNA probe. The cDNA fragment (524 base pair) obtained was then used to isolate cDNA clones by hybridization. We isolated two overlapping clones which were used to construct a full-length cDNA of 3392 base pairs. A single open reading frame of 1040 amino acids encodes a protein with a molecular mass of 116 kilodaltons containing the six known peptide sequences. The deduced amino acid sequence revealed no classical signal sequence or membrane-spanning domain. The alpha-mannosidase encoding cDNA can be expressed transiently in COS cells using the mammalian expression vector pXM, causing a 400-fold increase in alpha-mannosidase activity as well as a dramatic increase in immunoreactive polypeptide. The rat liver endoplasmic reticulum alpha-mannosidase bears striking homology to the vacuolar alpha-mannosidase from Saccharomyces cerevisiae.     

Location of rat liver iodothyronine deiodinating enzymes in the endoplasmic reticulum.

The iodothyronine-deiodinating enzymes (iodothyronine-5- and 5'-deiodinase) of rat liver were found to be located in the parenchymal cells. Differential centrifugation of rat liver homogenate revealed that the deiodinases resided mainly in the microsomal fraction. The subcellular distribution pattern of these enzymes correlated best with glucose-6-phosphatase, a marker enzyme of the endoplasmic reticulum. Plasma membranes, prepared by discontinuous sucrose gradient centrifugation, were found to contain very little deiodinating activity. Analysis of fractions obtained during the course of plasma membrane isolation showed that the deiodinases correlated positively with glucose-6-phosphatase (r larger than or equal to 0.98) and negatively with the plasma membrane marker 5'-nucleotidase (r ranging between -0.88 and -0.97). It is concluded that the iodothyronine-deiodinating enzymes of rat liver are associated with the endoplasmic reticulum.     

Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum.

The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function.     

Heterogeneity of smooth endoplasmic reticulum from rat liver studied by two-phase partitioning.

Smooth microsomal membranes, prepared from rat liver by sucrose-density-gradient centrifugation, were subfractionated by counter-current distribution in an aqueous two-phase system consisting of poly(ethylene glycol) and Dextran T500. A comparison of the distribution curves of marker enzymes, together with theoretically calculated curves, indicated the presence of at least five membrane subfractions, differing in the ratios of the marker enzymes. Glucose-6-phosphatase and arylesterase distributed in one manner, and NADPH-cytochrome c reductase and NADH-ferricyanide reductase in another. Evidence for further heterogeneities in the distribution of marker enzymes in smooth microsomes was obtained by analysing the membrane domain structure using a recently described method [Albertsson (1988) Q. Rev. Biophys. 21, 61-98]. Phenobarbital treatment did not influence the behaviour of the marker enzymes.     

Protein degradation by the phosphoinositide-specific phospholipase C-alpha family from rat liver endoplasmic reticulum.

A 60-kDa protein homologous to phosphoinositide-specific phospholipase C-alpha was purified to apparent homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the rough endoplasmic reticulum of rat liver through three sequential chromatographies on DEAE Toyopearl 650, AF-heparin Toyopearl 650M, and TSK gel G3000SW. The purified protein was monomeric, with an M(r) of 60,000. Eight types of protein were further separated from the 60-kDa protein and named ER60A-ER60H according to the order of their elution from a TSK gel DEAE-5PW column. They were essentially identical in terms of immunochemical properties and the NH2-terminal amino acid sequence. The partial amino acid sequence of ER60F showed homology to that of phosphoinositide-specific phospholipase C-alpha. ER60A-ER60H showed no phosphoinositide-specific phospholipase C activity. However, ER60A-ER60H catalyzed cleavage of themselves and the endoplasmic reticulum proteins protein disulfide-isomerase and calreticulin. Proteolytic degradation was inhibited by p-chloromercuribenzoate. These results indicate that ER60A-ER60H comprise a group of endoplasmic reticulum resident proteins and show thiol group-related proteolytic activity.     

Cryoresistance of rat liver endoplasmic reticulum membranes]

The effects of freezing of microsomes in liquid nitrogen and those of storage of microsomal suspensions at 2-4 degrees C and -3 - -5 degrees C for 24 hrs, on the enzymatic activities and hydrophobicity of membranes were studied. The hydrophobicity was determined by fluorescence of bound 1,8-anilino-naphthalene sulfonate. Rapid freezing of the microsomal suspension in liquid nitrogen followed by rapid warming did not change the hydrophobicity of the membranes, the rate of enzymatic lipid peroxidation, the level of cytochrome P-450 and the activity of NADH- and NADPH-cytochrome c reductase. A considerable decrease in the rate of enzymatic lipid peroxidation and membrane hydrophobicity was observed in the microsomes stored for 24 hrs at 2-4 degrees C. The 24-hr storage at -3 - -5 degrees C with subsequent thawing resulted in a rapid aggregation of the microsomes.     

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

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.     

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidylcholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A₂ of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphingomyelin which is not hydrolysed by the former enzyme.     

Location of rat liver iodothyronine deiodinating enzymes in the endoplasmic reticulum

The iodothyronine-deiodinating enzymes (iodothyronine-5- and 5′-deiodinase) of rat liver were found to be located in the parenchymal cells. Differential centrifugation of rat liver homogenate revealed that the deiodinases resided mainly in the microsomal fraction. The subcellular distribution pattern of these enzymes correlated best with glucose-6-phosphatase, a marker enzyme of the endoplasmic reticulum. Plasma membranes, prepared by discontinuous sucrose gradient centrifugation, were found to contain very little deiodinating activity. Analysis of fractions obtained during the course of plasma membrane isolation showed that the deiodinases correlated positively with glucose-6-phosphates (r >/ 0.98) and negatively with the plasma membrane marker 5′-nucleotidase (r ranging between ?0.88 and ?0.97). It is concluded that the iodothyronine-deiodinating enzymes of rat liver are associated with the endoplasmic reticulum.     

Lateral enzyme topology in the rough endoplasmic reticulum of rat liver

Summary Rough microsomes were subfractionated on the basis of different properties in order to investigate the nature and extent of the enzyme heterogeneity of these vesicles. A discontinuous gradient, containing monovalent cations allowed the separation of a ribosome-poor membrane fraction which was enriched in electron transport enzymes and relatively poor in phosphatases. Zonal centrifugation on a stabilizing gradient separated 3 fractions characterized by enrichment of electron transport enzymes, glucose-6-phosphatase and adenosinetriphosphatase, respectively. An essentially similar pattern was seen when ribosomes were removed with EDTA and the denuded vesicles subfractionated on a sucrose gradient. Rough microsomes from phenobarbitaltreated rats exhibited the same pattern both qualitatively and quantitatively. It appears that electron transport enzymes and two types of phosphatases are heterogeneously distributed among rough microsomal vesicles.This work has been supported by grants from the Swedish Medical Research Council. The authors wish to thank Mrs. Ulla-Britta Torndal for her valuable technical assistance     

Biochemically distinct vesicles from the endoplasmic reticulum fuse to form peroxisomes

As a rule, organelles in eukaryotic cells can derive only from pre-existing organelles. Peroxisomes are unique because they acquire their lipids and membrane proteins from the endoplasmic reticulum (ER), whereas they import their matrix proteins directly from the cytosol. We have discovered that peroxisomes are formed via heterotypic fusion of at least two biochemically distinct preperoxisomal vesicle pools that arise from the ER. These vesicles each carry half a peroxisomal translocon complex. Their fusion initiates assembly of the full peroxisomal translocon and subsequent uptake of enzymes from the cytosol. Our findings demonstrate a remarkable mechanism to maintain biochemical identity of organelles by transporting crucial components via different routes to their final destination.     

Specific lipids enhance the activity of UDP-GlcNAc: dolichol phosphate GlcNAc-1-phosphate transferase in rat liver endoplasmic reticulum membrane vesicles.

The rate of the reaction catalyzed by UDP-N-acetylglucosamine (GlcNAc):dolichol phosphate GlcNAc-1-phosphate transferase in rat liver endoplasmic reticulum vesicles was shown to be influenced by particular lipids. Utilizing in vitro assay conditions where the membrane vesicles retained latency of glucose-6-phosphatase activity, the addition of phosphatidylethanolamine, cardiolipin, or monogalactosyldiglyceride resulted in severalfold increases in the rate of dolichol pyrophosphate N-acetylglucosamine synthesis. Other phospholipids were not stimulatory. These rates were dependent on the concentrations of the exogenous lipids and of the substrate dolichol phosphate. In the presence of cardiolipin, the membrane-bound enzyme became more susceptible to inactivation by protease K and to inhibition by tunicamycin. Titration of cardiolipin-containing endoplasmic reticulum vesicles with adriamycin indicated that the majority of the cardiolipin was exposed on the outer surface. These results suggest that the particular lipids altered membrane structure in a way that allowed further access of the enzyme to substrate, inhibitor, and other molecules. Lipids observed in these studies to be stimulatory are known to exist in the macromolecular hexagonal phase and may therefore be affecting the GlcNAc-1-phosphate transferase by locally disrupting the bilayer structure of the membrane. As other dolichol-utilizing enzymes have been previously observed by other investigators to be similarly influenced by such lipids, the effects may be common to enzymes of the dolichol cycle.     

GTP hydrolysis by transitional endoplasmic reticulum from rat liver inhibited by all-trans-retinol

GTP hydrolysis by an endoplasmic reticulum fraction from rat liver enriched in part-rough, part-smooth transition elements was inhibited by all-trans-retinol half maximally at a concentration of about 10 micrograms/ml. Similar results were obtained with GTPase activity partially purified by ion-exchange (DE-52) chromatography. The inhibition was non-competitive and given by both retinol and retinaldehyde but not by retinoic acid or alpha-tocopheryl acetate. The hydrolysis of other nucleoside di- and triphosphates was much less affected by retinol. The activity was inhibited by detergents but at much higher concentrations than by retinol. The results suggest that enhancement of cell-free transfer from endoplasmic reticulum to Golgi apparatus by retinol observed previously at low concentrations of cytosol may be mediated through an interaction with GTP.