Golgi detection in mitotic and interphase cells by antibodies to secreted galactosyltransferase

Secreted galactosyltransferase from bovine milk was used to induce antibodies cross-reacting with corresponding intracellular enzymes in a variety of cell lines and tissues. In contrast to the original antigen, the reactive intracellular galactosyltransferase appears as individual species (apparent MW approx. 42000-46000) in SDS-polyacrylamide gel electrophoresis. In indirect immunofluorescence microscopy affinity-purified IgGs locate the galactosyltransferase in a distinct perinuclear and juxtanuclear position indicative for the Golgi region. The rearrangement of labelled structures upon colcemid or monensin treatment--drugs known to influence Golgi morphology and function--is further proof for a Golgi association. The fate and distribution of Golgi elements during mitosis is described at the light microscopical level using galactosyltransferase as easily identifiable marker. In addition we evaluate the utilization of wheat germ agglutinin (WGA) binding for Golgi identification on tissue culture cells and show that WGA is not a reliable marker for certain cell types such as MDCK.     

Subcellular localization of transferrin protein and iron in the perfused rat liver. Effect of Triton WR 1339, digitonin and temperature

The subcellular localization of 3H-labelled 59Fe-loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques. More than 96% of the 59Fe present in the liver of rats perfused with 59Fe-labelled transferrin is recovered in the parenchymal cells. Rat livers were perfused with 10 micrograms/ml 3H-labelled 59Fe-saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant (S) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients. 3H label distributes essentially around densities of 1.13-1.14 g/ml overlapping to a large extent with the distribution of galactosyltransferase, the marker enzyme of the Golgi complex. However, after treatment with low concentrations of digitonin the 3H label dissociates from galactosyltransferase and is shifted to higher densities, suggesting an association of transferrin with cholesterol-rich endocytic vesicles which could derive from the plasma membrane. 59Fe is mostly found in the supernatant fraction largely in the form of ferritin, as indicated by its reaction with antiferritin antibodies. In the mitochondrial fraction the density distribution of 59Fe suggests an association with lysosomes and/or mitochondria. In contrast to the lysosomal enzyme cathepsin B, the density distribution of 59Fe was only slightly affected by pretreatment of the rats with Triton WR 1339, suggesting its association with the mitochondria. At 15 degrees C, 59Fe and 3H labels are recovered together in low-density endocytic vesicles. On the basis of our results we suggest that, at low extracellular transferrin concentration, iron uptake by the liver involves endocytosis of the transferrin protein. The complex is interiorized in low-density acidic vesicles where iron is released. The iron passes into the cytosol, where it is incorporated into ferritin and into the mitochondria. The iron-depleted transferrin molecule would then be returned to the extracellular medium during the recycling of the plasma membrane.     

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.     

Fragmentation of re-formation of mitotic Golgi apparatus detected by a centrifugal method

The fragmentation/re-formation process of the Golgi apparatus during mitosis was studied by flotation centrifugation in a stepwise sucrose density gradient. The mitotic Golgi fraction was obtained from Chinese hamster ovary cells synchronized with thymidine and nocodazole. The Golgi apparatus detected by a marker enzyme, galactosyltransferase, was separated into two peaks by the flotation centrifugation. The amount of the Golgi recovered at the lower density peak was less in the mitotic cells than in the interphase cells. The separation profile of the mitotic Golgi returned to that of the interphase Golgi by further incubation of the mitotic cells. The re-formation of the fragmented Golgi was inhibited by nocodazole and vinblastine, but not by actinomycin D and cycloheximide.     

Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway

Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane. During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused. These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.     

Effect of acute ethanolic intoxication on the neuraminidase activity of rat liver golgi apparatus

Neuraminidase and galactosyltransferase were investigated in total Golgi apparatus and in the three fractions of increasing densities (GF₁, GF₂ and GF₂) isolated from the microsomal fraction of rat liver homogenates by flotation in a discontinuous sucrose density gradient (Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45–72). About 50% decreases in neuraminidase content (units/g liver) and specifixc activity (units/ mg protein) were observed in total Golgi as well as in the three fractions isolated at 45 min, 90 min, 180 min and 16 h after administration of a single oral dose of 50% aqueous ethanol (0.6 g/100 g body weight). Colchicine administration (intraperitoneal injection, 0.5 mg/100 g body weight) caused a similar loss of neuraminidase activity; however, the effect of ethanol plus colchicine was not additive. Golgi galactosyltransferase, on the other hand, experienced marked increases of activity following ethanol administration but, unlike the results reported by others (Gang, H., Lieber, C.S. and Rubin, E. (1973) Nat. New Biol. 243, 123–125), significant increases in total activity and specific activity were already quite evident at 90 min after ethanol ingestion. In contrast with the decreased values observed in Golgi, the total particle-bound neuraminidase was significantly elevated following ethanol administration. Ultrastructural studies revealed increased lysosomal content and detachment of polysomes from the rough endoplasmic reticulum. A model, which takes into account these enzymological and ultrastructural findings and their biological significance, is proposed.     

Effect of aging on changes in substrate and effector levels of rat-liver glycolytic and lipogenic enzymes during induction

The uptake and subcellular processing of radiolabelled prolactin has been studied in male and female rats. Analytical subcellular fractionation of liver homogenates from rats injected with 125I-prolactin showed that in female rats the prolactin was primarily internalised to low density (1.12 g X cm-3) membranes. Approx. 10-15% of the total label was found in high density membranes, similar in distribution to lysosomal marker enzymes. In the normal male rat, prolactin was internalised solely to lysosomal type membranes. However, in male rats treated with estrogen, the distribution of prolactin was very similar to that seem in the female, indicating that internalisation to low density membrane is dependent on the presence of prolactin receptors. Gel exclusion chromatography showed that the prolactin internalised to the lysosomal membranes was extensively degraded whereas that associated with the low density membrane remained intact. Use of digitonin, to establish the identity of the low density membrane gave inconclusive results, but suggested that the prolactin was associated with membrane bearing NADH pyrophosphatase rather than the classical Golgi marker, galactosyltransferase.     

Isolation of a Golgi rich fraction from rat ventral prostate

Unmodified procedures for isolation of fractions rich in Golgi elements from other tissues have not proved applicable to the rat ventral prostate because of the tendency of membranous material to aggregate. We have devised a new procedure whereby: 1) a Golgi rich fraction from rat ventral prostate was released by a gentle two-step homogenization and isolated by centrifugation through discontinuous sucrose density gradients; 2) the specific activity of UDP-galactose: glycoprotein galactosyltransferase increased 69-fold in this fraction; 3) the isolated Golgi fraction was reasonably free from mitochondria, lysosomes, endoplasmic reticulum and plasma membranes as shown by the relatively low activities of marker enzymes; 4) the specific activities of acid phosphatase and 5'-nucleotidase in the Golgi rich fraction was 4 times greater than that in prostate homogenate. Both enzymes are secretory products and their presence in Golgi elements is probably associated with their packaging in secretory granules.     

Phosphatidylinositol kinase and diphosphoinositide kinase of rat kidney cortex: properties and subcellular localization.

The properties of phosphatidylinositol kinase and diphosphoinositide kinase from rat kidney cortex were studied. The enzymes were completely Mg2+-dependent. Cutscum detergent activated phosphatidylinositol kinase, but diphosphoinositide kinase was inhibited by all detergents tested. The pH optima were 7.7 for phosphatidylinositol kinase and 6.5 for diphosphoinositide kinase. On subcellular fractionation of kidney-cortex homogenates by differential centriflgation, the distribution of phosphatidylinositol kinase resembled that of the marker enzymes for brush-border, endoplasmic-reticulum and Golgi membranes. Diphosphoinositide kinase distribution resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), diphosphoinositide phosphatase and triphosphoinositide phosphatase. Activities of both kinases were low in purified brush-border fragments. Diphosphoinositide kinase is probably localized in the Golgi complex.     

Isolation and characterization of a Golgi-rich fraction from the adrenal medulla.

1. A Golgi-rich fraction from bovine adrenal medulla was isolated by centrifugation through discontinuous sucrose density gradients. 2. The specific activity of UDPgalactose-N-acetylglucosamine galactosyl transferase was increased in this fraction. Therefore, this enzyme is a useful marker for Golgi in bovine adrenal medulla. 3. Golgi membranes were reasonably free from mitochondria, lysosomes, endoplasmic reticulum and chromaffin granules as shown by the relatively low activities of marker enzymes. 4. The negative staining techniques of electron microscopy revealed the presence of a system of tubules, vesicles and plate-like center regions which are similar to those structures previously described of the Golgi fraction isolated from the liver. 5. The specific activity of 5'-nucleotidase in the Golgi-rich fraction was 3.5 times greater than that in adrenal homogenates. However, the subcellular distribution patterns of galactosyl transferase and 5'-nucleotidase were similar. The possibility that 5'-nucleotidase might be a conspicious component of the Golgi apparatus is discussed.     

The subcellular distribution of rat liver serine-pyruvate aminotransferase.

1. The subcellular distribution of L-serine-pyruvate aminotransferase activity in rat liver was investigated. About 80% was recovered from cell-free homogenates in a 'total-particles' fraction and the remainder in the cytosol. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients showed a distribution for serine-pyruvate aminotransferase activity closely matching that observed for mitochondrial marker enzymes. 3. A study of the solubilization of enzymes from combined subcellular particles by digitonin at various concentrations also indicated a common subcellular location for serine-pyruvate aminotransferase and established mitochondrial enzymes. 4. The increase in liver serine-pyruvate amino-transferase activity induced by glucagon injection was accounted for as an increased mitochondrial activity.     

Biochemical studies on rat liver Golgi apparatus. II. Further characterization of isolated Golgi fraction.

Although the preparation of rat liver Golgi apparatus isolated by our method contains appreciable activities of NADH- and NADPH-cytochrome c reductases and glucose-6-phosphatase, these enzymes as well as thiamine pyrophosphatase of the extensively fragmented Golgi fraction are partitioned in aqueous polymer two-phase systems quite differently from those associated with microsomes. Similarly, the partition patterns of acid phosphatase and 5'-nucleotidase of the Golgi fragments differ from those of homogenized lysosomes and plasma membrane, respectively. It is concluded that most, if not all, of these marker enzymes in the Golgi fraction cannot be ascribed to contamination by the non-Golgi organelles. In sucrose density gradient centrifugation the NADH- and NADPH-cytochrome c reductase activities of the Golgi fraction behave identically with galactosyltransferase but differently from the reductase activities of microsomes, again indicating that the reductases are inherently associated with the Golgi apparatus. NADPH-cytochrome c reductase of the Golgi preparation is immunologically identical with that of microsomes. The marker enzymes mentioned above and galactosyltransferase behave differently from one another when the Golgi fragments are subjected to partitioning in aqueous polymer two-phase systems, suggesting that these enzymes are not uniformly distributed in the Golgi apparatus structure.     

CERAMIDE-GALACTOSYLTRANSFERASE AND CEREBROSIDE-SULPHOTRANSFERASE LOCALISATION IN GOLGI MEMBRANES ISOLATED BY A CONTINUOUS SUCROSE GRADIENT OF MOUSE BRAIN MICROSOMES

Abstract— A 17,000 g supernatant of mouse brain microsomes was subfractionated on a continuous sucrose gradient in order to localise ceramide galactosyltransferase (CGalt, EC 2.4.1.47) and cerebroside sulphotransferase (CST, EC 2.8.2.11), both enzymes involved in the synthesis of myelin lipids. The submicrosomal fractions were analysed for marker enzymes of myelin, plasma membranes, Golgi membranes, endoplasmic reticulum and lysosomes, and their protein distribution was studied. The results and EM studies give evidence that CGalT and CST are located in the Golgi membranes of the brain.     

Prespore cell fate bias in G1 phase of the cell cycle in Dictyostelium discoideum

By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.     

Uptake and processing of prolactin by alternative pathways in rat liver

The uptake and subcellular processing of radiolabelled prolactin has been studied in male and female rats. Analytical subcellular fractionation of liver homogenates from rats injected with ¹²⁵I-prolactin showed that in female rats the prolactin was primarily internalised to low density (1.12 g·cm^?3) membranes. Approx. 10–15% of the total label was found in high density membranes, similar in distribution to lysosomal marker enzymes. In the normal male rat, prolactin was internalised solely to lysosomal type membranes. However, in male rats treated with estrogen, the distribution of prolactin was very similar to that seen in the female, indicating that internalisation to low density membrane is dependent on the presence of prolactin receptors. Gel exclusion chromatography showed that the prolactin internalised to the lysosomal membranes was extensively degraded whereas that associated with the low density membrane remained intact. Use of digitonin, to establish the identity of the low density membrane gave inconclusive results, but suggested that the prolactin was associated with membrane bearing NADH pyrophosphatase rather than the classical Golgi marker, galactosyltransferase.     

Differentiation of mucous neck cells into parietal cells: a new concept of mitochondrial biogenesis

Parietal cells of the gastric fundic mucosa are small and contain only a few tiny mitochondria when they begin to differentiate from mucous neck cells. The canalicular ATPase activity characteristic of mature parietal cells is discrete in these young cells, whereas areas of very high activity are apparent in the Golgi complex, reticulum, nuclear envelope, mitochondrial wall, and plasma membrane. Close relations and contacts occur between mitochondria and these organelles, and the size and number of mitochondria increase progressively. These relations, as well as mitochondrial ATPase activity (a true differentiation marker), cease once the mitochondria become as numerous and large as those of a mature parietal cell. Our observations suggest that a secondary form of mitochondrial biogenesis, involving the massive participation of other organelles and independent of the classical mechanisms inherent in mitosis, occurs in parietal cells at the beginning of G1 phase during the 6 days of their maturation.     

Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells

Incubation of cultured cells at 20 degrees C blocks the transport of newly synthesized plasma membrane proteins, and the proteins accumulate intracellularly in a terminally glycosylated form. When baby hamster kidney cells are infected with the ts O45 mutant of vesicular stomatitis virus, and incubated at 20 degrees C, the terminally glycosylated spike glycoprotein G of the virus accumulates in the membranes of a tubular network localized on the trans side of the Golgi cisternae, the trans-Golgi network (TGN). We have used the G protein of ts O45 as a marker for the TGN and isolated a TGN fraction using a combination of conventional cell fractionation techniques and immunoisolation. The TGN was separated from the bulk of the endoplasmic reticulum, mitochondria, lysosomes, plasma membrane, and endosomes, while the activity of trans-Golgi marker galactosyltransferase copurified with the G protein. Using G protein as the TGN marker we have determined that the TGN was enriched 25-fold in the final fraction relative to the total homogenate. Several polypeptides (Mr 75,000, 87,000, 92,000, and 120,000) copurified with the G protein in the isolated TGN fraction and most likely represent resident markers of the compartment.     

Interaction of membranes of the Golgi complex with microtubules in vitro.

We have developed an in vitro assay for characterizing the binding of elements of the Golgi complex to microtubules. The binding assay comprises three distinct components, Golgi elements purified from Vero cells by subcellular fractionation, taxol-polymerized tubulin from bovine brain coupled to magnetic beads and cytosol from HeLa cells. Binding of Golgi elements to microtubules is quantitated by measuring the activity of the Golgi marker enzyme, galactosyltransferase, associated with the microtubule-coated beads retrieved with a magnet. In the presence of cytosol, 35 to 45% of the total input of galactosyltransferase activity (Golgi elements) bind to microtubules; only 3% of the Golgi elements bind to microtubules, however, in the absence of cytosolic factors. This binding is saturable at a cytosol concentration of approximately 5 mg/ml or at a high input of Golgi elements. Cytosol-stimulated binding of Golgi elements to microtubules is decreased to less than 15% when cytosol is pretreated with 2 mM N-ethylmaleimide (NEM) and it is abolished when cytosolic proteins are inactivated by heat or when microtubules have been coated with heat-stable microtubule-associated proteins (MAPs). Trypsinization of the membranes of the Golgi elements abolishes their ability to bind to microtubules. Furthermore, inactivation of cytoplasmic dynein by UV/vanadate treatment does not affect the binding. This suggests that the interaction of Golgi elements with microtubules depends on NEM-sensitive cytosolic factors and membrane-associated receptors, but not on the microtubule-based motor protein cytoplasmic dynein.     

Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus

Purified Golgi membranes of the human intestinal adenocarcinoma cell line Caco-2 were used as an antigen to produce a monoclonal antibody, G1/93, which specifically labels a tubulovesicular compartment near the cis side of the Golgi apparatus, including the first cis-cisterna itself, as visualized by single and double immunoelectron microscopy with antibodies against galactosyltransferase. The antigen recognized by G1/93 was identified as a protein with a subunit size of 53 kD. Pulse-chase experiments revealed that the 53-kD protein dimerizes immediately after synthesis followed by formation of oligomers of approximately 310 kD, probably homohexamers. The protein has a transmembrane topology with only a short cytoplasmic segment as assessed by protease protection experiments. Glycosidase digestion studies indicated that the protein is probably not glycosylated. The unique subcellular distribution of the G1/93 antigen in close vicinity to the cis-Golgi is in line with the notion that this protein may delineate the biosynthetic transport pathway from the endoplasmic reticulum to the Golgi apparatus. Moreover, G1/93 is a useful marker to identify the cis side of the Golgi apparatus in a variety of human cells.     

Characterization of tyrosylprotein sulfotransferase from rat liver and other tissues

The sulfoconjugation of tyrosyl residues is a widespread post-translational modification of biologically active peptides and proteins. In this paper we describe the characterization of a rat liver tyrosylprotein sulfotransferase that is capable of catalyzing the transfer of a sulfate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the synthetic polymer, poly-(Glu6,Ala3,Tyr1) (EAY; Mr 47,000) using a simple filter paper assay. Following sucrose density gradient centrifugation and comparison with known subcellular marker enzyme activities, rat liver tyrosylprotein sulfotransferase activity was shown to have a distribution similar to the Golgi enzyme, galactosyltransferase. Using the enriched Golgi preparation, rat liver tyrosylprotein sulfotransferase displayed a pH optimum of 6.7 and required the presence of 20 mM Mn2+ for maximal activity. Co2+ (20 mM) was able to produce 26% of the maximal stimulation observed with Mn2+, whereas other metal ions, such as Mg2+, Ca2+, and Co2+, were not effective in stimulating tyrosylprotein sulfotransferase activity. Whereas tyrosylprotein sulfotransferase activity was observed in the native membrane-bound state, EAY sulfation was maximally enhanced 3-fold when assayed in the presence of Lubrol Px. Under the optimal conditions for assaying the sulfation of EAY by a rat liver enriched Golgi fraction, significant degradation of the sulfate donor, PAPS, was observed. The addition of both NaF and 5'-AMP to the incubation mixture was found to effectively prevent PAPS degradation and increase the amount of product formed in the assay by 10-fold. Using the optimized conditions for the sulfation of EAY by rat liver tyrosylprotein sulfotransferase, membrane-bound sulfotransferase activity was also observed in the crude microsomal pellets of a variety of rat tissues, including lung, pituitary, and cerebellum, as well as in livers from different species.