Sphingomyelin synthase is absent from endosomes

Both the Golgi and the endosomes have recently been proposed as the main site of SM-synthase, the enzyme responsible for sphingomyelin (SM) biosynthesis. To settle this confusion, we studied the subcellular distribution of SM-synthase in human liver-derived HepG2 and baby hamster kidney BHK-21 cells. To discriminate between Golgi and endosomes we made use of 3,3-diaminobenzidine (DAB) cytochemistry. Cells were incubated with a conjugate of transferrin (Tf) and horseradish peroxidase (HRP), or with unconjugated HRP, to label the recycling pathway and the complete endocytic pathway (including lysosomes) with peroxidase activity, respectively. After cell homogenization, the peroxidase activity was used to induce a local deposition of DAB-polymer. The total SM-synthase activity was not affected by this procedure, and, in contrast to endosomes labeled with (125)I-Tf, organelles containing SM-synthase did not increase in buoyant density as determined by Percoll density gradient fractionation. Thus, little, if any, SM-synthase localizes to the endocytic pathway of HepG2 and BHK-21 cells. In experiments performed at low temperature to inhibit vesicular transport, we found less than 10% of newly synthesized short-chain SM at the cell surface. We conclude that most SM-synthase activity is present in the Golgi, and to a small extent at the cell surface.

A novel method for measuring protein expression at the cell surface

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3′-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into suspension by proteinase K and EDTA treatment at 0 degrees C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin concanavalin A (ConA). The suspended cells were encapsulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant 125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Surface 125I-labelled beta-Na+,K(+)-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, including endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum.

We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were also incubated with Tf/HRP. DAB cytochemistry caused approximately 40% of microsome-associated "complex" glycosylated [35S]alpha 1-antitrypsin ([35S]c-AT) to shift in a Percoll density gradient. Only part of the density shifted [35S]c-AT could be recovered by immunoprecipitation. A maximum effect was measured already after 10 min of Tf/HRP uptake. The density distribution of the "high mannose" glycosylated form of 35S-alpha 1-anti-trypsin [( 35S]hm-AT) was not affected by Tf/HRP. If in addition to Tf/HRP also an excess of non-conjugated transferrin was present in the medium, [35S]c-AT was not accessible for Tf/HRP, showing the involvement of the transferrin receptor (TfR) in the process. Furthermore, we show that if Tf/HRP and [35S]c-AT were located in different vesicles, the density distribution of [35S]c-AT was not affected by DAB-cytochemistry. Pulse-labeling with [35S]methionine was used to show that [35S]c-AT became accessible to endocytosed Tf/HRP minutes after acquirement of the complex configuration. A common intracellular localization of endocytosed Tf/HRP and secretory protein could be confirmed by immuno-electron microscopy: cryosections labeled with anti-albumin (protein A-colloidal gold) as well as DAB reaction product showed double-labeling in the trans-Golgi reticulum.

Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells.

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.

Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins.

Rat liver organelles involved in receptor-mediated endocytosis were labeled with a conjugate of galactosylated BSA to horseradish peroxidase [( 3H]galBSA-HRP), injected 10 min before sacrifice. These organelles were recovered at low density (1.11-1.13 g/ml) in sucrose gradients (Quintart, J., P. J. Courtoy, J. N. Limet, and P. Baudhuin, 1983, Eur. J. Biochem., 131:105-112). Upon incubation of such low density fractions in 3,3'-diaminobenzidine (DAB) and H2O2 and equilibration in a second sucrose gradient, galBSA-HRP-containing particles selectively shifted towards heavier densities (Courtoy, P. J., J. Quintart, and P. Baudhuin, 1984, J. Cell Biol., 98:870-876, companion paper), resulting in up to 250-to 300-fold purification with respect to the homogenate. The most purified preparations, wherein DAB-stained structures represented approximately 85% of the total volume of particles, contained only trace activities of enzymes usually regarded as markers for other subcellular entities. These minor activities could reflect either contamination or true enzyme association to the ligand-containing structures. Considering the latter hypothesis, at most 1.0% of alkaline phosphodiesterase I and 2.6% of 5'-nucleotidase (markers for plasma membrane), 3.6% of N-acetyl-beta-glucosaminidase (lysosomes), and 6.0% of galactosyltransferase (Golgi complex) from the homogenate would be associated with the whole population of ligand-containing organelles. After DAB cytochemistry on liver fixed 10 min after galBSA-HRP injection, ligand-containing structures accounted for 0.78-0.89% of the fractional volume of the hepatocytes and displayed a membrane area of 2,100 cm2/cm3, compared with 6,700 cm2/cm3 for the pericellular membrane. Altogether, our data support the hypothesis that these ligand-containing organelles are structurally distinct from plasma membrane, lysosomes, and Golgi complex.

Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.

Galactosylated BSA (galBSA) and its conjugate to horseradish peroxidase (galBSA-HRP) enter the galactose-specific pathway of hepatocytes. 10 min after intravenous injection, structures containing either ligand sediment mostly between 33,000 and 3 X 10(6) g X min (LP fraction) and have an equilibrium density of 1.11-1.13 g/ml in sucrose gradients (Quintart, J., P. J. Courtoy, J. N. Limet, and P. Baudhuin, 1983, Eur. J. Biochem., 131:105-112). Such low density fractions, prepared from rats given galBSA-HRP, were incubated for 30 min at 25 degrees C in 5.5 mM 3,3'-diaminobenzidine (DAB) and 11 mM H2O2 in buffered sucrose. Upon equilibration in a second sucrose gradient, the galBSA-HRP distribution shifted towards higher (approximately 1.19 g/ml) density, but the bulk of protein remained at low density. In the absence of H2O2, galBSA-HRP distribution was also found at low density. As observed by electron microscopy, particles equilibrating at higher density after DAB cytochemistry were largely made of vesicles or tubules filled with DAB reaction product. The density shift of galBSA-HRP-containing organelles after incubation with DAB and H2O2 is attributed to the trapping of HRP-oxidized DAB inside the host organelles. If the low density fractions isolated from a rat injected with [3H]galBSA-HRP were mixed in vitro with similar fractions from another rat given [14C]galBSA, the 3H distribution shifted after DAB cytochemistry, but the 14C distribution was essentially unaffected. By contrast, if both derivatives were injected simultaneously, a concomitant density shift was observed. In conclusion, the DAB-induced density shift was specific to ligand-HRP-containing organelles. The potentials of the method include the purification of HRP-containing particles and the study of their association to ligands, fluid-phase tracers, or marker enzymes.

Some Contributions of Phosphatase and 3,3'-Diaminobenzidine Cytochemistry to Cell Biology

This presentation will highlight cytochemical studies that have illuminated some aspects of structure and function of the endoplasmic reticulum (ER); these have been reviewed recently (1, 2). Phosphatase (Pase) cytochemistry has led to the formulation of new questions regarding secretory mechanisms in a number of endocrine and exocrine cells; it has also made the status of GERL as a distinct organelle considerably firmer. 3,31-diaminobenzidine (DAB) cytochemistry has revealed the presence of an organelle apparently ubiquitous in mammalian cells, the anucleoid peroxisomes(microperoxisomes). DAB cytochemistry has been utilized recently by Gonatas et al. (3) to demonstrate that internalized plasma membrane is transported to GERL.Our initial use of Pase cytochemistry to visualize cell organelles included nucleoside diphosphatase (NDPase), thiamine pyrophosphatase (TPPase), and acid Pase (AcPase). NDPase hydrolyzes the diphosphates of inosine, uridine, and guanosine but not cytidine or adenine diphosphates. Since the work of Yamasaku and Hayaishi (4) we have used TPPase and NDPase interchangeably.