Cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism

A number of studies have demonstrated that cholera toxin (CT) is found in detergent-insoluble, cholesterol-enriched domains (rafts) in various cells, including neurons. We now demonstrate that even though CT is associated with these domains at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism, at both early and late stages of neuronal development. CT transport to the Golgi apparatus, and its subsequent degradation, is inhibited by hypertonic medium (sucrose), and by chlorpromazine; the former blocks clathrin recruitment, and the latter causes aberrant endosomal accumulation of clathrin. Moreover, both internalization of the transferrin receptor (Tf-R), which occurs via a clathrin-dependent mechanism, and CT internalization, are inhibited to a similar extent by sucrose. In contrast, the cholesterol-binding agents filipin and methyl-beta-cyclodextrin have no effect on the rate of CT or Tf-R internalization. Finally, once internalized, CT becomes more detergent-soluble, and chlorpromazine treatment renders internalized CT completely detergent-soluble. We propose two models to explain how, despite being detergent-insoluble at the cell surface, CT is nevertheless internalized via a raft-independent mechanism in hippocampal neurons.     

Rate of Retrograde Transport of Cholera Toxin from the Plasma Membrane to the Golgi Apparatus and Endoplasmic Reticulum Decreases During Neuronal Development

Abstract: Various glycolipid-binding toxins are internalized from the cell surface to the Golgi apparatus. Prominent among these is cholera toxin (CT), which consists of a pentameric B subunit that binds to ganglioside GM1 and an A subunit that mediates toxicity. We now demonstrate that rhodamine (Rh)-CT can be further internalized from the Golgi apparatus to the endoplasmic reticulum (ER) in cultured hippocampal neurons and in neuroblastoma N18TG-2 cells and that the A subunit is essential for retrograde transport to the ER. In addition, the rate of internalization of Rh-CT to the Golgi apparatus and ER decreases dramatically as hippocampal neurons mature. The Golgi apparatus was labeled in almost all 1-day-old neurons after <1 h of incubation with Rh-CT but was labeled in <10% of 14-day-old neurons after 1 h. During the first 14 days in culture, there was a 15-fold increase in the number of ¹²⁵I-CT-binding sites per cell, indicating that the decrease in the rate of internalization of Rh-CT is not due to reduced levels of cell surface GM1 in older neurons. These results imply that the rate of retrograde transport of CT from the plasma membrane to the Golgi apparatus and ER is regulated during neuronal development and differentiation.     

The Scribble Polarity Protein Stabilizes E-Cadherin/p120-Catenin Binding and Blocks Retrieval of E-Cadherin to the Golgi

Several polarity proteins, including Scribble (Scrb) have been implicated in control of vesicle traffic, and in particular the endocytosis of E-cadherin, but through unknown mechanisms. We now show that depletion of Scrb enhances endocytosis of E-cadherin by weakening the E-cadherin-p120catenin interaction. Unexpectedly, however, the internalized E-cadherin is not degraded but accumulates in the Golgi apparatus. Silencing p120-catenin causes degradation of E-cadherin in lysosomes, but degradation is blocked by the co-depletion of Scrb, which diverts the internalized E-cadherin to the Golgi. Loss of Scrb also enhances E-cadherin binding to retromer components, and retromer is required for Golgi accumulation of Scrb, and E-cadherin stability. These data identify a novel and unanticipated function for Scrb in blocking retromer-mediated diversion of E-cadherin to the Golgi. They provide evidence that polarity proteins can modify the intracellular itinerary for endocytosed membrane proteins.     

Wheat germ agglutinin stains dispersed post-golgi vesicles after treatment with the cytokinesis inhibitor psychosine

The galactosylsphingosine psychosine (Psy) is one of the sphingolipids and induce the formation of multinuclear cells in several cell lines by inhibiting cytokinesis. In the present report, we show that intracellular organelles, including wheat germ agglutinin (WGA)-positive vesicles and early endosomes, are selectively dispersed by Psy. WGA is a conventional Golgi marker and WGA-positive vesicles appeared to co-localize with the Golgi apparatus in untreated cells. Psy treatment induced the dispersal of WGA-positive vesicles without affecting the structure of the Golgi apparatus, resulting in discrimination of WGA-positive vesicles from the Golgi apparatus. In sharp contrast to this effect of Psy, WGA-positive vesicles were not affected by brefeldin A treatment, which induced the disappearance of the Golgi apparatus. Immunostaining with anti-TGN46 antibodies revealed that a large portion of the WGA-positive vesicles were derived from the trans-Golgi network. Notably, the dispersed WGA-positive vesicles did not stain with anti-syntaxin 6, another marker of the trans-Golgi network. During cytokinesis, WGA-positive vesicles in the cytoplasm decreased, and WGA staining accumulated at the cleavage furrow, which was apparently inhibited by the presence of Psy. These data suggest that the transport of WGA-positive vesicles to the cleavage furrow is associated with the progression of cytokinesis.     

Cholera toxin toxicity does not require functional Arf6- and dynamin-dependent endocytic pathways

Cholera toxin (CT) and related AB(5) toxins bind to glycolipids at the plasma membrane and are then transported in a retrograde manner, first to the Golgi and then to the endoplasmic reticulum (ER). In the ER, the catalytic subunit of CT is translocated into the cytosol, resulting in toxicity. Using fluorescence microscopy, we found that CT is internalized by multiple endocytic pathways. Inhibition of the clathrin-, caveolin-, or Arf6-dependent pathways by overexpression of appropriate dominant mutants had no effect on retrograde traffic of CT to the Golgi and ER, and it did not affect CT toxicity. Unexpectedly, when we blocked all three endocytic pathways at once, although fluorescent CT in the Golgi and ER became undetectable, CT-induced toxicity was largely unaffected. These results are consistent with the existence of an additional retrograde pathway used by CT to reach the ER.     

Treponema denticola Msp-deduced peptide conjugate, P34BSA, promotes RhoA-dependent actin stress fiber formation independent of its internalization by fibroblasts

P34(BSA), a BSA conjugate of a synthetic 10-mer peptide deduced from Treponema denticola major outer sheath protein (Msp), stabilizes actin filaments in fibroblasts and retards cell motility. We reported previously that it is internalized by cells, binds and bundles actin filaments in vitro, and activates RhoA; yet, its site and mechanism of action were not defined. We have assessed P34(BSA)'s modes of interaction with and signaling to fibroblasts. At 4 degrees C, P34(BSA) was not internalized, but it bound to the plasma membrane and promoted actin stress fiber formation at approximately 80% capacity compared with 37 degrees C controls, casting doubt that cellular uptake is a critical step for its cytoskeleton-stabilizing property. In Rho G-LISA and co-immunoprecipitation assays, P34(BSA) was found to activate RhoA, even at 4 degrees C, to promote its interaction with guanosine nucleotide exchange factor p114RhoGEF. It also caused phosphorylation of cofilin. Upon RhoA inhibition, either by C3 transferase RhoA inhibitor or by transfection with a dominant negative RhoA construct, P34(BSA) did not achieve the stress fiber formation seen with P34(BSA) alone. By inhibiting phosphatidylinositol-3 kinase (PI 3-K) with LY294002, the P34(BSA) effects were completely blocked. Depletion of cholesterol with methyl-beta-cyclodextrin (MbetaCD) partially inhibited P34(BSA) signaling via the plasma membrane to the cytoskeleton. This suggests that multivalent P34(BSA) activation of lipid raft components requires active PI 3-K, and initiates the pathway through a RhoGEF and RhoA, which mediates stress fiber formation in fibroblasts. Hence, P34(BSA) may represent a novel tool to investigate RhoA-dependent processes, such as remodeling filamentous actin in eukaryotic cells.     

Golgi localization and phosphorylation of oxysterol binding protein in Niemann-Pick C and U18666A-treated cells.

Oxysterol binding protein (OSBP) translocation between Golgi and vesicular/cytoplasmic compartments is affected by conditions that alter cholesterol and sphingomyelin homeostasis, indicating a role in lipid and sterol regulation in this organelle. In this study, we show that OSBP dissociation from the Golgi apparatus was inhibited when LDL cholesterol efflux from lysosomes was blocked in Niemann-Pick C (NPC) or U18666A [3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one]-treated fibroblasts. Dissociation of OSBP from the Golgi apparatus in response to LDL was independent of de novo cholesterol biosynthesis. OSBP did not localize with filipin-stained lysosomal cholesterol, and the NPC defect did not alter OSBP expression or phosphorylation. However, OSBP in the Golgi apparatus was progressively dephosphorylated (as assessed by a molecular mass shift on SDS-PAGE) in U18666A-treated fibroblasts or Chinese hamster ovary cells as a result of combined inhibition of LDL cholesterol transport and de novo cholesterol synthesis. In vivo phosphopeptide mapping and mutagenesis of OSBP was used to identify the cholesterol-sensitive phosphorylation sites at serines 381, 384, and 387 that were responsible for the altered mobility on SDS-PAGE. NPC-1 protein-mediated release of LDL-derived cholesterol and de novo biosynthesis regulates OSBP localization and phosphorylation. This indicates that OSBP responds to or senses altered cellular sterol content and transport.     

Wheat germ agglutinin is selectively transported to multivesicular bodies.

Colloidal iron dextran particles bearing wheat germ agglutinin (WGA/FeDex) were bound by glycoconjugates expressed at the surface of HepG2 cells. Bound WGA/FeDex was internalized when cells were incubated at 37 degrees C and accumulated in intracellular structures which have the same buoyant density as the plasma membrane when examined on Percoll density gradients. The intracellular structures containing WGA/FeDex were identified as multivesicular bodies (MVB) by transmission electron microscopy. WGA/FeDex was not transported to lysosomes nor did it interfere with uptake and transport of GalBSA to lysosomes by the asialoglycoprotein receptor. WGA/FeDex was seen predominantly in non-coated invaginations at the cell surface, suggesting it may enter cells at a different site than GalBSA/FeDex. Highly enriched plasma membranes and MVBs containing superparamagnetic [125I]WGA/FeDex particles were prepared by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes prepared by HIMAC were enriched 30-fold for [125I]WGA/FeDex, 15-fold for alkaline phosphodiesterase I, and 9-fold for galactosyltransferase relative to the crude post-nuclear homogenate and consisted entirely of plasmalemmal sheets. Intracellular structures containing WGA/FeDex were enriched 35-fold for [125I]WGA/FeDex, 10-fold for alkaline phosphodiesterase I, and 10-fold for galactosyltransferase but did not contain lysosomal beta-galactosidase. WGA/FeDex has a different ultimate destination in HepG2 cells than ligands internalized by the asialoglycoprotein receptor and can be used to obtain highly enriched plasma membranes and MVBs from cultured cells.     

Effect of monensin on the neuronal ultrastructure and endocytic pathway of macromolecules in cultured brain neurons

1. The endocytic pathway of horseradish peroxidase (HRP) was investigated in the perikarya of cultured neurons by electron microscopy and enzyme cytochemistry. The tracer was observed in endocytic pits and vesicles, endosomes, multivesicular bodies, and lysosomes. It took approximate 15 min for the transfer of HRP from the exterior of the cell to the lysosomes. 2. Monensin induced distension of the Golgi apparatus and formation of intracellular vacuoles. When neurons were incubated with both monensin and HRP for 30 to 120 min, the number of HRP-labeled endosomes was greater than that in the monensin-free group, whereas the reverse was seen for HRP-positive lysosomes. The formation of HRP-positive lysosomes in monensin-treated cells was blocked by 47 to 79%. 3. These results indicate that the intracellular transport of the endocytosed macromolecule is pH dependent. It is also possible that the export of lysosomal enzymes is inhibited by monensin, resulting in an accumulation of the endosomes and a reduction of the lysosomes.     

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN).     

Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins

We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.     

Endocytosis of mannose-6-phosphate binding sites by mouse T-lymphoma cells

The endocytosis and intracellular transport of mannose-6-phosphate conjugated to bovine serum albumin (Man-6-P:BSA) by mouse T-lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man-6-P:BSA was labeled with fluorescein or 125I and used to locate both surface and intracellular Man-6-P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent- or 125I-labeled Man-6-P:BSA at 0 degree C revealed a uniform distribution of the Man-6-P binding sites over the cell surface. Competition experiments indicate that the Man-6-P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37 degrees C, endocytosis of Man-6-P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5-15-min incubation of cells at 37 degrees C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man-6-P:BSA in the Golgi region after 15-30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man-6-P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30-min incubation at 37 degrees C, the internalized Man-6-P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co-stained for acid phosphatase. These results demonstrate a temporal participation of clathrin-containing coated vesicles during the initial endocytosis of Man-6-P binding sites and that one step in the Man-6-P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.     

Important role for the V-type H(+)-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor

Our previous studies demonstrated that a green fluorescent protein-tagged parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor stably expressed in LLCPK-1 cells undergoes agonist-dependent internalization into clathrin-coated pits. The subcellular localization of the internalized PTH/PTHrP receptor is not known. In the present study, we explored the intracellular pathways of the internalized PTH/PTHrP receptor. Using immunofluorescence and confocal microscopy, we show that the internalized receptors localize at a juxtanuclear compartment identified as the Golgi apparatus. The receptors do not colocalize with lysosomes. Furthermore, whereas the internalized receptors exhibit rapid recycling, treatment with proton pump inhibitors (bafilomycin-A1 and concanamycin A) or brefeldin A, Golgi disrupting agents, reduces PTH/PTHrP receptor recycling. Together, these data indicate an important role for the vacuolar-type hydrogen-ATPase and the Golgi apparatus in postendocytic PTH/PTHrP receptor recovery.     

LYSOSOMAL PACKAGING IN DIFFERENTIATING AND DEGENERATING ANURAN LATERAL MOTOR COLUMN NEURONS

The role of the Golgi apparatus and the Golgi-endoplasmic reticulum-lysosome complex (GERL) in the genesis of lysosomes was examined in differentiating and degenerating motor neurons of anuran larvae. Acid phosphatase, aryl sulfatase, and thiolacetic acid esterase were utilized as marker enzymes for the lysosomal system, while nucleoside diphosphatase and thiamine pyrophosphatase labeled the inner saccule(s) of the Golgi apparatus. Reduced osmium tetroxide was routinely deposited in the outer Golgi saccule regardless of the state of neuronal maturation. In all young neurons, the disposition of acid hydrolase reaction product paralleled the formation of GERL, with no lytic activity in the Golgi apparatus per se. Hypertrophy of the Golgi apparatus and GERL was observed in the early phases of degeneration, and both organelles apparently exhibit extensive hydrolytic activity. Dense bodies, autophagic vacuoles, and primary lysosomes were found arising from GERL, while the Golgi apparatus may produce primary lysosomal granules during regression. On the other hand, in differentiating neurons, hydrolytic activity was restricted to GERL and an occasional dense body and autophagic vacuole. These studies illustrate a parallelism between the development of GERL and genesis of primary and secondary lysosomes during neuronal cytodifferentiation, and implicate GERL and possibly the Golgi apparatus in lysosomal packaging in degenerating neurons.     

Two lysosomal membrane proteins,LGP85 and LGP107, are delivered to late endosomes/lysosomes through different intracellular routes after exiting from the trans-Golgi network

Lysosomal membrane proteins are delivered from their synthesis site, the endoplasmic reticulum (ER) to late endosomes/lysosomes through the Golgi complex. It has been proposed that after leaving the Golgi they are transported either directly or indirectly (via the cell surface) to late endosomes/lysosomes. In the present study, we examined the transport routes taken by two structurally different lysosomal membrane proteins, LGP85 and LGP107, in rat 3Y1-B cells. Here we show that newly synthesized LGP85 and LGP107 are delivered to late endosomes/lysosomes via a direct route without passing through the cell surface. Interestingly, although LGP107 is delivered from the Golgi to early endosomes containing internalized horseradish peroxidase-conjugated transferrin (HRP-Tfn) en route to lysosomes, LGP85 does not pass through the HRP-Tfn-positive early endosomes. These results suggest, therefore, that LGP85 and LGP107 are sorted into distinct transport vesicles at the post-Golgi, presumably the trans-Golgi network (TGN), after which LGP85 is delivered directly to late endosomes/lysosomes, but significant fractions of LGP107 are targeted to early endosomes before transport to late endosomes/lysosomes. This study provides the first evidence that after exiting from the Golgi, LGP85 and LGP107 are targeted to late endosomes/lysosomes via a different pathway.     

Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P- 450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.     

Low density lipoprotein receptor and cation-independent mannose 6- phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells

Efficient transport of cell surface glycoproteins to the Golgi apparatus has been previously demonstrated for a limited number of proteins, and has been proposed to require selective sorting in the endocytic pathway after internalization. We have studied the endocytic fate of several glycoproteins that accumulate in different organelles in a variant clone of PC12, a regulated secretory cell line. The cation-independent mannose 6-phosphate receptor and the low density lipoprotein receptor, both rapidly internalized from the cell surface, and the synaptic vesicle membrane protein synaptophysin, were transported to the Golgi apparatus with equivalent, nonlinear kinetics. Transport to the Golgi apparatus (t1/2 = 2.5-3.0 h) was several times faster than turnover of these proteins (t1/2 greater than or equal to 20 h), indicating that transport of these proteins to the Golgi apparatus occurred on average several times for each protein. In contrast, Thy-1, a protein anchored in the membrane by a glycosylphosphoinositide group, was internalized and transported to the Golgi apparatus more slowly than the three transmembrane proteins. Since each of the transmembrane proteins studied showed the same t1/2 for transport to the Golgi apparatus, we conclude that transport of these proteins from the cell surface to the Golgi apparatus does not require sorting information specific to any one of these proteins. These results suggest that one of the functions of late endosomes is constitutive recycling of cell surface receptors through the Golgi apparatus if they fail to recycle to the cell surface directly from early endosomes, and that the late endosome recycling pathway is followed frequently by many rapidly internalized proteins.     

AP2 clathrin adaptor complex, but not AP1, controls the access of the major histocompatibility complex (MHC) class II to endosomes

Newly synthesized MHC II alpha- and beta-chains associated with the invariant chain chaperone (Ii) enter the endocytic pathway for Ii degradation and loading with peptides before transport to the cell surface. It is unclear how alphabetaIi complexes are sorted from the Golgi apparatus and directed to endosomes. However, indirect evidence tends to support direct transport involving the AP1 clathrin adaptor complex. Surprisingly, we show here that knocking down the production of AP1 by RNA interference did not affect the trafficking of alphabetaIi complexes. In contrast, AP2 depletion led to a large increase in surface levels of alphabetaIi complexes, inhibited their rapid internalization, and strongly delayed the appearance of mature MHC II in intracellular compartments. Thus, in the cell systems studied here, rapid internalization of alphabetaIi complexes via an AP2-dependent pathway represents a key step for MHC II delivery to endosomes and lysosomes.     

Ultrastructural distribution of NADPase within the Golgi apparatus and lysosomes of mammalian cells

Cytochemical studies with over 40 different mammalian cell types have indicated that NADPase activity is associated with the Golgi apparatus and/or lysosomes of all cells. In the majority of cases, NADPase is restricted to saccular elements comprising the medial region of the Golgi stack and an occasional lysosome. There is often weak NADPase activity in other Golgi compartments such as the trans Golgi saccules and/or elements of the trans Golgi network. In some cells, however, strong NADPase activity is found within these latter compartments, either exclusively in trans Golgi saccules or elements of the trans Golgi network, or in combination with medial Golgi saccules and each other including (1) medial Golgi saccules + trans Golgi saccules, (2) medial Golgi saccules + trans Golgi saccules + trans Golgi network, or (3) trans Golgi saccules + trans Golgi network. In some rare cases, no NADPase activity is detectable in either Golgi saccules or elements of the trans Golgi network, but it is observed in an occasional lysosome or throughout the lysosomal system of these cells. It is unclear at present if these variations in the distribution of NADPase across the Golgi apparatus, and between the Golgi apparatus and lysosomal system, are due to differences in targeting mechanisms or to the existence of "bottlenecks" in the natural flow of NADPase along the biosynthetic pathway toward lysosomes. While no clear pattern in the association of strong NADPase activity with lysosomes was apparent relative to the ultrastructural distribution of NADPase activity in Golgi saccules or elements of the trans Golgi network, the results of this investigation suggested that cells having NADPase localized predominantly toward the trans aspect of the Golgi apparatus (in trans Golgi saccules or elements of the trans Golgi network or both) have few NADPase-positive lysosomes. The only exception is hepatocytes which were classified as predominantly trans but had noticeable NADPase activity within medial Golgi saccules and elements of the trans Golgi network as well, and highly reactive lysosomes. Other cells showing highly reactive lysosomes including (1) Kupffer cells of liver and those forming the proximal convoluted tubules of the kidney, both of which also had strong NADPase activity within medial and trans Golgi saccules and elements of the trans Golgi network, (2) Leydig cells of the testis and interstitial cells of the ovary, which also showed strong NADPase activity within medial Golgi saccules, and (3) macrophages from lung, spleen and testis, and Sertoli cells from the testis all of which showed no Golgi associated NADPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

The relationships between the Golgi apparatus, GERL, and lysosomes of fetal rat liver Kupffer cells examined by ultrastructural phosphatase cytochemistry

Kupffer cells are the sinusoidal macrophages of the liver. Using ultrastructural phosphatase cytochemical methods, we examined the relationship between the Golgi apparatus, GERL, and lysosomes of Kupffer cells in fetal rat livers identified, in part, by their ability to phagocytize intravenously injected latex spheres. Thiamine pyrophosphatase (TPPase) activity was localized to the inner Golgi saccules and some vesicles in the Golgi region but not to GERL. A TPPase-like activity, demonstrable in lysosomes, was abolished by sodium fluoride but not suppressed by the alkaline phosphatase inhibitors L-cysteine and L-p-bromotetramisole. Acid phosphatase (AcPase) was localized by GERL, some coated vesicles, and in lysosomes, but not to the Golgi stacks. Continuities between GERL and lysosomes were observed. Phagosomes containing internalized latex spheres received TPPase and AcPase sequentially. TPPase was localized in phagosomes immediately after latex administration. AcPase activity was not found here until at least 10 minutes following the injection of the particulates. Our findings indicate that Kupffer cell lysosomes are derived from GERL, but also suggest that phagosomes may receive material packaged by the Golgi apparatus as well as GERL.