Arguments in favour of endosome maturation.
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Animals↗
Biomedical subjects
W Stoorvogel
Publications and source records attributed to W Stoorvogel.
Differences in the endosomal distributions of the two mannose 6-phosphate receptors.
Multiple immunolabeling of cryosections was performed to compare the subcellular distributions of the two mannose 6-phosphate receptors (MPRs) involved in the intracellular targeting of lysosomal enzymes: the cation-dependent (CD) and cation-independent (CI) MPR. In two cell types, the human hepatoma cell line HepG2 and BHK cells double transfected with cDNA's encoding for the human CD-MPR and CI-MPR, we found the two receptors at the same sites: the trans-Golgi reticulum (TGR), endosomes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the same extent in the same HA I-adaptor positive coated buds and vesicles. Endosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysis showed a relative enrichment of the CI-MPR in endosomes containing many internal vesicles. Two endosomal subcompartments were discerned, the central vacuole and the associated tubules and vesicles (ATV). We found an enrichment of CD-MPR over CI-MPR in the ATV. Lateral segregation of the two receptors within the plane of membranes was also detected on isolated organelles. Double immunolabeling for the CD-MPR and the asialoglycoprotein receptor, which mainly recycles between endosomes and the plasma membrane, revealed that these two receptors were concentrated in different subpopulations of endosomal ATV. The small GTP-binding protein rab4, which has been shown to mediate recycling from endosomes to the plasma membrane, was localized at the cytosolic face of many endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. These data suggest that the two MPRs exit the TGR via the same coated vesicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recycle MPRs to TGR.
Animals↗
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)
3,3'-Diaminobenzidine↗
Identification of subcellular compartments involved in biosynthetic processing of cathepsin D.
We have assigned the biosynthetic processing steps of cathepsin D to intracellular compartments which are involved in its transport to lysosomes in HepG2 cells. Cathepsin D was synthesized as a 51-kDa proenzyme. After formation of 51-55-kDa intermediates due to processing of N-linked oligosaccharides, procathepsin D was proteolytically processed to an intermediate 44-kDa and the mature 31-kDa enzyme. The intersection of the biosynthetic pathway of cathepsin D with the endocytic pathway was labeled with horseradish peroxidase and monitored biochemically by 3,3'-diaminobenzidine cytochemistry. Horseradish peroxidase was used either as a fluid-phase marker to label the entire endocytic pathway or conjugated to transferrin (Tf) to label endosomes only. Directly after biosynthesis cathepsin D was accessible neither to horseradish peroxidase nor Tf-horseradish peroxidase. Newly synthesized 51-55-kDa species of cathepsin D present in the trans-Golgi reticulum were accessible to both horseradish peroxidase and Tf-horseradish peroxidase. The accessibility of trans-Golgi reticulum to both endocytosed horseradish peroxidase and Tf-horseradish peroxidase was monitored by colocalization with a secretory protein, alpha 1anti-trypsin. The proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurred in compartments which were fully accessible to fluid-phase horseradish peroxidase. Tf-horseradish peroxidase had access to only 20% of 44-kDa cathepsin D while it had no access to 31-kDa cathepsin D. In contrast, the 31-kDa species was completely accessible to fluid-phase horseradish peroxidase. We conclude that proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurs in endosomes, whereas the processing of 44-31-kDa cathepsin D takes place in lysosomes.
Carcinoma, Hepatocellular↗
Late endosomes derive from early endosomes by maturation.
Endocytosed proteins destined for degradation in lysosomes are targeted mainly to early endosomes following uptake. Late endosomes are the major site for entry of newly synthesized lysosomal hydrolases via the cation-independent mannose 6-phosphate receptor into the degradative pathway. No consensus exists as to the mechanism of transport from early to late endosomes. We used asialoorosomucoid and transferrin to label selected parts of the degradative and receptor-recycling pathways, respectively, in the human hepatoma cell line HepG2. Intracellular mixing of sequentially endocytosed 125I- and HRP-labeled ligands was monitored by using 3,3'-diaminobenzidine-mediated density perturbation. The entire endocytic pathway of asialoorosomucoid, except for the lysosomes, remained fully accessible to subsequently endocytosed transferrin conjugated to HRP with unchanged kinetics. These results together with immunoelectron microscopic data support a model in which early endosomes gradually mature into late endosomes.
Asialoglycoproteins↗
A pool of intracellular phosphorylated asialoglycoprotein receptors which is not involved in endocytosis.
One proposed function of the asialoglycoprotein receptor in hepatocytes is to mediate the endocytosis of galactose and N-acetylgalactosamine-exposing glycoproteins. Recently we defined a pool of intracellular H1 subunits of the asialoglycoprotein receptor (ASGPR) in the human hepatoma cell line HepG2 which appeared not to be involved in endocytosis (Stoorvogel, W., Geuze, H. J., Griffith, J. M., Schwartz, A. L., and Strous, G. J. (1989) J. Cell Biol. 108, 2137-2148). In addition, a pool of stably phosphorylated intracellular ASGPR has been detected (Fallon, R. J., and Schwartz, A. L. (1988) J. Biol. Chem. 263, 13159-13166). In the current study we integrate these findings and provide evidence for the existence of two types of intracellular nonexchangeable compartments containing ASGPR. A transiently phosphorylated pool of ASGPR shuttles between the plasma membrane and endosomes, via a pathway identical to that of the transferrin receptor. The second pool comprises 20% of the total intracellular ASGPR, is stably phosphorylated at a serine residue, and is located in intracellular compartments devoid of recycling transferrin receptor. We refer to this ASGPR pool as the "silent pool." We furthermore show that the two receptor pools are confined to compartments exhibiting different buoyant densities on sucrose density gradients. ASGPR in the "silent pool" is fully glycosylated, suggesting a post-Golgi sorting mechanism for trafficking to this compartment. Possible functions of the "silent" ASGPR pool are discussed.
Asialoglycoprotein Receptor↗
Trafficking of the transferrin receptor.
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Animals↗
Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells.
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.
Asialoglycoprotein Receptor↗
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.
Biological Transport↗
Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles.
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.
Animals↗
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.
Asialoglycoprotein Receptor↗
Cloning of nitrate reductase genes from the cyanobacterium Anacystis nidulans.
Anacystis nidulans, a non-nitrogen-fixing cyanobacterium, can fulfill its nitrogen requirement by the assimilation of nitrate. The first step in the pathway, the reduction of nitrate to nitrite, is catalyzed by the molybdo-protein nitrate reductase. In this study, newly developed techniques for gene cloning in A. nidulans R2 were used for the isolation of two genes involved in nitrate reduction. One gene was cloned by complementation of the corresponding mutant; the other gene was picked up from a cosmid gene library by using a restriction fragment containing the transposon-inactivated gene as a probe. Both genes were unlinked single-copy chromosomal genes. Transformation studies provided evidence for the existence of a third locus involved in nitrate reduction.
Cloning, Molecular↗