Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

 Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway. Accepted: 26 January 1998     

Retinoid modulation of cell-free membrane transfer between endoplasmic reticulum and Golgi apparatus

Cell-free transfer of radiolabeled membrane proteins from part-rough, part-smooth transitional elements of the endoplasmic reticulum to Golgi apparatus immobilized to nitrocellulose in the presence of nucleoside triphosphate, an ATP-regenerating system and a cytosol fraction was promoted by retinol. At an optimum concentration of 1 microgram/ml, the rate and amount of transfer was approximately doubled over 1 to 2 h of incubation in the cell-free system. The transition vesicles induced to form in the cell-free system were concentrated by preparative free-flow electrophoresis in order to study separately the steps of vesicle formation from transitional endoplasmic reticulum and the steps of vesicle fusion with Golgi apparatus. The retinol effect was on vesicle formation as evidenced by an approx. 2-fold increase in transition vesicle numbers, as determined by electron microscope morphometry, and amount from protein determinations on the isolated fractions enriched in transition vesicles. The retinol response in the complete transfer could be eliminated by addition of concentrated cytosol, including cytosol depleted of retinol. An interaction of retinol with some component of the vesicle formation process, possibly involving guanine nucleotides, is indicated.     

Zopfochytrium is a new genus in the Chytridiales with distinct zoospore ultrastructure

While surveying chytrid diversity in lakes and streams, we found on cellulosic bait a chytrid that had both monocentric and polycentric thallus forms. We brought this chytrid into axenic culture from three sites in eastern North America, studied its thallus development and zoospore ultrastructure, and compared its 28S rDNA sequence with those of other members of the Chytridiomycota. Thallus morphology matched that described for the rare chytrid, Cladochytrium polystomum Zopf. Sporangia were spherical and produced numerous long discharge tubes. After discharge, zoospores remained in spherical clusters at the tips of the inoperculate openings of discharge tubes. After 10–30 min zoospores either swam away or encysted in place. Zoospore ultrastructural features included a cell coat, flagellar plug, and paracrystalline inclusion, features typical of members of the Chytridiales. However, the flagellar apparatus structure and organellar organization differed from that of zoospores previously described. Based on its molecular phylogeny and its zoospore ultrastructural features, we classify C. polystomum as a member of the Chytridiaceae in the Chytridiales. Because its thallus development and its ribosomal DNA sequences diverged decidedly from those of Cladochytrium tenue Nowak, the type species of Cladochytrium, we erected Zopfochytrium as a new genus for this chytrid.     

Mitosis, Cytokinesis and Colony Formation in Pediastrum boryanum

Uninucleate cells of Pediastrum become multinucleate by a seriesof synchronous mitoses. Mitotic nuclei are enclosed by a perinuclearenvelope of endoplasmic reticulum. Cytoplasmic cleavage of themultinucleate cells leads to the production of uninucleate,biflagellate zoospores (zooids) which are subsequently releasedinto a lenticular vesicle through a rupture in the outer layerof the parental cell wall. Within the vesicle, presumably derivedfrom part of the inner layer of parental wall, the zooids swarmactively before aggregating in a planar array. Bands of microtubulesunderlie the plasmalemma of the zooids which, when the zooidsaggregate, are usually coplanar with the newly formed colony.The role of microtubules in patterned colony formation and inthe development of the characteristic horns on peripheral cellsof colonies of Pediastrum is discussed.     

Development of the discharge apparatus in the fungus Entophlyctis

Correlative light and electron microscopic observations were used to reconstruct the morphological events involved in the development of the discharge apparatus of Entophlyctis zoosporangia. A discharge plug formed as vesicles containing fibrillar material fused with the plasma membrane and deposited their matrices between the plasma membrane and zoosporangial wall. At the apex of the enlarging plug, the zoosporangial wall lost its microfibrillar appearance, became diffuse, and left an inoperculate discharge pore. The discharge plug exuded through this pore and then expanded into a sphere which rested at the tip of the discharge papilla or tube. After the release of the discharge plug, the number of fibrilla containing vesicles decreased and abundant endoplasmic reticulum appeared in the cytoplasm below the plug. Granular material then accumulated at the interface of the discharge plug and the plasma membrane. This was the endo-operculum. A single layer of endoplasmic reticulum subtended the area of plasma membrane which the endo-operculum covered. Later, dictyosomes appeared in the cytoplasm below the endo-operculum. Fusion of Golgi vesicles with the plasma membrane below the endo-operculum coincided with the initiation of cytoplasmic cleavage. This sequence of events indicates that, unlike the discharge plug, the endo-operculum does not originate by vesicular addition of preformed material.     

Pattern formation and the fine structure of the developing cell wall in colonies of Pediastrum boryanum

A single-layered disc of peripheral pronged cells and central prongless cells impart the typical gear shape to colonies of Pediastrum, while the walls of each cell have a characteristic reticulate triangular pattern. The two-layered wall forms in the cells during colony formation following zoospore aggregation and adhesion. The uniformly thin outer layer reflects contours resulting from differential thickening in the reticulate pattern of the inner, thicker, more fibrillar and granular wall layer. The reticulate pattern thus imparted to the outer wall layer persists in empty zoosporangia following the release of zoospores. Columns of electron-dense material extend through the outer wall layer except at the ridges and centers of the reticulum. Following mitosis and cleavage, the resulting zoospores are extruded within a vesicle membrane consisting of the inner wall layer. Separation of this membrane from the parent cell occurs in material of the inner layer adjacent to the outer wall. Vesicles containing swarming zoospores also contain a granular material which appears to become associated with the aggregating and adhering cells of new colonies. Microtubules occur in zoospores prior to adherence but are absent during wall deposition.     

CELL SHAPE AND WALL PATTERN IN RELATION TO CYTOPLASMIC ORGANIZATION IN PEDIASTRUM SIMPLEX

P. simplex is a single-pronged, fenestrated species of Pediastrum. Comparison is made in regard to cell differentiation and structure with P. boryanum, a 2-pronged, unfenestrated species, with emphasis on the origin of cell wall pattern and the regulation of cell shape. The characteristic wall pattern is initiated with the deposition of plaques of wall material of the outer wall layer when zoospores have assembled in the colony. The pattern is postulated to be templated in the plasma membrane. The inner, thicker wall layer is fibrillar and deposited from vesicles derived from the golgi apparatus. In P. simplex 2–4 dictyosomes are present in contrast to the single dictyosome of P. boryanum. The dictyosomes lie at the concave inner face of the nucleus. Blebs of its ribosome-free outer membrane are contributed to the forming face of the golgi apparatus. Parallel microtubules underlie the plasma membrane in the aggregating zoospores and disappear after the initiation of wall formation. The possible role of microtubules and other organelles in the determination of cell shape in Pediastrum is discussed.     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.     

Ultrastructure of the pea cotyledon Golgi apparatus: Origin of dense vesicles and the action of brefeldin A

Summary We have followed the action of brefeldin A (BFA) on the Golgi apparatus of developing pea cotyledons, the cells of which are actively engaged in the synthesis and deposition of storage proteins. The Golgi apparatus of normal cells is characterized by the presence of three different types of vesicle: smooth-surfaced secretory vesicles, dense vesicles which carry the storage proteins, and clathrin-coated vesicles (CCV). The dense vesicles originate at the cis cisternae and undergo a maturation as they pass through the Golgi stack, presumably as a result of cisternal progression. CCV bud off from dense and smooth vesicles, which may be attached to one another, at the trans pole of the Golgi apparatus. BFA eliminates the CCV and leads, initially, to an increase in the number and length of the cisternae. Dense vesicles are still to be seen, and many show an increase in diameter. Longer BFA treatments result in a trans-driven vesiculation and an accumulation of vesicles within the vicinity of single cisternae. The vesicles were sometimes seen to be connected to one another via a network of tubules. As judged by immunocytochemistry with gold-coupled legumin and vicilin antisera, some of the dilated vesicles originate directly from dense vesicles by swelling whereas others probably arise by dilation of Golgi cisternae since they possess a layer of flocculent storage proteins at their periphery. By contrast the centre of the dilated vesicles labels positively with antibodies against complex glycans, indicating that the ability to segregate storage proteins from cell wall or lytic vacuole glycoproteins is lost during extended BFA treatment. The effects of BFA are reversible when cotyledons are further incubated on Gamborg's medium for 5 h without the inhibitor.Dedicated to Professor R. Kollmann on the occasion of his 65th birthday.     

Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)     

The occurrence of the Golgi apparatus in mouse oocytes. Demonstration of thiamine pyrophosphatase activity

Location of thiamine pyrophosphatase activity as a marker of the Golgi apparatus was studied ultracytochemically in mouse oocytes with germinal vesicle (OGV), oocytes at metaphase I (OMI) and oocytes at metaphase II (OMII), and further in cells of the respective cumulus oophorus serving as comparative objects. TPPase activity in cumulus oophorus cells and in OGV was found exclusively in the Golgi apparatus. In OMI the reaction product of TPPase activity was observed in isolated smooth vesicles, and in only one case in structures identifiable as the Golgi apparatus. In OMII the occurrence of TPPase activity was also recorded in isolated smooth vesicles in cortical cytoplasm and further, exceptionally, in smooth concentrically arranged vesicles or tubules. The TPPase activity was not present in vesicular complexes. The results have shown that after the resumption of meiosis the occurrence of the reaction product of TPPase activity drops abruptly due to the reduction of the Golgi apparatus. Changes affecting the Golgi apparatus after the resumption of meiosis are related to the loss of the nucleus after the germinal vesicle breakdown.     

Lipoprotein secretion by rat liver Golgi apparatus. Lipoprotein particles and lipase activity.

Lipoprotein particles of the size range of very low density lipoproteins in smooth endoplasmic reticulum, peripheral elements of the Golgi apparatus, and secretory vesicles of the immature Golgi apparatus face are 55 to 80 nm in diameter. Particles in mature secretory vesicles are smaller (45 nm). Concomitant with the change in particle size, the lumina of mature vesicles increase in electron density. A technique to fractionate immature and mature secretory vesicles was based on precipitation of a cupric-ferrocyanide complex (Hatchett's brown) through the action of a NADH-ferricyanide oxido-reductase resistant to glutaraldehyde which is characteristic of the membranes of mature secretory vesicles and of the plasma membrane of liver. Mature secretory vesicle fractions so isolated were enriched in cholesterol and depleted in triglycerides relative to immature vesicles on a phospholipid basis. Lipase activity was present in secretory vesicle fractions of the Golgi apparatus as shown by biochemical analysis and by cytochemistry. Cytochemical studies showed lipase to be present in both mature and immature vesicles but most evident in immature vesicles. The findings suggest that some very low density lipoprotein particles are converted to particles of smaller diameter during transit through Golgi apparatus. A lipase-mediated hydrolysis of triglycerides may relate to the transformation.     

Differentiation and continuity of the Golgi apparatus during carposporogenesis inPolysiphonia (Rhodophyta)

Summary Changes in the morphology of the Golgi apparatus during carposporogenesis occur gradually and are observed first in proximal cisternae. As successive cisternae mature, the appearance of the Golgi apparatus is gradually altered. Morphological changes are observed in the contents of differentiating cisternae and detached vesicles, and in the mode of vesicle detachment. The reprogramming of the Golgi apparatus between consecutive developmental stages does not appear to occur abruptly. Cisternae transitional between successive stages are observed to have features characteristic of both. Several unusual morphological features of the Golgi apparatus and detached vesicles are discussed.     

ATP-dependent fusion of liposomes with the Golgi apparatus of perforated cells

We examined the interactions of lipid vesicles (liposomes) labeled with various fluorescent markers with the intracellular membranes of semi-intact ("perforated") fibroblasts. When incubations were performed in the presence of an ATP-regenerating system, both vesicle lipids and entrapped water soluble markers were transferred to the Golgi apparatus of treated cells, indicative of membrane fusion. Fusion occurred using unilamellar vesicles 30-80 nm in diameter and composed of phosphatidylcholine alone, but was inhibited when equimolar amounts of either phosphatidylserine or sphingomyelin were present in the vesicles. Lipid vesicle-Golgi membrane fusion was also inhibited by pretreatment of the perforated cells with N-ethylmaleimide. These findings suggest that lipid vesicles may be useful for delivery of labeled lipids, macromolecules, and dyes to the Golgi apparatus, and for modeling the interactions of transport vesicles with the Golgi apparatus.     

Vesicle production and fusion during lobe formation inMicrasterias visualized by high-pressure freeze fixation

Summary Two different types of Golgi vesicles involved in wall formation can be visualized during lobe growth inMicrasterias when using high-pressure freeze fixation followed by freeze substitution. One type that corresponds to the dark vesicles (DV) described in literature seems to arise by a developmental process occurring at the Golgi bodies with the single vesicles being forwarded from one cisterna to the next. The other vesicle type appears to be produced at thetrans Golgi network without any visible precursors at the Golgi cisternae. A third type of vesicle, produced by median andtrans cisternae, contains slime; these are considerably larger than those previously mentioned and they do not participate in wall formation. The distribution of the two types of cell wall vesicles at the cell periphery and their fusion with the plasma membrane are shown for the first time, since chemical fixation is too slow to preserve a sufficient number of vesicles in the cortical cytoplasm. The results indicate that fusions of both types of vesicles with the plasma membrane are possible all over the entire surface of the growing half cell. However, the DVs are much more concentrated at the growing lobes, where they form queues several vesicles deep behind zones on the plasma membrane thought to be specific fusion sites. The structural observations reveal that the regions of enhanced vesicle fusion correspond in general to the sites of calcium accumulation determined in earlier studies. By virtue of the absence of the DVs in the region of cell wall indentations the second type of wall forming vesicle appears prominent; they too fuse with the plasma membrane and discharge their contents to the wall.     

Tubules of the trans Golgi apparatus visualized by immunoelectron microscopy

 Tubules constitute an integral part of the Golgi apparatus and have been shown to form a complex and dynamic network at its trans side. We have studied in detail structural features of the trans Golgi network and its relationship with the cisternal stack in thin sections of Lowicryl K4M embedded human absorptive enterocytes by immunolectron microscopy. Immunoreactive sites for α1,3 N-acetylgalactosaminyltransferase and blood group A substance were detectable troughout the cisternal stack and the entire trans Golgi network. Furthermore, the entire trans Golgi network was reactive for CMPase activity. Evidence for two kinds of tubules at the trans side of the Golgi apparatus was found: tubules that laterally connect adjacent and distant cisternal stacks, and others extending from central and lateral portions of trans cisternae to form the complex and extensive trans Golgi network. Trans cisternae showed often the peeling-off phenomenon and were continuous with the trans Golgi network. Both, trans cisternae and tubules of the trans Golgi network exhibited regionally buds and vesicles with a lace-like, non clathrin coat, previously reported by others in NRK cells, which contained glycoproteins with terminal N-acetylgalactosamine residues. These buds and vesicle are therefore involved in constitutive exocytosis. Accepted: 12 January 1998     

Involvement of the Golgi apparatus in sorting of materials to opposite ends of frog rod retinal photoreceptors

We have studied the rod cells of retinas of Rana pipiens by phosphatase cytochemistry and immunocytochemistry. We find that the Golgi apparatus of these cells, although different in its intracellular distribution from that of other neurons, has a cis-trans organization like that of other neurons as regards morphological features and the distribution of phosphatase activities. Antibodies against opsin bind to several sacs of the rod Golgi apparatus, especially those at the trans side of the Golgi stack. This suggests that Golgi involvement in the packaging of opsin for eventual delivery to the photoreceptive outer segments of the cell involves passage through trans Golgi systems. Proteins destined for the opposite end of the cell--the presynaptic terminal--also seem to pass through trans Golgi systems, as is indicated both by immunocytochemical localization of the synaptic vesicle protein p38 (synaptophysin) and by the presence of thiamine pyrophosphatase activity in some of the synaptic vesicles. Our findings suggest that sorting of membrane proteins destined for opposite ends of the photoreceptor takes place in systems at or near the trans Golgi face.     

ANALYSIS OF MUCILAGE SECRETION AND EXCRETION IN MICRASTERIAS (CHLOROPHYTA) BY MEANS OF IMMUNOELECTRON MICROSCOPY AND DIGITAL TIME LAPSE VIDEO MICROSCOPY1

Two different, independent, and alternative modes of mucilage excretion were found in the unicellular green alga Micrasterias denticulata Bréb. under constant culture conditions. The cells were capable of either excreting mucilage over all their cell surface or they extruded mucilage from one of their polar ends, which enabled directed movement such as photoorientation or escape from unfavorable environmental conditions. By means of a polyclonal antibody raised against Micrasterias mucilage, the secretory pathway of Golgi derived mucilage vesicles from their origin to their discharge was analyzed by means of conventional and energy filtering TEM. Depending on the stage of the cell cycle, mucilage vesicles were subjected to maturation processes. This may occur either after they have been pinched off from the dictyosomes (e.g. during cell growth) or when still connected to trans‐Golgi cisternae, as in the case of interphase cells. Only fully grown mature vesicles contained mucilage in its final composition as indicated by antibody labeling. After fusion of mucilage vesicles with vacuoles, no immunolabeling was found in vacuoles, indicating that the vesicle content was digested. Mucilage vesicles fused with the plasma membrane in areas of cell wall pores but were also able to excrete mucilage at any site directly through the respective cell wall layer. This result disproves earlier assumptions that the pore apparatus in desmids are the only mucilage excreting areas at the cell surface. Both mechanisms, excretion through the pores and through the cell wall, lead to formation of mucilage envelopes covering the entire cell surface.     

Xylosylation and glucuronosylation reactions in rat liver Golgi apparatus and endoplasmic reticulum

We have studied in rat liver the subcellular sites and topography of xylosylation and galactosylation reactions occurring in the biosynthesis of the D-glucuronic acid-galactose-galactose-D-xylose linkage region of proteoglycans and of glucuronosylation reactions involved in both glycosaminoglycan biosynthesis and bile acid and bilirubin conjugation. The specific translocation rate of UDP-xylose into sealed, "right-side-out" vesicles from the Golgi apparatus was 2-5-fold higher than into sealed right-side-out vesicles from the rough endoplasmic reticulum (RER). Using the above vesicle preparations, we only detected endogenous acceptors for xylosylation in the Golgi apparatus-rich fraction. The specific activity of xylosyltransferase (using silk fibroin as exogenous acceptor) was 50-100-fold higher in Golgi apparatus membranes than in those from the RER. Previous studies had shown that UDP-galactose is translocated solely into vesicles from the Golgi apparatus. In these studies, we found the specific activity of galactosyltransferase I to be 40-140-fold higher in membranes from the Golgi apparatus than in those from the RER. The specific translocation rate of UDP-D-glucuronic acid into vesicles from the Golgi apparatus was 10-fold higher than into those from the RER, whereas the specific activity of glucuronosyltransferase (using chondroitin nonasaccharide as exogenous acceptor) was 12-30-fold higher in Golgi apparatus membranes than in those from the RER. Together, the above results strongly suggest that, in rat liver, the biosynthesis of the above-described proteoglycan linkage region occurs in the Golgi apparatus. The specific activity of glucuronosyltransferase, using bile acids and bilirubin as exogenous acceptor, was 10-25-fold higher in RER membranes than those from the Golgi apparatus. This suggests that transport of UDP-D-glucuronic acid into the RER lumen is not required for such reactions.