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HeLa cell membranes were studied for the distribution and orientation of the Golgi marker enzyme uridine diphosphate-galactose:beta-D-N-acetylglucosamine beta, 1-4 transferase (GT). Short pulse labeling in the presence of [35S]methionine resulted in two precursor species (Mr = 44,000 and 47,000), present in a microsomal fraction with a density of 1.18 g/ml in sucrose, presumably derived from the rough endoplasmic reticulum. Processing of the N-linked oligosaccharide(s) occurred only after the precursor molecules migrated to lighter density fractions, presumably derived from the Golgi complex. The mature GT molecules (Mr = 54,000) contain O-linked oligosaccharides as shown by beta-elimination of metabolically incorporated [3H]galactose. The O-glycosylation occurred mainly in the light density fractions. The topology of GT was studied on membrane fractions after labeling with [35S]methionine as well as immunocytochemically on ultrathin cryosections at the electron microscope level. Our results indicate that both the antigenic determinants of GT as well as polypeptide chain are present intramembraneously and at the luminal side of the membranes of the Golgi complex and rough endoplasmic reticulum.

The effect of monensin on the metabolism and glycosylation of the trans-Golgi enzyme galactosyltransferase (GT) was studied in HeLa cells. The synthesis of GT was not affected by monensin; however, in the presence of the drug GT precursor molecules (Mr 54 000 and 47 000) were transported to the Golgi system at a slower rate than in control cells. the half-life of GT as an intrinsic membrane protein of the trans-Golgi system was prolonged in the presence of monensin. Mature GT (Mr 54 000) contained in addition to one N-linked oligosaccharide several O-linked oligosaccharides; the majority of them consisted of the disaccharide galactose-N-acetylgalactosamine containing one or two sialic acid residues. We conclude that GT resides at an intracellular location where significant sialylation occurs. The sialylation of O-linked oligosaccharides was not hampered by the presence of monensin. Two-dimensional gel electrophoresis combined with neuraminidase treatment revealed that GT consists of a number of species in a pI range between 4 and 7. Removal of sialic acid residues resulted in a less acidic pI range of GT but the charge heterogeneity persisted. Immunoelectron microscopy with antibodies against GT showed that monensin affects primarily the GT-containing cisternae.

A semiquantitative method using immunocytochemistry on ultrathin cryosections and the protein A-gold technique was performed to study the effect of insulin on the cellular distribution of the glucose transporters in cultured 3T3-L1 adipocytes. In basal cells a substantial portion of the label was present in a tubulovesicular structure at the trans side of the Golgi apparatus, likely to represent the trans-Golgi reticulum, and in small vesicles present in the cytoplasm. Treatment with insulin induced a rapid translocation of transporters from the tubulovesicular structure to the plasma membrane. The transporter labeling of the plasma membrane increased three-fold and that of the tubulovesicular structure decreased by half. There was no effect of insulin on the degree of label in the small cytoplasmic vesicles. Removal of insulin from stimulated cells rapidly reversed the distribution of transporters to that seen in basal cells. This study thus provides the first morphological evidence for the occurrence of transporter translocation in insulin action and identifies for the first time the intracellular location of the responsive transporters.

Malaisse-Lagae demonstrated in 1975 that peri-insular (PI) cells and tele-insular (TI) cells produce amylase (Am) and chymotrypsinogen (Ch) in a different ratio. These biochemical measurements are in contradiction with recent observations of Bendayan (1985), who found that the Am/Ch ratio measured with the protein A-gold technique applied to ultrathin Epon sections was the same in PI and TI cells. We have previously shown (Posthuma et al., 1984) that experimentally induced changes in Am and Ch content of rat pancreas are quantitatively reflected by immuno-gold labeling of zymogen granules in cryosections. Here we applied the same technique to compare the Am/Ch labeling density ratios in PI and TI pancreatic cells. To ascertain constancy of experimental conditions, we used ultrathin cryosections from tissue blocks consisting of TI and PI tissue elements. Consecutive sections of these blocks were alternatively immunolabeled for Am and Ch, using protein A-gold as marker. The density of gold particles over zymogen granules of both PI and TI cells was measured. It appeared that the Am/Ch labeling density ratio was significantly lower in PI than in TI cells. This difference resulted from a lower Am labeling as well as higher Ch labeling density over zymogen granules in PI cells.

Substantial information about the ASGP-R has accumulated in the 10 years following the initial studies of this receptor by Ashwell and Morell. Many of its biochemical properties, its structure, and its orientation within the plasma membrane are now known. The pathways of ASGP ligand and receptor, with the CURL organelle being a central component, are summarized in Fig. 18. The major pathway of the ligand through the cell, beginning with binding at the cell surface and ending with degradation in lysosomes, has been investigated in detail. Recently, alternate routes of the ligand such as the ligand recycling pathway have been observed. With regard to the itinerary of the receptor, there is now biochemical, kinetic, and morphological evidence to support receptor recycling. The new concept of CURL as an important intracellular organelle has originated from studies of ASGP-R recycling. Its importance in the dissociation and segregation of ligand and receptor as well as in receptor recycling is now evident. In addition, there has been a concurrent investigation of other receptor systems that participate in receptor-mediated endocytosis, providing parallels and contrasts to the ASGP-R of hepatocytes. Many critical issues still exist in the cell biology of the ASGP-R. What are the structural requirements of the receptor for ligand binding and subsequent endocytosis of the receptor-ligand complex? Very little is known about the interactions between the receptor and the lipid bilayer in which it resides. How does the receptor move laterally in the plasma membrane? Are there proteins or glycolipids closely associated with the ASGP-R and, if so, what is their function? What is the mechanism that causes receptor clustering into coated pits? Although the existence of a pathway for ligand recycling has been demonstrated, there are still many issues to be addressed. What signals a particular ligand molecule for recycling? Is it a stochastic process? What is the function of this route of ligand movement? How are the various ligand pathways coordinated and regulated? In addition, there are many unanswered questions regarding the receptor pathway. How does CURL mediate the sorting of ASGP-R from ligand? How are receptors with different destinations (e.g., ASGP-R and IgA receptor) sorted in CURL? What is the mechanism of ASGP-R degradation and how is it regulated? Finally, how does the Golgi function in the ASGP system and what is the relationship between the Golgi and CURL? Future investigation of these issues will require further observations with existing techniques as well as new approaches.(ABSTRACT TRUNCATED AT 400 WORDS)

Using double-label quantitative immunoelectron microscopy on ultrathin cryosections of rat liver, we have compared the endocytotic pathways of the receptors for asialoglycoprotein (ASGP-R), mannose-6-phosphate ligands (MP-R), and polymeric IgA (IgA-R). All three were found within the Golgi complex, along the entire plasma membrane, in coated pits and vesicles, and within a compartment of uncoupling of receptors and ligand ( CURL ). The receptors occurred randomly at the cell surface, in coated pits and vesicles. Within CURL tubules ASGP-R and MP-R were colocalized , but IgA-R and ASGP-R displayed dramatic microheterogeneity. Thus, in addition to its role in uncoupling and sorting recycling receptor from ligand, CURL serves as a compartment to segregate recycling receptor (e.g. ASGP-R) from receptor involved in transcytosis (e.g. IgA-R).

Immunogold double-labeling and ultrathin cryosections were used to compare the subcellular distribution of albumin, mannose 6-phosphate receptor (MPR), galactosyltransferase, and the lysosomal enzymes cathepsin D, beta-hexosaminidase, and alpha-glucosidase in Hep G2 cells. MPR and lysosomal enzymes were found throughout the stack of Golgi cisternae and in a trans-Golgi reticulum (TGR) of smooth-surfaced tubules with coated buds and vesicles. The trans-Golgi orientation of TGR was ascertained by the co-localization with galactosyltransferase. MPR was particularly abundant in TGR and CURL, the compartment of uncoupling receptors and ligands. Both TGR and CURL also contained lysosomal enzymes, but endogenous albumin was detected in TGR only. The coated buds on TGR tubules contained MPR, lysosomal enzymes, as well as albumin. MPR and lysosomal enzymes were also found in coated pits of the plasma membrane. CURL tubules seemed to give rise to smooth vesicles, often of the multivesicular body type. In CURL, the enzymes were found in the lumina of the smooth vesicles while MPR prevailed in the tubules. These observations suggest a role of CURL in transport of lysosomal enzymes to lysosomes. When the cells were treated with the lysosomotropic amine primaquine, binding of anti-MPR to the cells in culture was reduced by half. Immunocytochemistry showed that MPR accumulated in TGR, especially in coated buds. Since these buds contain endogenous albumin and lysosomal enzymes also, these data suggest that coated vesicles originating from TGR provide for a secretory route in Hep G2 cells and that this pathway is followed by the MPR system as well.

After receptor-mediated uptake, asialoglycoproteins are routed to lysosomes, while transferrin is returned to the medium as apotransferrin. This sorting process was analyzed using 3,3'-diaminobenzidine (DAB) cytochemistry, followed by Percoll density gradient cell fractionation. A conjugate of asialoorosomucoid (ASOR) and horseradish peroxidase (HRP) was used as a ligand for the asialoglycoprotein receptor. Cells were incubated at 0 degree C in the presence of both 131I-transferrin and 125I-ASOR/HRP. Endocytosis of prebound 125I-ASOR/HRP and 131I-transferrin was monitored by cell fractionation on Percoll density gradients. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation gave rise to a density shift of 125I-ASOR/HRP-containing vesicles due to HRP-catalyzed DAB polymerization. An identical change in density for 125I-transferrin and 125I-ASOR/HRP, induced by DAB cytochemistry, is taken as evidence for the concomitant presence of both ligands in the same compartment. At 37 degrees C, sorting of the two ligands occurred with a half-time of approximately 2 min, and was nearly completed within 10 min. The 125I-ASOR/HRP-induced shift of 131I-transferrin was completely dependent on the receptor-mediated uptake of 125I-ASOR/HRP in the same compartment. In the presence of a weak base (0.3 mM primaquine), the recycling of transferrin receptors was blocked. The cell surface transferrin receptor population was decreased within 6 min to 15% of its original size. DAB cytochemistry showed that sorting between endocytosed 131I-transferrin and 125I-ASOR/HRP was also blocked in the presence of primaquine. These results indicate that transferrin and asialoglycoprotein are taken up via the same compartments and that segregation of the transferrin-receptor complex and asialoglycoprotein occurs very efficiently soon after uptake.

An exploration was conducted as to whether the relative concentration of two intracellular proteins could be evaluated quantitatively from their labeling densities in ultrathin cryosections labeled with the immunogold technique. As a model rat pancreatic cells were used in which the content of amylase (Am) and chymotrypsinogen (Ch) was experimentally altered. Rats were fed either normal laboratory chow or food containing soybean trypsin inhibitor (STI), which affects the Am/Ch ratio in the tissues. The changes in Am and Ch protein levels and enzyme activities were measured biochemically in cell suspension homogenates or in zymogen granule fractions. Within 5 days a maximal change in the Am/Ch was observed as a result of adaptation to the STI diet. The Am/Ch ratio determined biochemically was compared with that from counts of gold particles bound to the respective protein in immunogold-labeled cryosections. The two data sets matched fairly well, indicating that the intensity of the immunoreaction is a reliable reflection of antigen concentration in this system.

Ubiquitin, a 76 amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Free (unconjugated) ubiquitin was localized in hepatoma cells using affinity purified anti-ubiquitin antibodies and colloidal gold immunoelectron microscopy. The anti-ubiquitin antibodies recognize only unconjugated ubiquitin. Ubiquitin is found within the cytoplasm, nucleus, the microvilli, autophagic vacuoles and lysosomes.

We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.

The intracellular distributions of the cation-independent mannose 6-phosphate receptor (MPR) and a 120-kD lysosomal membrane glycoprotein (lgp120) were studied in rat hepatoma cells. Using quantitative immunogold cytochemistry we found 10% of the cell's MPR located at the cell surface. In contrast, lgp120 was not detectable at the plasma membrane. Intracellularly, MPR mainly occurred in the trans-Golgi reticulum (TGR) and endosomes. lgp120, on the other hand, was confined to endosomes and lysosomes. MPR was present in both endosomal tubules and vacuoles, whereas lgp120 was confined to the endosomal vacuoles. In cells incubated for 5-60 min with the endocytic tracer cationized ferritin, four categories of endocytic vacuoles could be discerned, i.e., vacuoles designated MPR+/lgp120-, MPR+/lgp120+, MPR-/lgp120+, and vacuoles nonimmunolabeled for MPR and lgp120. Tracer first reached MPR+/lgp120-, then MPR+/lgp120+, and finally MPR-/lgp120+ vacuoles, which are assumed to represent lysosomes. To study the kinetics of appearance of endocytic tracers in MPR-and/or lgp120-containing pools in greater detail, cells were allowed to endocytose horse-radish peroxidase (HRP) for 5-90 min. The reduction in detectability of MPR and lgp120 antigenicity on Western blots, due to treatment of cell homogenates with 3'3-diaminobenzidine, was followed in time. We found that HRP reached the entire accessible pool of MPR almost immediately after internalization of the tracer, while prolonged periods of time were required for HRP to maximally access lgp120. The combined data suggest that MPR+/lgp120+ vacuoles are endocytic vacuoles, intermediate between MPR+/lgp120-endosomes and MPR-/lgp120+ lysosomes, and represent the site where MPR is sorted from lgp120 destined for lysosomes. We propose that MPR is sorted from lgp120 by selective lateral distribution of the receptor into the tubules of this compartment, resulting in the retention of lgp120 in the vacuoles and the net transport of lgp120 to lysosomes.

We used high-resolution immunocytochemistry on ultrathin frozen sections labeled with colloidal gold to study the subcellular distribution of the asialoglycoprotein receptor in rat liver. The receptor was localized along the entire hepatocyte plasma membrane, including the bile capillary membrane, but was scarce intracellularly. Sinusoidal lining (Kupffer) cells and blood cells showed no immunoreactivity. In liver cells of rats injected with 1 to 100 micrograms of asialoorosomucoid (ASOR) 2-15 min before tissue fixation, endocytotic internalization of receptors at the blood front was conspicuous. At all times in this interval, receptor was present in approximately 100-nm vesicles and larger vacuoles adjacent to the sinusoidal plasma membrane. No other significant intracellular receptor was noted during the 15-min exposure to ASOR; in particular, lysosomes and Golgi complex were not labeled. Our observations, in combination with data from the literature which demonstrate that, under these conditions, the ligand is transferred further to the Golgi complex-lysosome region, suggest that the receptor and ligand are dissociated in the vicinity of the plasma membrane, after which the receptor rapidly returns to the cell surface.

Complexes of protein-A with 5 and 16 nm colloidal gold particles (PA/Au5 and PA/Au16) are presented as sensitive and clean immunoprobes for ultrathin frozen sections of slightly fixed tissue. The probes are suitable for indirect labeling and offer the opportunity to mark multiple sites. The best procedure for double labeling was to use the smaller probe first, i.e., antibody 1 - PA/Au5 - antibody 2 - PA/Au16. When this was done, no significant interference between PA/Au5 and PA/Au16 occurred. Using this double-labeling procedure we made an accurate comparison between the subcellular distributions of amylase as a typical secretory protein and of GP-2 a glycoprotein, characteristic for zymogen granule membrane (ZGM) preparations. We prepared two rabbit antibodies against GP-2. One antibody (R x ZGM) was obtained by immunizing with native membrane material. The specificity of R x ZGM was achieved by adsorption with the zymogen granule content subfraction. The other, R x GP-2, was raised against the GP-2 band of the SDS polyacrylamide profile of ZGM. We found that the carbohydrate moiety of GP-2 was involved in the antigenic determinant for R x ZGM, while R x GP-2 was most likely directed against GP-2 polypeptide backbone. THe immunocytochemical observations showed that GP-2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane, the Golgi membranes, and its association with the membranes of the zymogen granules. On the other hand, GP-2 was present in the contents of the zymogen granules and in the acinar and ductal lumina. Also, a GP-2-like glycoprotein was found in the cannulated pancreatic secretion (Scheffer et al., 1980, Eur. J. Cell Biol. 23:122-128). Hence, GP-2 should be considered as a membrane-associated secretory protein of the rat pancreas.

The receptor for asialoglycoproteins (ASGPR) was localized in human hepatoma Hep G2 cells by means of quantitative immunoelectron microscopy. Without ligand added to the culture medium, we found 34% of the total cellular receptors on the plasma membrane, 37% in compartment of uncoupling receptor and ligand (CURL), and 21% in a trans-Golgi reticulum (TGR) that was defined by the presence of albumin after immuno-double labeling. A small percent of the ASGPR was associated with coated pits, the Golgi stacks, and lysosomes. After incubation of the cells with saturating concentrations of the ligand asialo-orosomucoid (ASOR), the number of cell surface receptors decreased to 20% of total cellular receptors, whereas the receptor content of CURL increased by a corresponding amount to 50%. The ASGPR content of TGR remained constant. In contrast, after treatment of the cells with 300 microM of the weak base primaquine (PMQ), cell surface ASGPR had decreased dramatically to only 4% of total cellular receptors whereas label in the TGR had increased to 42%. ASGPR labeling of CURL increased only to 47%. The labeling of other organelles remained unchanged. This affect of PMQ was independent of the presence of additional ASOR. Implications for the intracellular pathway of the ASGPR are discussed.

Using immunogold labeling of ultrathin cryosections, we have studied the localization of secretory, lysosomal, and membrane proteins in the Golgi complexes of several cell types. All proteins were present in the stacks of Golgi cisternae, illustrating that the cisternae comprise a ubiquitous way station for proteins with multiple destinations. The labeling patterns support the concept that peripheral Golgi vesicles represent the main site of secretory protein concentration. Of the membrane proteins studied, the Golgi enzyme galactosyltransferase was confined to the trans-most few cisternae, whereas the receptors for asialoglycoproteins and for polymeric immunoglobulin A occurred in most cisternae, with increasing concentration approaching the trans side. The findings are discussed in relation to a cisternal cis to trans progression of Golgi cisternae and membrane specificity.

An ultrastructural analysis of human cytotoxic T lymphocyte-target cell (CTL-TC) interaction has been undertaken to enable a better understanding of the killing mechanism. Attention was focused on granules in the CTL, which are known to contain lethal compounds. Within the membrane-delimited cytotoxic granule an electron-dense core as well as numerous membrane vesicles were identified. In CTL-TC conjugates, specific membrane interactions take place, allowing the formation of intercellular clefts into which the granule cores and internal vesicles are released. T cell surface membrane molecules known to be involved in CTL-TC interaction (T cell receptor, CD3 and CD8) are present on the membranes of the granule cores and internal vesicles, facing outward. An explanation for this localization of the membrane may be found in the fact that the granule is connected with an endocytotic pathway. Moreover, the lumen of the granule is rich in the enzyme cathepsin D, which indicates an association with a lysosomal compartment. Exocytosed vesicles and cores are seen to adhere to the plasma membrane of the TC. Although the exact contents of the granule vesicles and core remain to be identified, we suggest that specific interaction of CTL membrane molecules on the cytolytic granule components with molecules on the plasma membrane of the TC may ensure the unidirectional delivery of the lethal hit.

In this study, we re-investigated the localization of albumin in hepatocytes using immunocytochemistry (ICC) on ultrathin cryo-sections of rat livers, which were immersion or perfusion-fixed with paraformaldehyde or a mixture of paraformaldehyde and glutaraldehyde. The sections were indirectly labelled with rhodamine-conjugated IgG or with 8 nm protein A-colloidal gold particles. We demonstrate that perfusion-fixation is obligatory for a proper localization of albumin. After perfusion fixation, all hepatocytes of fed and fasted rats were uniformly labelled. At the subcellular level albumin was localized in hepatocytes in the membrane-bound compartments of the entire secretory pathway, i.e. the rough and smooth ER, peripheral Golgi vesicles, all Golgi cisternae and the secretory granules. Concentration of albumin appeared mainly to occur at the cis-Golgi side.

Human hepatoma cells, infected by vesicular stomatitis virus, offer a good system to study simultaneously the intracellular localization of a well defined transmembrane glycoprotein (VSV-G), a secretory glycoprotein (transferrin), and a nonglycosylated secretory protein (albumin). We used monospecific antibodies in combination with 5- and 8-nm colloidal gold particles complexed with protein A to immunolabel these proteins simultaneously in thin frozen sections of hepatoma cells. VSV-G, transferrin, and albumin are present in the same rough endoplasmic reticulum cisternae, the same Golgi compartments, and the same secretory vesicles. In the presence of the ionophore monensin intracellular transport is blocked at the trans cisternae of the Golgi complex, and VSV-G, transferrin, and albumin accumulate in dilated cisternae, which are apparently derived from the trans-Golgi elements. Glycoproteins, synthesized and secreted in the presence of monensin, are less acidic than those in control cultures. This is probably caused by a less efficient contact between the soluble secretory proteins and the membrane-bound glycosyltransferases that are present in the most monensin-affected (trans) Golgi cisternae.

Amylase (Am) and chymotrypsinogen (Chtg) were demonstrated in rat and guinea pig exocrine pancreatic cells by immunofluorescence and immunoferritin cytochemistry on thin and ultrathin frozen sections. We describe two observations indicating that Am and Chtg may behave differently in the pre-Golgi phase of their intracellular transport. Firstly, aggregates of material within the RER cisternae of the guinea pig (so-called intracisternal granules) reacted strongly with anti-Chtg, but showed no affinity for anti-Am. Secondly, in both rat and guinea pig, the increase in labeling intensity from cytoplasm (RER) to secretory granules was larger for Chtg than for Am. We hypothesize that the two proteins do not travel in-parallel towards the Golgi complex. Compared with Chtg, Am would lag behind in the RER cisternae.

Present knowledge of the in situ intracellular localization of acid hydrolases is mainly based on enzyme-cytochemical observations. In the preputial gland cells beta-glucuronidase and acid phosphatase were thus demonstrated in lysosome-like secretory granules and the GERL system. We applied immunocytochemistry to localize beta-glucuronidase at the earliest sites of biosynthesis. Lysosomal beta-glucuronidase was purified from preputial gland by column chromatography and SDS gel electrophoresis. Antibodies were raised in rabbit and affinity-purified preparations were used for immunocytochemistry on thin frozen sections of perfusion fixed preputial glands. Indirect procedures were applied with a second antibody labelled with rhodamine for fluorescence, and 5 or 8 nm protein A-gold probes for electron microscopy. beta-Glucuronidase occurred in all cells, except for the precursor cells, and was localized throughout the endoplasmic reticulum and perinuclear space, in all Golgi cisternae and storage granules, and in autophagic vacuoles. Thus in the preputial gland cell, beta-glucuronidase is present in both the lysosomal and the secretory system.

In the present study, we have localized by immunocytochemistry at the LM and EM level, procollagen type III (PIIIP), fibronectin (FN) and heparan sulfate proteoglycan (HSPG). Intracellularly, PIIIP was observed in both parenchymal and endothelial cells. In parenchymal cells, PIIIP was found in Golgi derived vesicles. This observation suggests that PIIIP synthesis is a normal function of liver parenchymal cells. In endothelial cells, vesicles, which could not be identified, were seen to contain PIIIP. This result does not allow to conclude, whether sinusoidal endothelial cells secrete or take up PIIIP. Extracellularly, PIIIP was present around portal and central veins, in the space of Disse and between adjacent parenchymal cells. In the space of Disse, almost all interstitial collagen fibrils reacted with the anti PIIIP antibodies. This observation leads to the conclusion that most fibrils of the space of Disse contain type III in addition to type I collagen molecules. By immunofluorescence, FN was seen mainly along the sinusoids in discrete dots. By EM, FN was found to be present in diffuse material closely associated with the sinusoidal membrane of the parenchymal cells and in strands connecting adjacent parenchymal cells, parenchymal and endothelial cells or parenchymal cells and collagen fibrils. FN was also present in vascular and ductular basal laminae. Strong HSPG reaction was observed around bile ducts. Moderate reaction was seen around blood vessels and in the space of Disse. In the latter location, the ultrastructural distribution of HSPG resembles that of FN, i.e. HSPG is present in diffuse material and in strands.

The weak bases chloroquine, primaquine, NH4Cl and the ionophore monensin exert similar but not identical effects on sorting, transport and processing of cathepsin D in several human cell lines (fibroblasts, HepG2 cells, U937, monocytes). The drugs inhibit the segregation of newly synthesized cathepsin D from the secretory route. The kinetics of transport of nonsegregated cathepsin D precursor along the secretory route is retarded resulting in a delayed hypersecretion. Higher concentrations of the drugs can arrest the intracellular transport completely. The extent of inhibition of segregation varies among the different human cell types tested. Thus, in fibroblasts the secretion can be stimulated to exceed 80%, while in U937 cells the secretion cannot be enhanced above 50% although both cell types have the same basal rate of secretion (approximately 10% of the synthesized cathepsin D). We suggest that pH-independent sorting mechanisms contribute to the targeting of cathepsin D in U937 cells. Processing of the cathepsin D remaining in cells is characteristically changed depending on the drug. The proteolytic processing is strongly inhibited by chloroquine and is rather insensitive to monensin. Unlike the other drugs, monensin blocks the formation of complex oligosaccharides in cathepsin D and allows for extensive secretion solely of molecules that are sensitive to endo H.

The occurrence of the circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was monitored during sporogonic development in Anopheles stephensi mosquitoes. Using a monoclonal anti-CS protein antibody (3Sp2) and immunogold labeling on ultrathin cryosections it was found that CS protein is synthesized in immature oocysts from day 6 onwards when there are not yet signs of sporozoite formation. The CS protein is rapidly incorporated in the oocyst plasmalemma, which subsequently invaginates into the parasite. In the oocyst only the external sporozoite membrane contains CS protein. The inner pellicle membranes, rhoptries and micronemes do not react with monoclonal antibody (MoAb) 3Sp2.

The acid hydrolases alpha-glucosidase, beta-galactosidase, N-acetyl-beta-D-hexosaminidase, beta-glucocerebrosidase and cathepsin D were studied immunocytochemically in normal and mutant human cells using monoclonal and affinity-purified polyclonal antibodies. For light microscopy, Rhodamine or Fluorescein-labelled conjugates were used, and for electron microscopy protein A-gold conjugates were employed. With the double labelling procedure, it was found that in normal fibroblasts every lysosome contained all the enzymes studied. The method described also enabled us to demonstrate the presence or absence of mutant enzyme protein in fibroblasts derived from patients with a genetic lysosomal enzyme deficiency. Immunoreactive acid hydrolases or their precursor forms were found in the rough endoplasmic reticulum, the cisternae of the Golgi complex, Golgi associated vesicles and lysosomes. This is in agreement with the present concept that the Golgi complex plays an essential role in the processing and targeting of lysosomal enzymes.

In rats infused with asialoglycoprotein for 60 min, receptor-mediated endocytosis of the ligand occurred exclusively in hepatic parenchymal cells. We have used double-label immunoelectron microscopy on ultrathin cryosections of rat liver to identify the site at which the asialoglycoprotein receptor and its ligand dissociate following their common endocytosis. Asialoglycoprotein receptor, ligand and clathrin were identified and quantitated by the use of monospecific antibodies followed by gold-protein A complexes. Both receptor and ligand were found associated with the membrane of clathrin-coated vesicles close to the cell surface. We identified other vesicles that contained ligand accumulated within the lumen. The membranes of these latter vesicles contained little receptor, but receptor was concentrated in tubular extensions that were largely free of ligand. We call this organelle CURL (compartment of uncoupling of receptor and ligand). CURL vesicles appear to transform into secondary lysosomes, wherein the ligand is degraded. The tubular vesicles are, we propose, an intermediate in recycling the receptor to the cell surface.

We used the immunogold method on ultrathin cryosections to measure intracellular amylase (Am) concentrations in subcellular compartments of rat exocrine pancreatic cells. Previously, the quantitation procedure was characterized in a model system consisting of Am dispersed at known concentration in a matrix of gelatin. Variations in labeling efficiency, due to differences in matrix density, were equalized by embedding in 30% polyacrylamide (PAA). Here we applied these model conditions to rat pancreas and established intracellular Am concentrations [Am]. Specimen blocks were composed of tissue and a reference layer of gelatin mixed with a known Am concentration ([Am]r), both fixed in glutaraldehyde. Cryosections of the PAA embedded blocks were immunogold labeled for Am. The labeling density was measured in the reference layer (LDr) and in structures in exocrine cells that were involved in Am synthesis and transport (LDs). In each of these structures the Am concentration ([Am]s) was calculated from: [Am]s = [Am]r. LDs/LDr In this way we measured average concentrations ranging from 63 mg/ml in rough endoplasmic reticulum to 261 mg/ml in secretory granules. Concentration of Am appeared to occur mainly in the most cis- and the most trans-Golgi cisternae. To check whether sterical hindrance was an inherent bias to the [Am] measurements in compartments that contained high concentrations of the enzyme, the labeling efficiency for Am in intact isolated secretory granules in gelatin and embedded in PAA, was compared with the efficiency when the granules were lysed and approximately 50 times diluted in gelatin before PAA embedment. It appeared that Am was detected with similar efficiency under both conditions. This demonstrated that sterical hindrance did not cause errors in the measurements of cellular Am concentrations.

In rat hepatocytes the Golgi complex contains a considerable amount of receptors for asialoglycoproteins (ASGP-R). To establish whether the presence of ASGP-R in the Golgi complex originate from de novo synthesis isolated rat hepatocytes were incubated with 100 micrograms/ml cycloheximide to stop protein synthesis. Provided that protein synthesis was completely inhibited by cycloheximide, uptake and degradation of ligand (asialo-orosomucoid) were unaffected. Also intracellular transport of newly synthesized proteins, as determined by monitoring biosynthesis and intracellular transport of albumin and ASGP-R, was not affected. After culturing the cells for 3.5 h in the presence of cycloheximide, no more albumin could be detected in the Golgi complex with immunofluorescence microscopy. However, immunocytochemical assessment showed that the ASGP-R was still in the Golgi complex. These results suggest that the Golgi complex contains a pool of ASGP-R which is independent of neosynthesis for several hours.