Localization of GTP-stimulated core glycosylation to fused microsomes.

Purified rough microsomes from liver maximally incorporated N-acetyl-[3H]glucosamine into endogenous acceptors from UDP-N-acetyl-[3H]glucosamine substrate, providing the associated ribosomes were removed and 0.5 mM GTP was added. These conditions also led to the coalescence of microsomes into large fused membranes. By measurement of membrane profiles on electron micrographs, a correlation was observed between GTP-stimulated glycosylation and microsomal membrane length (r2 = 0.92). Membrane fusion was not observed in the absence of GTP, with sugar transfer inhibited by greater than 90% for acid-resistant acceptors (protein), and approximately 50% for acid-labile acceptors (lipid-linked intermediates). When radiolabeled acceptors were localized by electron microscope radioautography, high concentrations of silver grains (83 grains/100 microns membrane length) were observed over fused membranes with lower grain densities observed over unfused membranes in the same preparation (20 grains/100 microns). These studies directly link microsomal membrane fusion to GTP-stimulated core glycosylation. The observations extend the suggestion of Godelaine et al. (1979, Eur. J. Biochem. 96:17-26) that physiological levels of GTP promote the translocation of substrate across endoplasmic reticulum membranes which, we propose, occurs via a membrane fusion phenomenon.

Localization of thyroiglobulin antigenicity in rat thyroid sections using antibodies labeled with peroxidase or 125I-radioiodine.

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling. Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin. It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.

Coalescence of microsomal vesicles from rat liver: a phenomenon occurring in parallel with enhancement of the glycosylation activity during incubation of stripped rough microsomes with GTP.

Rough microsomes from rat liver have been subjected to various treatments and incubated afterwards with UDP-N-acetyl-[14C]glucosamine and GDP-mannose in the presence of GTP (0.5 mM), or of other nucleotides. In agreement with earlier results from this laboratory, the preparations previously treated to strip off the ribosomes and incubated in the presence of GTP assembled dolichol-linked oligosaccharides and transferred these oligosaccharides to endogenous protein acceptors much more actively than untreated preparations, or stripped preparations incubated in the absence of GTP. Thin-section and freeze-fracture electron microscopy have revealed that pyrophosphate-treated preparations incubated with GTP are aggregated and contain numerous vesicles as large as 1-4 micrometer, or more. Such large vesicles were not present before incubation and thus were considered to have been formed through coalescence of regular-sized ones. Like glycosylation, the coalescence phenomenon depends upon the removal of ribosomes, because it occurred whether ribosomes had been stripped, at least partly, with pyrophosphate, KCl, or puromycin, but not when rough microsomes had been washed with 0.25 M sucrose or with KCl and MgCl2. Like glycosylation, it also depends on the addition of GTP and was not induced by ATP, UTP, CTP, and nonhydrolysable analogues of GTP. Rough microsomes coalesced, however, when pyrophosphate-treated preparations were incubated with GTP in the absence of nucleotide sugars, or in the presence f tunicamycin, indicating that the coalescence phenomenon does not result from the glycosylation of some membrane constituents.

Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes.

We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with [3H]sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography. The data demonstrate that dispersed fragments of the rat liver Golgi complex (i.e., unstacked vesicles and tubules) reconstitute into stacked saccules when microinjected into Xenopus cytoplasm. After the formation of stacked saccules, reconstituted Golgi fragments transport constituents into a portion of the exocytic pathway of the host cell by a microtubule-regulated process.

Galactose transfer to endogenous acceptors within Golgi fractions of rat liver.

The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al. procedure (1973. J. Cell Biol. 59:45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used. The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid. The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation). The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method. Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay. The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles.

Membrane fusion and glycosylation in the rat hepatic Golgi apparatus.

When purified Golgi fractions were incubated with UDP-[3H]galactose in the absence of Triton-X-100, radioactivity was incorporated into an endogenous lipid and several peptide acceptors. Electron microscope analysis of Golgi fractions incubated in the endogenous galactosyl transferase assay medium revealed extensive fusion of Golgi saccules. Systematic removal of constituents in the galactosyl transferase assay medium showed enhanced (minus beta-mercaptoethanol) or reduced (minus ATP, minus sodium cacodylate buffer or minus MnCl2) fusion of Golgi membranes compared to the complete medium, Stereologic analysis revealed a correlation between membrane fusion and galactosyl transferase activity (r = 0.99, P less than 0.001). Electron microscope radioautography was carried out after incubation of Golgi fractions with UDP-[3H]galactose. Silver grains were not observed over trans elements of Golgi but were revealed mainly over large fused saccules with the number of silver grains being proportionate to membrane fusion (r = 0.92, P less than 0.001). Bilayer destabilization at points of Golgi membrane fusion may act to translocate galactose across the Golgi membrane and thereby provide a fusion regulated substrate for terminal glycosylation.

Roles for alpha(2)p24 and COPI in endoplasmic reticulum cargo exit site formation.

A two-step reconstitution system for the generation of ER cargo exit sites from starting ER-derived low density microsomes (LDMs; 1.17 g/cc) is described. The first step is mediated by the hydrolysis of Mg(2+)ATP and Mg(2+)GTP, leading to the formation of a transitional ER (tER) with the soluble cargo albumin, transferrin, and the ER-to-Golgi recycling membrane proteins alpha(2)p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enriched therein. Upon further incubation (step two) with cytosol and mixed nucleotides, interconnecting smooth ER tubules within tER transforms into vesicular tubular clusters (VTCs). The cytosolic domain of alpha(2)p24 and cytosolic COPI coatomer affect VTC formation. This is deduced from the effect of antibodies to the COOH-terminal tail of alpha(2)p24, but not of antibodies to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTPgammaS, inhibition by Brefeldin A (BFA), or depletion of beta-COP from cytosol. Therefore, the p24 family member, alpha(2)p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs and the generation of ER cargo exit sites.

Role of p97 and syntaxin 5 in the assembly of transitional endoplasmic reticulum.

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc(-1)) isolated from rat liver homogenates reconstitute tER by Mg(2+)GTP- and Mg(2+)ATP-hydrolysis-dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.

GTP stimulates fusion between homologous and heterologous nuclear membranes.

The tissue and species specificity of GTP-stimulated nuclear membrane fusion has been examined. The fusion capacity of the membranes of nuclei isolated from two different tissue sources and three different animal species was determined. In all cases the incubation of isolated nuclei in the presence of 0.5 mM GTP led to the pairing of nuclei and formation of continuous outer membranes between the nuclei as a result of membrane fusion. Experiments using mixtures of nuclei from the different sources demonstrated that hybrid nuclear membranes could be formed as a result of the fusion between the outer membranes of heterologous nuclear pairs. The results suggest that the capacity for nuclear membranes to fuse in the presence of GTP is highly conserved when viewed on an evolutionary basis.

Accumulation of polyunsaturated free fatty acids coincident with the fusion of rough endoplasmic reticulum membranes.

The accumulation of polyunsaturated free fatty acids (PUFAs) was observed coincident with GTP-dependent fusion of liver rough microsomes. Whereas 0.5 mM NADPH led to a parallel reduction (greater than 50%) in membrane fusion and PUFA accumulation, indomethacin (50 microM) either had little effect or slightly augmented both processes. CTP was observed to stimulate accumulation of PUFAs and diacylglycerol (DAG). Therefore PUFAs may be relevant for GTP-dependent membrane fusion and together with DAG may play a role in fusion stimulated in the presence of CTP.

Effect of organic mercury on the electrical resistance of phosphatidylserine bilayers.

Acidic phospholipids have been shown to form complexes with methyl mercury chloride, at physiological pH, in vitro. To check if this interaction had any effect on the physical properties of membranes made with these lipids, the specific resistance of phosphatidylserine bilayers was monitored, as a function of time, in the absence and in the presence of methyl mercury chloride in the bathing solution. While the resistance of the bilayer remained constant in the absence of the toxic, it dropped an average of 17% in four hours in the presence of 100 microM methyl mercury chloride. Such observations suggest that the physical integrity of these membranes is modified by the interaction with organic mercury. This result may be relevant to the observed degeneration of nerve membranes in Minamata disease.

Freeze-fracture analysis of the effects of intermediates of the phosphatidylinositol cycle on fusion of rough endoplasmic reticulum membranes.

While searching for the identity of the effector of the putative GTP-binding protein involved in fusion of rough endoplasmic reticulum (RER) cell-free incubation conditions were found permitting fusion in a GTP-independent manner. Membrane fusion was obtained using medium required to study synthesis of phosphatidylinositol (PI). We now report on the effects of various co-factors and intermediates of the PI cycle on the interaction of rough microsomes. By freeze-fracture, fusion of rough microsomes was defined as the appearance of fracture-planes of membrane larger than those of unincubated membrane. Cytosine triphosphate (CTP, 3 mM) in the presence of 2 mM MnCl2 was most effective in stimulating fusion. Guanosine triphosphate (GTP) at the same concentration, could substitute for CTP to stimulate fusion, ATP, ITP, UTP and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) could not. When combined together in the same medium CTP potentiated the effect of GTP. Arachidonic acid (20 micrograms/ml) also stimulated fusion in the presence of MnCl2. This led to the appearance of large fracture-planes of membrane with a heterogeneous distribution of intramembranous particles. Other saturated fatty acids at the same concentration did not stimulate fusion. Phosphatidylinositol (PI, 50 micrograms) and 2 mM MnCl2 had a similar effect as arachidonic acid and MnCl2 in stimulating fusion. The PI effect was largely augmented in the presence of CTP. Our results are consistent with the concept that metabolism of phospholipids may modulate GTP-dependent fusion of RER membranes.

Properties of a GTP sensitive microdomain in rough microsomes.

Stripped rough microsomes (SRM) fuse when incubated with physiological concentrations of GTP and MgCl2. In order to examine further to what extent such fusions are associated with other membrane functions of rough endoplasmic reticulum, we have evaluated the role of cytosolically exposed peptide constituents of SRM in fusion, and the possible relationship of GTP/MgCl2-induced fusion in protein transport across endoplasmic reticulum (ER) membranes, and in ER-Golgi interactions. Controlled proteolytic digestion of SRM led to the loss of fusion capability at 15 micrograms/ml trypsin--a concentration which maintained the latency of intraluminal mannose-6-phosphatase. Hence, a cytosolically exposed protein(s) regulated fusion. Based on ribonuclease-induced ribosome capping experiments, it was further concluded that the cytosolic oriented protein(s) was sequestered beneath the ribosome. As co-translational cell free translocation of placental lactogen across SRM was similar in control membranes compared to those rendered incapable of fusing, it was concluded that the fusion phenomenon may not be related to translocation. Under conditions promoting homologous fusion of SRM or Golgi membranes, mixtures of the two membranes showed no heterologous membrane fusion as assessed morphologically or by the transport of newly synthesized membrane glycoprotein. These experiments attest to the specificity of cytosolically exposed protein(s) in regulating nucleotide/divalent cation-induced membrane fusion.

Terminal glycosylation in rat hepatic Golgi fractions: heterogeneous locations for sialic acid and galactose acceptors and their transferases.

Endogenous acceptors for N-acetylglucosamine (GlcNAc), galactose (Gal) or sialic acid (NeuAc) transfer were labeled to high activities when purified hepatic Golgi fractions were incubated with the corresponding radiolabeled nucleotide sugar in the absence of detergent. The in vitro conditions which were optimal for the endogenous glycosylation of GlcNAc and Gal acceptors (Mn2+, ATP) also promoted fusion within a subset of Golgi membranes. Electron microscope radioautography revealed that the majority of NeuAc acceptors were associated with unfused Golgi membranes, whereas the majority of Gal acceptors were localized to fused membranes. GlcNAc acceptors were approximately equally distributed between fused and unfused membranes. Under conditions in which Golgi membrane fusion was absent (-Mn2+), only NeuAc transfer was active. The majority of endogenous NeuAc acceptors were consequently assigned to the more trans regions of the hepatic Golgi apparatus as concluded from a combination of radioautography (NeuAc transfer) and acid NADPase cytochemistry (reactive medial and trans Golgi saccules). The distribution of NeuAc and Gal transferases was assessed after Percoll gradient centrifugation of disrupted Golgi fractions. The median density of NeuAc transferase was lower than that of Gal transferase. The studies are indicative of distinct Golgi components harboring the majority of acceptors and enzymes for terminal glycosylation.

Modulation of GTP-dependent fusion by linoleic and arachidonic acid in derivatives of rough endoplasmic reticulum from rat liver.

The effect of modulation of the content of unsaturated free fatty acids on GTP-dependent fusion of stripped rough microsomes from rat liver was determined. Cytidine monophosphate, CDP and CTP were all observed to be able to stimulate free fatty acid accumulation and coincident membrane fusion. GTP was required for membrane fusion in the presence of cytidine nucleotide but was not required for free fatty acid accumulation. In the presence of GTP and cytidine nucleotide, the addition of ATP and CoA led to the synthesis of triacyglycerol and marked inhibition of both free fatty acid accumulation and membrane fusion. Delipidated bovine serum albumin also inhibited both free fatty acid accumulation and membrane fusion. Analysis by gas chromatography indicated that linoleic acid and arachidonic acid were the most actively fluctuating of the accumulated free fatty acids. Comparison by quantitation indicated a high correlation between GTP-dependent membrane fusion and changes in amount of unesterified linoleic acid and arachidonic acid. The results suggest that polyunsaturated free fatty acids may be required for GTP-dependent membrane fusion.

Chlorambucil drug resistance in chronic lymphocytic leukemia: the emerging role of DNA repair.

Various mechanisms have been implicated in nitrogen mustard drug resistance. The role of these mechanisms in the development of chlorambucil drug resistance in chronic lymphocytic leukemia (CLL) is discussed. We review these mechanisms with emphasis on the emerging role of DNA repair, and specifically, recombinational repair. Inhibition of these repair processes may lead to new therapies, not only in CLL, but in other malignancies as well.

Electrophoretic protein blots as aids in choosing fixatives for immunocytochemistry.

We used electrophoretic protein blots prepared from polyacrylamide gels to test the effect of different fixatives on the antigenicity of rough endoplasmic reticulum (RER) peptides from rat liver. Protein blots were prepared by the procedure of Towbin et al. (Proc Natl Acad Sci USA 76:4350, 1979), treated with different fixatives, rinsed to inactivate non-specific reactive sites, and then reacted with rabbit polyclonal anti-rat liver RER antibodies, followed by peroxidase-conjugated anti-rabbit antibodies. On the basis of differences in immunostaining densities as determined by densitometry, we found that RER peptides displayed differential sensitivities to various fixatives. Anti-rat liver RER antibodies and the immunogold technique were applied to methacrylate sections of in vitro fixed rat liver rough microsomes. Specific labeling was observed over the microsomes and was shown by quantitation to vary in a similar manner to the immunostaining of specific peptides in protein blots following different fixations. We conclude that protein blots may serve as useful tools for screening the effects of different fixatives on cell antigenicity, and therefore may be helpful in immunocytochemical studies.

Synthesis of CDP-diacylglycerol by rat liver rough microsomes as visualized by electron microscopic autoradiography: relationship to GTP-stimulated membrane fusion.

Using detergent-free conditions of incubation for the analysis of liponucleotide synthesis, we compared GTP-dependent formation of CDP-diacylglycerol (CDP-DG) and membrane fusion in RNA-depleted rough microsomes from rat liver. After incubation of stripped rough microsomes (SRM) in the presence of GTP and [5-3H]-CTP, radioactivity was recovered in lipid extracts and identified by thin-layer chromatography as a single spot which co-migrated with CDP-DG. The nucleotide requirement for CDP-DG synthesis and that for membrane fusion were observed to be identical. We next carried out an electron microscopic autoradiographic analysis on incubated membranes to determine the site of incorporation of [5-3H]-CTP. Silver grains were observed directly over the unilamellar membranes of natural vesicles. In confirmation of the biochemical data, quantitation of silver grain density indicated more grains over membranes incubated in the presence of GTP than over those incubated in the absence of this nucleotide. For membranes incubated in the presence of GTP, the grain density was similar over fused and unfused membranes in the same preparation. When SRM were incubated with the enzyme co-factors required for synthesis of phosphatidylinositol, a GTP-independent membrane fusion was observed by both transmission and freeze-fracture electron microscopy. Together with the biochemical and autoradiographic data, this suggests that phospholipid metabolism may be activated by GTP and lead to the fusion of RER membrane.

Subcellular distribution of acid NADPase activity within the parenchymal cells of rat liver.

We examined the distribution of acid NADPase activity in rat hepatocytes by cytochemistry and subcellular fractionation. Cytochemical studies revealed strong NADPase activity in many lysosomes and Golgi saccules situated between the medial and trans aspect of the stack. Light, spotty deposits of reaction product also were observed frequently within elements of the Golgi-associated endoplasmic reticulum lysosome (GERL) system. Biochemical studies with liver homogenates indicated that NADPase activity was distributed across various subcellular fractions in a pattern resembling typical nonspecific acid phosphatase activity, as monitored using beta-glycerophosphate as substrate. Thus, by differential centrifugation, most cellular NADPase activity was found in the large granule (ML) fraction (61%). The microsomal (P) fraction showed about 17% whereas the nuclear (N) and cytosol (S) fractions each accounted for about 11% of the remaining NADPase activity, respectively. Separation of the ML fraction into endosomes and secondary lysosomes revealed most of the acid NADPase activity associated with lysosomes. An alternate one-step method for isolation of fractions indicated that 3% of the recovered acid NADPase activity, but 55% of the Golgi marker enzyme galactosyl transferase, was associated with a purified intact Golgi fraction. Cytochemical studies with ML and intact Golgi fractions indicated that bona fide reaction product was localized to lysosomes and to the same saccules that were reactive in vivo. We propose that saccules making up the trans half of the Golgi stack may represent a site for accumulation of a biogenetic precursor of the lysosomal acid NADPase in hepatocytes.

Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjection of rat liver microsomes into Xenopus oocytes.

Fragments of rough and smooth endoplasmic reticulum purified from rat liver were injected into Xenopus oocyte cytoplasm. Light and electron microscopy, cytochemistry, immunocytochemistry, and enzyme assay were employed to determine the fate of heterologous membranes in the host cytoplasm. The in vivo-incubated microsomes disappeared in a time-dependent manner. Within 3 hr, rough microsomes were replaced by flattened ER cisternae and smooth microsomes were replaced by a network of anastomosing tubules. Polyclonal antibodies against rat liver microsomes and protein A-gold complexes were applied to glycol methacrylate sections of microinjected oocytes. Specific labeling was observed over discrete rough and smooth ER cisternae 3 hr after microinjection. Endogenous ER was not labeled by this technique, and label was not observed when sections were treated with pre-immune antibodies. Diaminobenzidene cytochemistry of microinjected rat lacrimal gland microsomes revealed enzyme activity in heterologous microsomes after 3 hr of in vivo incubation. Control injected microsomes (inactivated by heat denaturation) became associated with autophagic vacuoles, coincident with changes in lysosomal activity. Freshly isolated un-denatured microsomes did not provoke changes in lysosomal activity, and glucose-6-phosphatase activity associated with microinjected membranes could be detected 21 hr after in vivo incubation. Since rat liver microsomes reconstitute after in vivo incubation into cytoplasmic structures resembling those from which they were derived, we conclude that the microinjected membrane fragments act as templates for their own three-dimensional organization.