Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides

Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc₃Man₉(GlcNAc)₂, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associated with the Golgi. These include mannosidase I (removes 1-2 mannose residues from Man_6-9[GlcNAc]₂), mannosidase II (removes mannose residues from GlcNAcMan₅[GlcNAc]₂), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). We have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltransferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.     

Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol- dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.     

Distribution of glycosyltransferases among Golgi apparatus subfractions from liver and hepatomas of the rat.

Glycosyltransferase activities of highly purified fractions of Golgi apparatus, plasma membrane and endoplasmic reticulum, all from the same homogenates, were analyzed and compared. Additionally, Golgi apparatus were unstacked and the individual cisternae separated into fractions enriched in cis, median and trans elements using the technique of preparative free-flow electrophoresis. Golgi apparatus from both liver and hepatomas were enriched in all glycosyltransferases compared to endoplasmic reticulum and plasma membranes. However, Golgi apparatus from hepatomas showed both elevated fucosyltransferase and galactosyltransferase activities but reduced sialyltransferase and dipeptidyl peptidase IV (DPP IV) activities compared to liver. Activity of N-acetylglucosaminyltransferase was approximately the same in both liver and hepatoma Golgi apparatus. With normal liver, sialyl- and galactosyltransferase activities and DPP IV showed a marked cis-to-trans gradient of activity. Fucosyltransferase was concentrated in two regions of the electrophoretic separations, one corresponding to cis cisternae and one corresponding to trans cisternae. N-Acetylglucosaminyltransferase activity was more widely distributed but the endogenous acceptor activity was predominantly cis. With hepatoma Golgi apparatus, the pattern for DPP IV was similar to that for liver but those of sialyl- and galactosyltransferases differed markedly from liver. Instead of activity increasing cis to trans, the activities for sialyl- and galactosyltransferases decreased. For fucosyltransferases, activity dependent on exogenous acceptor was medial whereas with endogenous acceptor, two activity peaks, cis and trans, still were observed. For N-acetylglucosaminyltransferase the pattern for hepatoma was similar to that for liver. The results indicate alterations in the distribution of glycosyltransferase activities within the Golgi apparatus in hepatotumorigenesis that may reflect altered cell surface glycosylation patterns.     

Intracellular localization of GDP-l-fucose: Glycoprotein and CMP-sialic acid: Apolipoprotein glycosyltransferases in rat and pork livers

A GDP-l-fucose:glycoprotein fucosyltransferase which transfers l-fucose to terminal β-N-acetyl-d-glucosaminyl residues of sialidase-, β-galactosidase-treated α₁-acid glycoprotein and a CMP-sialic acid:glycoprotein sialyltransferase acting on sialidase-treated apolipoprotein-Ala₁ from human very low density lipoprotein have been shown to be concentrated in rat liver Golgi apparatus preparations at enrichments of 40- and 45-fold, respectively, and in pork liver Golgi-rich fractions at enrichments of 35- and 20-fold, respectively. A second fucosyltransferase acting on sialidase-treated α₁-acid glycopretein was absent from rat liver and was enriched only 13-fold in a pork liver Golgi-rich fraction. The smooth-surfaced microsome fraction was the only other rat liver subcellular fraction with appreciable levels of the GDP-l-fucose: β-N-acetyl-d-glucosaminide fucosyltransferase and the lipoprotein sialyltransferase (enrichments of 2.6- and 5.2-fold, respectivley). This enrichment could not be attributed to the plasma membrane content of the smooth microsome fraction since plasma membrane fractions from rat liver were shown to have relatively low concentrations of these two transferases (enrichments of 0.3 or less). Rat liver plasma membrane was also shown to have similarly low relative specific activities for three other glycosyltransferases (sialyl-, galactosyl-, and N-acetylglucosaminyl-). The accurate determination of the glycosyltransferase activities of the plasma membrane fraction required the use of relatively low concentrations of plasma membrane and relatively high concentrations of nucleotide-sugars in order to avoid interference by the high nucleotide-sugar pyrophosphatase and hydrolase activities of this fraction.     

Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.     

Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b alpha 2----8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis

Using a sucrose density gradient fractionation of a highly purified Golgi apparatus from rat liver, we determined the sub-Golgi distribution of CMP-NeuAc:GM3 ganglioside alpha 2----8sialyltransferase (GM3-SAT) and CMP-NeuAc:GT1b ganglioside alpha 2----8sialyltransferase (GT1b-SAT), in comparison with that of the other glycosyltransferase activities involved in ganglioside biosynthesis. While GM3-SAT was recovered in several density fractions, GT1b-SAT was mainly found on less dense sub-Golgi membranes; this indicates that these two activities are physically separate. Moreover, with regard to the monosialo pathway, CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase, UDP-GalNAc:GM3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GM2 ganglioside beta 1----3galactosyltransferase, and CMP-NeuAc:GM1 ganglioside alpha 2----3sialyltransferase were resolved from more dense to less dense fractions, respectively. In the disialo pathway, UDP-GalNAc:GD3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GD2 ganglioside beta 1----3galactosyltransferase and CMP-NeuAc:GD1b ganglioside alpha 2----3sialyltransferase co-distributed with the corresponding activities of the monosialo pathway. These last results indicate that many Golgi glycosyltransferases involved in ganglioside biosynthesis are localized in the order in which they act.     

Cerebroside Sulfotransferase in Golgi-Enriched Fractions from Rat Brain

Abstract: Golgi-enriched fractions were prepared from brainstems of 17-day-old rats by first floating off myelin, then fractionating the remaining pellet by a series of differential and density gradient centrifugations in sucrose. Fractions enriched in Golgi membranes were recovered at 0.46/0.76 m and 0.76/0.87 m interfaces on the final sucrose gradient as indicated by morphology and the biochemical markers thiamine pyrophosphatase and [³H]fucose-labeled glycoprotein. Morphology of the two fractions indicated very little contamination with myelin lamellae; however, the presence of significant levels of 2', 3'-cyclic nucleotidase in the lighter fraction suggested a contribution from oligodendroglial or myelin-related membranes. Cerebroside sulfotransferase was highly enriched in the lighter Golgi-enriched fraction relative to the denser fraction, the post-34, 880 x g microsomes, and the myelin-like fraction. In contrast, ceramide galactosyl transferase was more evenly distributed among the fractions. Our results show a more highly localized distribution of sulfatide synthesis than of galactocerebroside synthesis, probably in Golgi membranes or oligodendroglia-related membranes with similar properties.     

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.     

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.     

The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time

The increased polylactosamine glycosylation of LAMP-2 in MDCK cells cultured for 1 day relative to cells cultured for 3 days has been correlated with its slower rate of Golgi transit (Nabi and Rodriguez- Boulan, 1993, Mol. Biol. Cell., 4, 627-635). To determine if the differential polylactosamine glycosylation of LAMP-2 is a consequence of glycosyltransferase expression levels, the activities of beta1- 6GlcNAc-TV, beta1-3GlcNAc-T(i), beta1-2GlcNAc-TI, beta1, 4Gal-T, alpha2- 6sialyl-T, and alpha2-3sialyl-T were assayed and no significant differences in the activities of these enzymes in 1 and 3 day cell extracts were detected. During MDCK epithelial polarization, the Golgi apparatus undergoes morphological changes and apiconuclear Golgi networks were more evident in 3 day cells. Treatment with nocodazole disrupted Golgi networks and generated numerous Golgi clusters in both 1 day and 3 day cells. In the presence of nocodazole the differential migration of LAMP-2 in 1 and 3 day MDCK cells was maintained and could be eliminated by treatment with endo-beta-galactosidase, indicating that gross Golgi morphology did not influence the extent of LAMP-2 polylactosamine glycosylation. Nocodazole treatment did, however, result in the faster migration of LAMP-2 which was not due to modification of core N-glycans as the precursor form of the glycoprotein migrated with an identical molecular size. Following incubation at 20 degrees C, which prevents the exit of proteins from the trans-Golgi network, the molecular size of LAMP-2 increased to a similar extent in both 1 and 3 day MDCK cells. Extending the time of incubation at 20 degrees C did not influence the size of LAMP-2, demonstrating that its glycosylation is modified not by its retention within the Golgi but rather by its equivalent slower Golgi passage at the lower temperature in both 1 and 3 day cells. An identical effect was observed in nocodazole treated cells, demonstrating that Golgi residence time determines the extent of LAMP-2 polylactosamine glycosylation, even in isolated Golgi clusters.      

Sterol glucosylation by the plasma membrane from cotyledons of Phaseolus aureus

Membrane fractions were isolated from dark grown cotyledons of Phaseolus auneus by differential and sucrose density gradient centrifugation. Endoplasmic reticulum-, Golgi apparatus- and plasma membrane-rich fractions were identified by their respective enzymic activities and tested for their ability to transfer glucose from UDP-glucose to endogenous sterols to form steryl glucosides. The glucosyltransferase activity was shown to be located mainly at the plasma membrane.ABBREVIATIONS SG steryl glucoside - ASG acylated steryl glucoside - UDP-glc Uridine diphosphoglucose     

Systematic Analysis of proteoglycan modification sites in Caenorhabditis elegans by scanning mutagenesis

Proteoglycan modification is essential for development and early cell division in Caenorhabditis elegans. The specification of proteoglycan attachment sites is defined by the Golgi enzyme polypeptide xylosyltransferase. Here we evaluate the substrate specificity of this xylosyltransferase for its downstream targets by using reporter proteins containing proteoglycan modification sites from C. elegans syndecan/SDN-1. The N terminus of the SDN-1 contains a Ser-Gly proteoglycan site at Ser(71), flanked by potential mucin and N-glycosylation sites. However, Ser(71) was exclusively used as a proteoglycan site in vivo, based on mapping studies with a Ser(71) reporter protein, glycosyltransferase RNA interference, and co-expression of worm polypeptide xylosyltransferase. To elucidate the substrate requirements of this enzyme, a library of 42 point mutants of the Ser(71) reporter was expressed in tissue culture. The nematode proteoglycan modification site in SDN-1 required serine (not threonine), two flanking glycine residues (positions -1 and +1), and either one proximal acidic N-terminal amino acid (positions -4, -3, and -2) or a pair of distal N-terminal acidic amino acids (positions -6 and -5). C-terminal acidic amino acids, although present in many proteoglycan modification sites, had minimal impact on xylosylation at Ser(71). Proline inhibited glycosylation when present at -1, +1, or +2. The position of glycine, proline, and acidic amino acids allows the glycosylation machinery to discriminate between mucin and proteoglycan modification sites. The key residues that define proteoglycan modification sites also function with the Drosophila polypeptide xylosyltransferase, indicating that the specificity in the glycosylation process is evolutionarily conserved. Using a neural network method, a preliminary proteoglycan predictor has been developed.     

Golgi proteins persist in the tubulovesicular remnants found in brefeldin A-treated pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) and causes dismantling of the Golgi cisternae with relocation of resident Golgi proteins to the ER in many cultured cell lines. We examined the effects of BFA on Golgi organization and the distribution of Golgi markers in the rat exocrine pancreas. Immediately after BFA addition, Golgi stacks began to disorganize and Golgi cisternae to vesiculate, and by 15 min no intact Golgi cisternae remained. However, even after prolonged BFA incubation, clusters of small vesicles surrounded by transitional elements of the ER persisted both in the Golgi region and dispersed throughout the apical cytoplasm. These vesicles were morphologically heterogeneous in the density of their content and in the presence of cytoplasmic coats. Immunogold labeling demonstrated that some vesicles within the clusters contained gp58, a cis Golgi marker, and some contained alpha-mannosidase II, a middle/trans Golgi marker in this cell type. Neither marker was detected in the rough ER by immunogold or immunofluorescence labeling. When AlF4- was added during BFA treatment some of the vesicles in the clusters appeared coated. When microsomes were subfractionated into Golgi (light) and rough ER (heavy) fractions on sucrose density gradients, greater than 65% of alpha-mannosidase II and galactosyltransferase activities were found in light fractions (1.14-1.16 g/ml) in both control and BFA-treated lobules. In both cases equally low enzyme activity was recovered in heavier fractions (1.2-1.23 g/ml) containing RNA and alpha-glucosidase activity. However, 5 to 8% of the total recovered RNA consistently codistributed with the Golgi enzyme peak. These results indicate that BFA rapidly inhibits secretion and causes dismantling of the Golgi stacks in pancreatic acinar cells, but clusters of vesicles consisting of bona fide Golgi remnants persist even with prolonged exposure to BFA. Many of the vesicles contain Golgi markers by immunolabeling. By cell fractionation Golgi membrane enzyme activities are recovered in equal amounts in light (Golgi) fractions in both controls and BFA-treated specimens. These findings indicate that in the exocrine pancreas there is a dissociation of BFA's effects on the exocytic pathway: there is a block in transport and Golgi organization is disrupted, but remnant Golgi vesicles and tubules persist and retain Golgi membrane antigens and enzyme activities.     

Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [(14)C]lysine were assayed for hydroxy[(14)C]lysine and hydroxy[(14)C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[(14)C]lysine and glucosylgalactosylhydroxy[(14)C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [(14)C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. (14)C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5'-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.     

Localization of glycoprotein glycosyltransferases in the Golgi apparatus of rat and mouse testis

Golgi fractions prepared from rat testis have been shown to be enriched in the following glycoprotein glycosyltransferases: N-acetylglucosaminyltransferase, 47-fold, galactosyltransferase, 33-fold, and N-acetylglucosaminide fucosyltransferase, 15-fold. Appreciably lower transferase levels were obtained in other subcellular fractions. In the mouse, Golgi fractions were prepared from testis homogenates, testis cell suspensions and partially purified testis germinal cells; these fractions were also enriched in the above glycoprotein glycosyltransferases. Electron microscopic analysis indicated that a major portion of the total transferase activity was located in the Golgi apparatus of both rat and mouse testis although these experiments could not rule out the possible presence of some transferase activity in other organelles.     

Production of complex multiantennary N-glycans in Nicotiana benthamiana plants

In recent years, plants have been developed as an alternative expression system to mammalian hosts for the production of therapeutic proteins. Many modifications to the plant glycosylation machinery have been made to render it more human because of the importance of glycosylation for functionality, serum half-life, and the safety profile of the expressed proteins. These modifications include removal of plant-specific β1,2-xylose and core α1,3-fucose, and addition of bisecting N-acetylglucosamine, β1,4-galactoses, and sialic acid residues. Another glycosylation step that is essential for the production of complex human-type glycans is the synthesis of multiantennary structures, which are frequently found on human N-glycans but are not generated by wild-type plants. Here, we report both the magnICON-based transient as well as stable introduction of the α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV isozymes a and b) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) in Nicotiana benthamiana plants. The enzymes were targeted to the Golgi apparatus by fusing their catalytic domains to the plant-specific localization signals of xylosyltransferase and fucosyltransferase. The GnT-IV and -V modifications were tested in the wild-type background, but were also combined with the RNA interference-mediated knockdown of β1,2-xylosyltransferase and α1,3-fucosyltransferase. Results showed that triantennary Gn[GnGn] and [GnGn]Gn N-glycans could be produced according to the expected activities of the respective enzymes. Combination of the two enzymes by crossing stably transformed GnT-IV and GnT-V plants showed that up to 10% tetraantennary [GnGn][GnGn], 25% triantennary, and 35% biantennary N-glycans were synthesized. All transgenic plants were viable and showed no aberrant phenotype under standard growth conditions.     

Intracellular localization of heat shock proteins in maize

The intracellular distribution of the maize root heat shock proteins (hsp) was studied as a step toward understanding their physiological function. Linear sucrose density centrifugation was employed to separate organelles so the relative quantities of hsp in different subcellular compartments could be analyzed in a single preparation. Gradient fractions were assayed for the presence of hsp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and for marker enzyme activities. Analyses of 15 to 60% gradients showed five hsp to be organelle associated. Hsp 25 and 72 were in fractions containing closely equilibrating Golgi and endoplasmic reticulum marker activities, while hsp 18, 29, and 72 were in fractions containing overlapping plasma membrane, mitochondria, and glyoxysomal marker activities. Hsp larger than 72 kilodaltons were not present in gradient fractions. A second fractionation scheme achieved better separation of the two sets of closely equilibrating organelles. When a 13,000g centrifugation step to remove mitochondria was employed prior to gradient centrifugation, hsp 29 was absent from the gradient fractions. If the buoyant density of the endoplasmic reticulum was shifted by either maintaining the ribosomes on the membrane or removing them, a corresponding shift in the equilibrium positions of hsp 25 and 72 occurred. Hsp 18 and 70 remained in plasma membrane-containing fractions irrespective of these treatments.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid

Detailed investigations by quantitative centrifugal fractionation were conducted to determine the subcellular distribution of protein-bound sialic acid in rat liver. Homogenates obtained from perfused livers were fractionated by differential centrifugation into nuclear fraction, large granules, microsomes, and final supernate fraction, or were used to isolate membrane preparations enriched in either plasma membranes or Golgi complex elements. Large granule fractions, microsome fractions, and plasma membrane preparations were subfractionated by density equilibration in linear gradients of sucrose. In some experiments, microsomes or plasma membrane preparations were treated with digitonin before isopycnic centrifugation to better distinguish subcellular elements related to the plasma membrane or the Golgi complex from the other cell components; in other experiments, large granule fractions were obtained from Triton WR-1339-loaded livers, which effectively resolve lysosomes from mitochondria and peroxisomes in density gradient analysis. Protein-bound sialic acid and marker enzymes were assayed in the various subcellular fractions. The distributions obtained show that sialoglycoprotein is restricted to some particular domains of the cell, which include the plasma membrane, phagolysosomes, and possibly the Golgi complex. Although sialoglycoprotein is largely recovered in the microsome fraction, it has not been detected in the endoplasmic reticulum-derived elements of this subcellular fraction. In addition, it has not been detected either in mitochondria or in peroxisomes. Because the sialyltransferase activities are associated with the Golgi complex, the cytoplasm appears compartmentalized into components which biogenetically involve the Golgi apparatus and components which do not.     

Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the dimerization of lipoprotein lipase

Lipoprotein lipase (LPL), an enzyme playing the central role in triglyceride metabolism, is a glycoprotein and a homodimer of identical subunits. Dimerization and proper processing of oligosaccharide chains are important maturation steps in post-translational regulation of enzyme activity. Indirect evidences suggest that dimerization of LPL occurs in endoplasmic reticulum (ER) or Golgi. In this study, we investigated the dimerization status of LPL in 3T3-L1 adipocytes, using sucrose density gradient ultracentrifugation and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of ER-Golgi protein transport. In the presence of CCCP, no increase of cellular LPL activity was detected during 2 h of recovery period after the depletion of LPL with heparin and cycloheximide. Only endoglycosidase H (endo H)-sensitive subunits were found in CCCP-treated cells after endo H digestion, suggesting that inactive LPL was retained in ER. In the presence of castanospermine, an inhibitor of ER glucosidase I, LPL subunits of both control and CCCP-treated cells had same molecular weight, indicating that complete oligosaccharides were transferred to LPL subunits in the presence of CCCP. In sucrose density gradient ultracentrifugation, all the LPL protein synthesized in the presence of CCCP was found at the dimeric fractions as in control cells. Most of LPL protein in control cells showed high affinity for heparin, and there was no difference between the control and CCCP-treated cells. These results suggest that dimerization and acquisition of high affinity for heparin of LPL can occur in ER of CCCP-treated cells without acquisition of catalytic activity.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells I. Intracellular Site of Synthesis and Transport

The distribution of arabinose-containing macromolecules in suspension-culturedtobacco cells was examined using sucrose density gradients.Exogenously applied ¹⁴Carabinose was scarcely converted intoother sugars, and concentrated in the Golgi-rich fraction (1.15g/cm³) and then secreted to the cell wall. ¹⁴C-Arabinose wasalso incorporated in a lower sucrose density fraction (1.11g/cm³), which contains small vesicles presumably originatedfrom the Golgi apparatus. The arabinose-containing macromoleculesin this fraction was more easily solubilized in water than thosein the Golgi-rich fraction. Alkaline hydrolysis of the macromoleculesindicated that cell-wall glycoprotein is a major component ofthe macromolecules and that the degree of glycosylation is slightlygreater in the lower density fractions than in the Golgi-richfraction. Based on these results, a scheme is suggested in whichthe glycoproteins and polysaccharides are glycosylated in theGolgi apparatus and secreted to the cell wall via secretionvesicles in the low density fraction. The possibility of ¹⁴C-arabinose-containingmacromolecules, in the early phase of synthesis, being a markerof the plant Golgi apparatus is also proposed. (Received September 21, 1980; Accepted January 27, 1981)