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Our previous studies have shown that treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 μg/kg) at gestational day (GD) 15 reduces the pituitary synthesis of luteinizing hormone (LH) during the late fetal and early postnatal period, leading to the imprinting of defects in sexual behaviors at adulthood. However, it remains unclear how the attenuation of pituitary LH is linked to sexual immaturity. To address this issue, we performed a DNA microarray analysis to identify the gene(s) responsible for dioxin-induced sexual immaturity on the pituitary and hypothalamus of male pups, born of TCDD-treated dams, at the age of postnatal day (PND) 70. Among the reduced genes, we focused on gonadotropin-releasing hormone (GnRH) in the hypothalamus because of published evidence that it has a role in sexual behaviors. An attenuation by TCDD of GnRH expression emerged at PND4, and no subsequent return to the control level was seen. A change in neither DNA methylation nor histone acetylation accounted for the reduced expression of GnRH. Intracerebroventricular infusion of GnRH to the TCDD-exposed pups after reaching maturity restored the impairment of sexual behaviors. Supplying equine chorionic gonadotropin, an LH-mimicking hormone, to the TCDD-exposed fetuses at GD15 resulted in a recovery from the reduced expression of GnRH, as well as from the defects in sexual behavior. These results strongly suggest that maternal exposure to TCDD fixes the status of the lowered expression of GnRH in the offspring by reducing the LH-assisted steroidogenesis at the perinatal stage, and this mechanism imprints defects in sexual behaviors at adulthood.

Ubiquitylation of membrane proteins triggers their endocytosis at the plasma membrane and subsequent lysosomal degradation through multivesicular bodies (MVBs). A dominant-negative mutant SKD1/Vps4B caused an accumulation of ubiquitylated membrane proteins in MVBs. We have identified 22 membrane proteins whose trafficking is potentially regulated by ubiquitylation. Nine of them, including transferrin receptor (TfR), are indeed ubiquitylated and/or accumulated in MVBs in the cells expressing mutant Vps4. While the recycling route and iron-regulated expression of TfR are well characterized, the mechanism by which the degradation of TfR is regulated is largely unknown. We show that an excess of iron enhances both TfR's ubiquitylation and degradation in lysosomes. Probably, the up-regulated expression of ferritin, an endogenous iron-chelating molecule, attenuated the iron-induced degradation of TfR. Exogenously introduced lysine-less TfR, compared to the wild-type one, showed resistance to the iron-induced ubiquitylation and degradation, when endogenous TfR, which most certainly heterodimerizes with exogenous ones, was depleted with siRNA. These data suggest that the iron-induced ubiquitylation and degradation of TfR along with MVB pathway physiologically plays an important role in iron homeostasis.

Our previous studies have demonstrated that maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a reduction in gonadotropin biosynthesis in the fetal pituitary, resulting in the attenuated expression of steroidogenic proteins in the fetal gonads and the impairment of sexual behaviors in adulthood. However, the mechanism of the attenuation remains unknown. To address this issue, we investigated whether TCDD affects the pituitary production of gonadotropins, using cultured pituitary. In the absence of gonadotropin-releasing hormone (GnRH), a regulator of gonadotropin biosynthesis, TCDD did not affect the expression of gonadotropin mRNAs both in fetal and postnatal pituitaries. On the other hand, in the presence of GnRH, TCDD interfered with the synthesis of gonadotropin β-subunit mRNAs only in the fetal pituitary. A protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) and a PKA activator (8-bromoadenosine-3' 5'-cyclic monophosphate) induced the expression of gonadotropin mRNAs in the fetal pituitary. Among the subunits, only the induction of β-subunit was reduced by TCDD treatment. These results suggest that TCDD reduces gonadotropin biosynthesis via damage to GnRH-stimulated PKC and PKA signaling in a β-subunit- and fetal age-specific manner.

Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, but physiological functions of this protein in the brain remains unclear. In this study, we investigate the behavioral effect of disrupting the gene encoding cathepsin E in mice. We found that the cathepsin E-deficient (CatE-/-) mice were behaviorally normal when housed communally, but they became more aggressive compared with the wild-type littermates when housed individually in a single cage. The increased aggressive response of CatE-/- mice was reduced to the level comparable to that seen for CatE+/+ mice by pretreatment with an NK-1-specific antagonist. Consistent with this, the neurotransmitter substance P (SP) level in affective brain areas including amygdala, hypothalamus, and periaqueductal gray was significantly increased in CatE-/- mice compared with CatE+/+ mice, indicating that the increased aggressive behavior of CatE-/- mice by isolation housing followed by territorial challenge is mainly because of the enhanced SP/NK-1 receptor signaling system. Double immunofluorescence microscopy also revealed the co-localization of SP with synaptophysin but not with microtubule-associated protein-2. Our data thus indicate that cathepsin E is associated with the SP/NK-1 receptor signaling system and thereby regulates the aggressive response of the animals to stressors such as territorial challenge.

SKD1 belongs to the AAA-ATPase family and is one of the mammalian class E Vps (vacuolar protein sorting) proteins. Previously we have reported that the overexpression of an ATPase activity-deficient form of SKD1 (suppressor of potassium transport growth defect), SKD1(E235Q), leads the perturbation of membrane transport through endosomes and lysosomes, however, the molecular mechanism behind the action of SKD1 is poorly understood. We have identified two SKD1-binding proteins, SBP1 and mVps2, by yeast two-hybrid screening and we assign them as mammalian class E Vps proteins. The primary sequence of SBP1 indicates 22.5% identity with that of Vta1p from Saccharomyces cerevisiae, which was recently identified as a novel class E Vps protein binding to Vps4p. In fact, SBP1 binds directly to SKD1 through its C-terminal region (198-309). Endogenous SBP1 is exclusively localized to cytosol, however it is redirected to an aberrant endosomal structure, the E235Q compartment, in the cells expressing SKD1(E235Q). The ATPase activity of SKD1 regulates both the membrane association of, and assembly of, a large hetero-oligomer protein complex, containing SBP1, which is potentially involved in membrane transport through endosomes and lysosomes. The N-terminal half (1-157) of human SBP1 is identical to lyst-interacting protein 5 and intriguingly, SKD1 ATPase activity significantly influences the membrane association of lyst protein. The SKD1-SBP1 complex, together with lyst protein, may function in endosomal membrane transport. A primary sequence of mVps2, a mouse homologue of human CHMP2A/BC-2, indicates 44.4% identity with Vps2p/Did4p/Chm2p from Saccharomyces cerevisiae. mVps2 also interacts with SKD1 and is localized to the E235Q compartment. Intriguingly, the N-terminal coiled-coil region of mVps2 is required for the formation of the E235Q compartment but not for binding to SKD1. We propose that both SBP1 and mVps2 regulate SKD1 function in mammalian cells.

LIM kinase (LIMK) plays a critical role in stimulus-induced remodeling of the actin cytoskeleton by linking signals from the Rho family GTPases to changes in cofilin activity. Recent studies have shown an important role for LIMK1 signaling in tumor cell invasion through regulating actin dynamics. In this study, we investigate the role of LIMK1 in intracellular vesicle trafficking of lysosomes/endosomes. We analyzed by confocal immunofluorescence microscopy the cellular distribution of lysosomal proteins and the endocytosis of an endocytic tracer, epidermal growth factor (EGF), in LIMK1-transfected cells. We found in these cells an abnormal dispersed translocation of lysosomes stained for LIMPII and cathepsin D throughout the cytoplasm. The small punctate structures that stained for these lysosomal proteins were redistributed to the periphery of the cell. Computational 3D-image analysis of confocal immunofluorescence micrographs further demonstrated that these vesicles did not colocalize with the transferrin receptor, an early endosomal marker. Furthermore, LIMPII-positive lysosomes did not colocalize with early endosomes labeled with endocytosed Texas red-transferrin. These results indicate that there is no mixing between dispersed lysosomes and early endosomes in the LIMK1-transfected cells. Moreover, LIMK1 overexpression resulted in a marked retardation in the receptor-mediated internalization of Texas red-labeled EGF in comparison with mock-transfected cells. At 30 min after internalization, most of the Texas red-EGF staining overlapped with LIMPII-positive late endosomes/lysosomes in mock-transfected cells, whereas in LIMK1 transfectants only a small fraction of internalized EGF colocalized with LIMPII-positive structures in the perinuclear region. Taken together, the findings presented in this paper suggest that LIMK1 has a role in regulating vesicle trafficking of lysosomes and endosomes in invasive tumor cells.

It has been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) fibroblasts and U18666A-treated cells causes impairment of retrograde trafficking of the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300) from late endosomes to the trans-Golgi network (TGN). In apparent conflict with these results, here we show that as in normal fibroblasts, MPR300 localizes exclusively to the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This localization can explain why several lysosomal properties and functions, such as intracellular lysosomal enzyme activity and localization, the biosynthesis of cathepsin D, and protein degradation, are all normal in NPC fibroblasts. These results, therefore, suggest that the accumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval of MPR300 from endosomes to the TGN. Furthermore, treatment of normal and NPC fibroblasts with chloroquine, which inhibits membrane traffic from early endosomes to the TGN, resulted in a redistribution of MPR300 to EEA1 and internalized transferrin-positive, but LAMP-2-negative, early-recycling endosomes. We propose that in normal and NPC fibroblasts, MPR300 is exclusively targeted from the TGN to early endosomes, from where it rapidly recycles back to the TGN without being delivered to late endosomes. This notion provides important insights into the definition of late endosomes, as well as the biogenesis of lysosomes.

Small GTPase Rho and its downstream effectors, ROCK family of Rho-associated serine-threonine kinases, are thought to participate in cell morphology, motility, and tumor progression through regulating the rearrangement of actin cytoskeleton. Here we present evidence that transfection of human breast cancer cells with cDNA encoding a dominant active mutant of ROCK causes dispersal of lysosomal vesicles throughout the cytoplasm without perturbing the machinery of the endocytic pathway. The intracellular distribution of lysosomes and endocytosed transferrin, an early endosomal marker, were further assessed by confocal immunofluorescence microscopy. In the active ROCK transfected cells the lysosomal proteins, cathepsin D, LIMPII, and LAMP1, were found throughout the cytoplasm in dispersed small vesicles, which were not accessible to the endocytosed Texas Red-labeled transferrin. 3D-image analysis of lysosomal distribution in the active ROCK-transfectants revealed abundant punctate signals in the peripheral region of the basal plasma membrane. Cells expressing vector alone did not exhibit these alterations. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, induced LIMPII-positive/transferrin negative large vacuoles in the perinuclear region, and disappearence of the dispersed small vesicular structures. To our knowledge, this is the first evidence that increasing ROCK expression contributes to selective cellular dispersion of lysosomes in invasive breast cancer cells.

LGP85 (LIMP II) is a type III transmembrane glycoprotein that is located primarily in the limiting membranes of lysosomes and late endosomes. Despite being the abundant molecule of these compartments, whether LGP85 merely resides as one of the constituents of these membranes or plays a role in the regulation of endosome and lysosome biogenesis remains unclear. To elucidate these questions, we examined the effects of overexpression of LGP85 on the morphology and membrane traffic of the endosomal/lysosomal system. Here we demonstrate that overexpression of LGP85 causes an enlargement of early endosomes and late endosomes/lysosomes. Such a morphological alteration was not observed by overexpression of other lysosomal membrane proteins, LGP107(LAMP-1) or LGP96 (LAMP-2), reflecting a LGP85-specific function. We further demonstrate that overexpression of LGP85 impairs the endocytic membrane traffic out of these enlarged compartments, which may be correlated with or account for the accumulation of cholesterol observed in these compartments. Interestingly, co-transfection of LGP85 and the dominant-negative form of Rab5b (Rab5bS34N) abolished the formation of large vacuoles, suggesting that the GTP-bound active form of Rab5b is involved in the enlargement of endosomal/lysosomal compartments induced by overexpression of LGP85. Thus,these findings provide important new insights into the role of LGP85 in the biogenesis and the maintenance of endosomes/lysosomes. We conclude that LGP85 may participate in reorganizing the endosomal/lysosomal compartments.

Small GTPase RhoA regulates signal transduction from receptors in the membrane to a variety of cellular events related to cell morphology, motility, cytoskeletal dynamics, cytokinesis, and tumour progression, but it is unclear how RhoA regulates intracellular membrane dynamics of lysosomes. We showed previously by confocal immunofluorescence microscopy that the transfection of dominant active RhoA in MM1 cells causes the dispersal translocation of lysosomes stained for cathepsin D throughout the cytoplasm. Y-27632, a selective inhibitor of p160ROCK, impeded the cellular redistribution of lysosomes and promoted reclustering of lysosomes toward the perinuclear region. Here we have further investigated whether the acidic lysosomal vesicles dispersed throughout the cytoplasm are applied to the early endosomes in the endocytic pathway, and we demonstrate that the dispersed lysosomes were accessible to endocytosed molecule such as dextran, and their acidity was not changed, as determined by increased accumulation of the acidotropic probe LysoTracker Red. Brefeldin A did not induce the tabulation of these dispersed lysosomes, but it caused early endosomes to form an extensive tubular network. The dispersed lysosomes associated with cathepsin D and LIMPII were not colocalized with early endosomes, and these vesicles were not inaccessible to the endocytosed anti-transferrin receptor antibody. Moreover, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, induced a dramatic change in LIMPII-containing structures in which LIMPII-positive swollen large vacuoles were increased and small punctate structures disappeared in the cytoplasm. These swollen vacuoles were not doubly positive for LIMPII and transferrin receptor, and were not inaccessible to the internalized anti-transferrin receptor antibody. Therefore, our novel findings presented in this paper indicate that RhoA activity causes a selective translocation of lysosomes without perturbing the machinery of endocytic pathway.

Cathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [(32)P]phosphate, (32)P-labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of (32)P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated (35)S- or (32)P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al. (1993) J. Biochem. 113, 389-394]. Treatment of intracellular (35)S-labeled procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the (32)P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH(4)Cl treatment and about 90% of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intracellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized (35)S-labeled cathepsin B was retained in the cells in mature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism(s) other than the Man 6-P-dependent pathway.

To prevent neoplasia, cells of multicellular organisms activate cellular disposal programs such as apoptosis in response to deregulated oncogene expression, making the suppression of such programs an essential step for potentially neoplastic cells to become established as clinically relevant tumors. Since the mutation of ras proto-oncogenes, the most frequently mutated proto-oncogenes in human tumors, is very rare in some tumor types such as glioblastomas and gastric cancers, we hypothesized that mutated ras genes might activate a cell death program that cannot be overcome by these tumor types. Here we show that the expression of oncogenically mutated ras gene induces cellular degeneration accompanied by cytoplasmic vacuoles in human glioma and gastric cancer cell lines. Cells dying as a result of oncogenic Ras expression had relatively well-preserved nuclei that were negative for TUNEL staining. An immunocytochemical analysis demonstrated that the cytoplasmic vacuoles are derived mainly from lysosomes. This oncogenic Ras-induced cell death occurred in the absence of caspase activation, and was not inhibited by the overexpression of anti-apoptotic Bcl-2 protein. These observations suggested that oncogenic Ras-induced cell death is most consistent with a type of programmed cell death designated 'type 2 physiological cell death' or 'autophagic degeneration', and that this cell death is regulated by a molecular mechanism distinct from that of apoptosis. Our findings suggest a possible role for this non-apoptotic cell death in the prevention of neoplasia, and the activation of the non-apoptotic cell death program may become a potential cancer therapy complementing apoptosis-based therapies. In addition, the approach used in this study may be a valuable way to find genetically-regulated cell suicide programs that cannot be overcome by particular tumor types.

Lysosomal membrane glycoproteins carry targeting information in cytoplasmic regions. Two distinct targeting motifs in these regions, GY (glycine-tyrosine) and LI (leucine-isoleucine), have been identified and characterized. Accumulating evidence suggests that the adaptor complexes (AP-1, AP-2, and AP-3) recognize this information in cytoplasmic tails of transmembrane proteins. Here we report two different in vitro analyses (affinity beads and surface plasmon resonance) which revealed specific interaction between the cytoplasmic tail of LGP85 and AP-1 but not so with AP-2. We also noted requirement of the LI motif of the LGP85 tail in binding to the AP-1 complex. Our data and others which indicated the binding of AP-3 to the LIMP II (synonym of LGP85) tail suggest that the cytoplasmic tail of LGP85 interacts with AP-1 at the trans-Golgi network (TGN) and AP-3 at late endosomes, respectively. We propose that this sequential interaction between the lysosomal targeting signal and distinct APs along its transport pathway is responsible for the critical sorting of lysosomal membrane proteins and/or the potential proofreading system of mistargeted molecules.

The localization of cathepsin L and rat salivary cystatin-3 (RSC-3) in rat osteoclasts (rat femoral and alveolar bones) treated with or without E-64 (control) was examined immunocytochemically. In osteoclasts pretreated with E-64, immunoreactivity for cathepsin L was very weak extracellularly compared to that in the control osteoclasts. However, it was strong intracellularly. The localization of RSC-3 was unclear in the control osteoclasts, while in E-64 treated osteoclasts, both the clear zone and ruffled border areas showed a very strong immunoreaction. At the electron-microscopic level, in normal osteoclasts, numerous immunoreaction products for cathepsin L were found extracellularly in the bone matrix under the ruffled border, while few intracellular products were observed. In contrast, in the E-64-treated osteoclasts, only a few immunoreaction products were found extracellularly, while intracellularly cathepsin L was found in numerous endosome-lysosomal vacuoles. In the immunoreaction for RSC-3, the cytoplasm of the ruffled border was positive, and the tips of the RSC-3-positive ruffled border appeared to enter deeply into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in the vacuoles (probably autophagolysosomes). Thus, in E-64-treated osteoclasts, inhibition of the extracellular release of cathepsin L was demonstrated. In addition, intralysosomal accumulation of RSC-3 and deep penetration of the RSC-3-positive ruffled border into the bone matrix were found. These findings suggest that RSC-3 is associated with the inhibition of cathepsin L in both the lysosomes (in the osteoclasts) and bone matrix.

To investigate the intracellular transport mechanisms of lysosomal cathepsin D in yeast cells, we produced cathepsin D in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D. Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuoles with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein. To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells. Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction. Immunofluorescence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles. Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.

Organophosphate compounds are known to cause the selective release of rat liver microsomal beta-glucuronidase into plasma. To investigate the alterations of molecular forms and oligosaccharide moieties of liver beta-glucuronidase in organophosphate compound-administered rats, beta-glucuronidase was isolated from microsomal, Golgi, lysosomal, and serum fractions. In SDS-polyacrylamide gel electrophoresis, a single polypeptide band was observed on gels in Golgi and serum beta-glucuronidases. This result indicated that Golgi and serum beta-glucuronidases of treated rats did not undergo post-translational proteolytic processing, in contrast to those in control rat livers. Biochemical characterization of the isolated beta-glucuronidases by employing lectin affinity chromatography revealed that interaction of serum and Golgi enzymes with Ricinus communis agglutinin- and wheat germ agglutinin-Sepharose was fairly strong, and that microsomal and lysosomal enzymes were poorly retained on those columns. These results suggested that the serum and Golgi beta-glucuronidases are sialoglycoproteins. A clearance study also showed that infused serum beta-glucuronidase was slowly cleared from plasma with a half-life of about 60 min, but the asialo-serum enzyme was rapidly cleared with a half-life of about 5 min. These results imply that microsomal beta-glucuronidase undergoes extensive modification of the oligosaccharide moieties by terminal glycosyltransferases at the trans Golgi when it is destined for secretion into serum in response to treatment with an organophosphate compound.

Organophosphate compounds are known to cause a selective increase of beta-glucuronidase activity in rat serum. Previous data suggested that increase of serum beta-glucuronidase activity was well correlated with decrease of that activity in rat liver microsomal fraction, thereby, suggesting a role of the microsomal enzyme in mediating the organophosphate effect. To investigate further the intracellular sorting pathway of beta-glucuronidase in dibutyl phosphate-treated rats, liver subcellular fractions were prepared at 12 or 48 h after in vivo administration of [3H]leucine and it was established that microsomal beta-glucuronidase was the origin of the increased serum enzyme. To characterize the intracellular secretory pathway of beta-glucuronidase in dibutyl phosphate-treated rats, Golgi subfractions were isolated and a time course study was carried out. At 30 min after administration of dibutyl phosphate, specific activity of beta-glucuronidase in GF-2 (Golgi intermediate fraction) and GF-3 (Golgi heavy fraction) was significantly increased to the maximum. Furthermore, colchicine pretreatment of rats caused a delay of the peak of specific activity for 30 min in GF-2 and GF-3, and accumulation of enzyme activity in Golgi subfractions was observed. Colchicine pretreatment also had an inhibitory effect on release of beta-glucuronidase into serum until 30 min after dibutyl phosphate injection. The electrophoretic pattern of microsomal beta-glucuronidase on polyacrylamide gel was found to show two major bands of microsomal enzyme type and lysosomal enzyme type in dibutyl phosphate-treated rats. Taken together, these findings indicate that microsomal beta-glucuronidase follows the intracellular secretory pathway and is secreted into serum via Golgi complex in response to dibutyl phosphate.

Lysosomal cysteine proteinases of cathepsins B and H were isolated to a homogeneous state from rat liver by employing Sephadex G-75, DEAE-Sephacel, CM-Sephadex, and Mono S column chromatography. Each of the purified cathepsins B and H was demonstrated to be composed of a mixture of a single-chain form and the processed two-chain form upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To investigate the proteolytic maturation of lysosomal cathepsins B and H, turnover kinetics of these enzymes were studied by comparing the specific radioactivities of the incorporated [3H]leucine into either the single-chain form or two-chain form in vivo. The specific radioactivity derived from each protein band of lysosomal cathepsin H in SDS-PAGE at 1, 3, 6, 12, 24 and 48 h after the injection of a radiolabel showed that the peak of specific radioactivity of the single-chain form of cathepsin H appeared at 6 h and that after 6 h, the radiolabel was sequentially incorporated into the two-chain form, while the radiolabel in the single-chain form started to gradually decrease, suggesting that the single-chain form was processed to generate the mature enzyme after the enzyme was incorporated into the lysosomes. In contrast, in the case of cathepsin B, the appearance of a radiolabel in the single-chain form or in the two-chain form was observed almost concomitantly without time lag, indicating that the processing of cathepsin B occurred very rapidly in the lysosomes.

The membrane-association of early biosynthetic form of cathepsin D has been demonstrated in hepatoma cells, and this membrane-association is not mediated by mannose 6-phosphate residues, implying that a mannose 6-phosphate receptor-independent mechanism operates in the sorting of cathepsin D. In this paper, to demonstrate whether cathepsin D is associated with the lysosomal membranes, an in vitro binding experiment was carried out employing lysosomal cathepsin D or microsomal procathepsin D isolated from rat liver. Immunoblotting analysis revealed that an intermediate form of cathepsin D was associated with the lysosomal membranes; this lysosomal membrane-associated cathepsin D was released from the membranes by washing with Na2CO3 (pH 10.6) but not with solutions containing mannose 6-phosphate. This suggested that cathepsin D associates with the membranes by ionic-interaction, and that the membrane-associated cathepsin D resides as a peripheral membrane protein in the lysosomal membrane fraction. To confirm that the intermediate form of cathepsin D specifically interacts with the lysosomal integral membrane proteins, the lysosomal membrane fraction was treated with trypsin and the binding experiment was conducted. The result showed that the binding capacity of cathepsin D to the lysosomal membranes was apparently abolished and cathepsin D did not rebind to the membranes. These data suggest that the intermediate form of cathepsin D is preferentially recognized by the lysosomal membranous protein which complements the mannose 6-phosphate receptor-dependent intracellular sorting mechanism.

To investigate the effect of ethanol or colchicine on the intracellular proteolytic processing of lysosomal beta-glucuronidase, which is considered to occur in the Golgi complex in the intracellular sorting pathway, three rat liver Golgi subfractions, GF-1, GF-2, and GF-3, were isolated from ethanol- or colchicine-treated rats, and the electrophoretic patterns of the extracted Golgi beta-glucuronidase on polyacrylamide gel were examined. The isolated Golgi subfractions from the drug-treated rats gave a better yield of fraction than that from the control rats. The enzymatic characterization of these three subfractions showed no significant contamination by other subcellular structures such as plasma membranes, microsomes, or lysosomes, and no inhibitory effect of the drugs was observed. On the other hand, suppressed galactosyltransferase activity, a marker enzyme of the Golgi complex, was detected in the colchicine-treated rats. The electrophoretic pattern of Golgi beta-glucuronidase on polyacrylamide gel revealed one major band which moved to the same position as the lysosomal enzyme type in the control rats. In contrast, in the ethanol- and colchicine-treated rats, Golgi beta-glucuronidase was found to have two major bands stained for enzyme activity resulting from a mixture of microsomal- and lysosomal-type enzymes. These results suggested that the post-translational modification step, during conversion from a microsomal-type enzyme to a lysosomal-type enzyme, was apparently inhibited. Taken together, these findings indicated that ethanol or colchicine administration to rats caused an inhibitory effect on the intracellular post-translational modification of Golgi beta-glucuronidase destined for targeting to the lysosomes.

The localization of cathepsin L or type I collagen in the osteoclasts (rat femur) treated with or without E-64 (control) was examined immunocytochemically to investigate how E-64 affects the osteoclasts. Using a light microscope in the E-64-treated osteoclasts, the immunoreactivity for cathepsin L was extracellularly very weak compared with that in the control osteoclasts, but was strong intracellularly. The intracellular immuno-reactivity for type I collagen was found in the vacuoles in the E-64-treated osteoclasts but not in any vacuoles in the control osteo-clasts. On the other hand, the extracellular immunoreactivity along the resorption lacunae of the E-64-treated osteoclasts was somewhat weaker than that of the control osteoclasts. Using electron microscopy in the E-64-treated osteoclasts, only a small number of extracellular immunoreaction products for cathepsin L were seen along the resorption lacunae. In addition, intracellular cathepsin L was deposited in the endosome-lysosomal vacuoles which were well-developed by E-64. Gold particles indicating type I collagen appeared on the bone matrix, and they were also detected in the vacuoles and vesicles in the E-64-treated osteoclasts. However, they were not detected in the organelles of the control osteoclasts. Thus, in the E-64-treated osteoclasts, the extracellular release of cathepsin L was insufficient to suppress the degradation of collagen. In addition, the undegraded collagen seemed to be endocytosed. The above findings thus suggest that bone resorption is inhibited by this incomplete degradation of collagen.

Background Selenium-binding protein 1 (Selenbp1) is suggested to play a role in tumor suppression, and may be involved in the toxicity produced by dioxin, an activator of aryl hydrocarbon receptors (AhR). However, the mechanism or likelihood is largely unknown because of the limited information available about the physiological role of Selenbp1. Methods To address this issue, we generated Selenbp1-null [Selenbp1 (−/−)] mice, and examined the toxic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in this mouse model. Results Selenbp1 (−/−) mice exhibited only a few differences from wild-type mice in their apparent phenotypes. However, a DNA microarray experiment showed that many genes including Notch1 and Cdk1, which are known to be enhanced in ovarian carcinoma, are also increased in the ovaries of Selenbp1 (−/−) mice. Based on the different responses to TCDD between C57BL/6J and DBA/2J strains of mice, the expression of Selenbp1 is suggested to be under the control of AhR. However, wasting syndrome by TCDD occurred equally in Selenbp1 (−/−) and (+/+) mice. Conclusions The above pieces of evidence suggest that 1) Selenbp1 suppresses the expression of tumor-promoting genes although a reduction in Selenbp1 alone is not very serious as far as the animals are concerned; and 2) Selenbp1 induction by TCDD is neither a pre-requisite for toxicity nor a protective response for combating TCDD toxicity. General significance Selenbp1 (−/−) mice exhibit little difference in their apparent phenotype and responsiveness to dioxin compared with the wild-type. This may be due to the compensation of Selenbp1 function by a closely-related protein, Selenbp2.

Localization of cathepsin L in the synovial lining cells of the normal rat temporomandibular joint was investigated by the avidin-biotin-peroxidase complex method for semithin (1 μm) cryosections and the colloidal gold-labelled IgG method for ultrathin sections of LR gold resin. At the light-microscopic level, type A (macrophage-like) and B (fibroblast-like) cells formed the synovial lining layer. Extensive immunoreactivity for cathepsin L was observed in many granules and vacuoles of type A cells, while in the type B cells, immunoreactivity was found in very few granules. In the sublining layer, macrophages and a few fibroblasts were positive for cathepsin L. By electron microscopy, at the peripheral cytoplasm of the type A cells close to the lateral intercellular spaces and joint cavity, numerous coated vesicles and vacuoles (probably early endosomes) indicating endocytotic function were found. Gold particles indicating cathepsin L were localized in the vesicles (primary lysosomes) in the perinuclear cytoplasm and in the larger amorphous vacuoles (1 μm dia) as phagolysosomes. In type B cells, gold particles were limited to the vesicles only (primary lysosomes). The cathepsin L-positive primary lysosomes were numerous in a few fibroblasts in the sublining layer. These results indicate that type A cells contain a large amount of cathepsin L, and suggest that these cells endocytose surplus substances such as collagen and proteoglycan fragments in normal rat TMJ, effecting their digestion and degradation by the action of this proteolytic cathepsin.

We investigated the effect of insulin-like growth factors II and I (IGFII and IGFI) on septal primary cultures from mouse embryonic day 15 brains. The addition of IGFII to septal cultures enhanced total choline acetyltransferase (ChAT) activity in a dose-dependent manner. Maximal stimulation of ChAT activity was observed at 10 ng/ml IGFII. The effect of IGFII on ChAT activity was completely blocked by anti-IGFII/M-6-P receptor antibodies, whereas the antisera alone had no effect on the enzyme activity. Double-labeled immunohistochemical studies revealed that most ChAT-positive neurons expressed IGFII/M-6-P receptor immunoreactivity. These results indicate that the trophic effect of IGFII results from the direct action of this molecule through the IGFII/M-6-P receptor in septal cholinergic neurons. IGFI also stimulated ChAT activity, but with less potency than IGFII. Antibodies against the IGFII/M-6-P receptor inhibited approximately 50% of the IGFI response, suggesting that the effect of IGFI is mediated in part by the IGFII/M-6-P receptor. Thus, it appears that IGFII and IGFI are potent trophic factors for central cholinergic neurons and could potentially play a significant role in the differentiation, maintenance and regeneration of these neurons.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disruptor, causes reproductive and developmental toxic effects in pups following maternal exposure in a number of animal models. Our previous studies have demonstrated that TCDD imprints sexual immaturity by suppressing the expression of fetal pituitary gonadotropins, the regulators of gonadal steroidogenesis. In the present study, we discovered that all TCDD-produced damage to fetal production of pituitary gonadotropins as well as testicular steroidogenesis can be repaired by co-treating pregnant rats with α-lipoic acid (LA), an obligate co-factor for intermediary metabolism including energy production. While LA also acts as an anti-oxidant, other anti-oxidants; i.e., ascorbic acid, butylated hydroxyanisole and edaravone, failed to exhibit any beneficial effects. Neither wasting syndrome nor CYP1A1 induction in the fetal brain caused through the activation of aryl hydrocarbon receptor (AhR) could be attenuated by LA. These lines of evidence suggest that oxidative stress makes only a minor contribution to the TCDD-induced disorder of fetal steroidogenesis, and LA has a restorative effect by targeting on mechanism(s) other than AhR activation. Following a metabolomic analysis, it was found that TCDD caused a more marked change in the hypothalamus, a pituitary regulator, than in the pituitary itself. Although the components of the tricarboxylic acid cycle and the ATP content of the fetal hypothalamus were significantly changed by TCDD, all these changes were again rectified by exogenous LA. We also provided evidence that the fetal hypothalamic content of endogenous LA is significantly reduced following maternal exposure to TCDD. Thus, the data obtained strongly suggest that TCDD reduces the expression of fetal pituitary gonadotropins to imprint sexual immaturity or disturb development by suppressing the level of LA, one of the key players serving energy production.

Ubiquitylation of membrane proteins triggers their endocytosis at the plasma membrane and subsequent lysosomal degradation through multivesicular bodies (MVBs). A dominant-negative mutant SKD1/Vps4B caused an accumulation of ubiquitylated membrane proteins in MVBs. We have identified 22 membrane proteins whose trafficking is potentially regulated by ubiquitylation. Nine of them, including transferrin receptor (TfR), are indeed ubiquitylated and/or accumulated in MVBs in the cells expressing mutant Vps4. While the recycling route and iron-regulated expression of TfR are well characterized, the mechanism by which the degradation of TfR is regulated is largely unknown. We show that an excess of iron enhances both TfR's ubiquitylation and degradation in lysosomes. Probably, the up-regulated expression of ferritin, an endogenous iron-chelating molecule, attenuated the iron-induced degradation of TfR. Exogenously introduced lysine-less TfR, compared to the wild-type one, showed resistance to the iron-induced ubiquitylation and degradation, when endogenous TfR, which most certainly heterodimerizes with exogenous ones, was depleted with siRNA. These data suggest that the iron-induced ubiquitylation and degradation of TfR along with MVB pathway physiologically plays an important role in iron homeostasis.

An enzyme hydrolyzing flavine-adenine dinucleotide (FAD) to flavine mononucleotide (FMN) and adenosine monophosphate (AMP) was purified about 460-fold over the isolated lysosomal membranes with 9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and the absence of SDS. Purification procedures included: preparation of crude lysosomal membranes, solubilization with Triton X-100, WGA-Sepharose, Con A-Sepharose, hydroxylapatite chromatography, gel filtration with Superdex 200, DEAE ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme, estimated by gel filtration with Superdex 200, was approximately 560 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular weight of 140,000. The pH optimum for FAD hydrolysis was 8.5 with an apparent Km of 0.1 mM and the isoelectric point was pH 7.3. The activity was inhibited by o-phenanthroline, EDTA, DTT, and NEM and was slightly stimulated by Zn ion, but was not affected by Ca or Mg ions. The purified FADase contained N-linked complex type oligosaccharide chains lacking neuraminic acids. The NH2 terminal 21 amino acid residues of the purified FADase were Ser-Pro-Cys-Val-Cys-Asp-Pro-Val-Val-Val-Cys-Lys-Val-Val-Pro-Cys-Thr-Leu- Ala-Leu .

The localization of vasa homolog protein in the spermatogenic cells of mice, rats, and guinea pigs was studied by immunofluorescence and electron microscopies with the antibody against mouse vasa homolog (MVH) protein. By immunofluorescence microscopy, four types of granular staining patterns were identified: (1) fine particles observed in diplotene and meiotic cells, (2) small granules associated with a mitochondrial marker and appearing in pachytene spermatocytes after stage V, (3) strands lacking the mitochondrial marker in late spermatocytes, and (4) large irregularly shaped granules in round spermatids. Immunoelectron microscopy defined the ultrastructural profiles of these MVH protein-positive granules: the first type consisted of small dense particles, the second had intermitochondrial cement (IMC), the third type, consisting of strands, had loose aggregates of either material dissociated from IMC or 70-90-nm particles, and the fourth had typical chromatoid bodies (CBs). The results suggest that MVH proteins function in these components of nuage. MVH protein-positive structures other than CBs disappeared during meiosis and CB appeared first in early spermatids. The results suggest that the formation of nuage is discontinued between spermatocytes and spermatids. The formation of nuage in spermatocytes and of CB in spermatids is discussed.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.

Sialoglycoprotein with a molecular mass of 85 kDa (LGP85) was purified from rat liver lysosomal membranes with a 0.9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included: preparation of lysosomal membranes, elimination of LGP107 and LGP96 with immunoaffinity columns, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. LGP85 contains about 22.8% carbohydrate and the carbohydrate moiety is composed of mannose, galactose, fucose, glucosamine, galactosamine, and neuraminic acid, in a molar ratio of 40:20:2:23:3:13. Susceptibility to neuraminidase and immunoreactivity of the protein in intact tritosomes were examined to study the topology of the protein in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the protein were not observed in intact tritosomes until the tritosomes had been disrupted by osmotic shock. These observations suggest that both oligosaccharide chains and the main protein portion of the protein are located on the interior surface of the tritosomal membranes. Subcellular localization of LGP85 was determined using enzyme immunoassay. The lysosomes seem to be the major location. LGP85 in the lysosomes was divided into the membrane bound form (90%) and the soluble form (10%). Immunoelectron microscopy clearly confirmed that the localization of LGP85 is mainly confined to lysosomes.

Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.

A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal targetting signal.

Autophagy is an intracellular protein transport process leading to the degradation of organelles and long-lived proteins in eukaryotes. The down-regulation of autophagy observed in cancer cells has been associated with tumor progression. This study investigated autophagy induced by resveratrol, a natural compound, in human glioma cells. Glioma cells were exposed to resveratrol, and the cell growth and autophagic level were evaluated. Resveratrol inhibited growth and induced cell death in U373 glioma cells. When treated with resveratrol, glioma cells stably expressing GFP fused to LC3, recruited more GFP-LC3-labeled autophagosomes, and the percentage of cells with GFP-LC3-labeled autophagosomes increased. Furthermore, in resveratrol-treated glioma cells, pretreatment with P38 or ERK1/2 inhibitors reduced the autophagic level, suggesting that resveratrol-induced autophagy was positively regulated by P38 and the ERK1/2 pathway. The Akt/mTOR pathway was not involved in resveratrol-induced autophagy. Our results suggest that resveratrol has an anticancer effect on glioma cells by inducing autophagy.

Autophagy has multiple physiological functions, including protein degradation, organelle turnover and the response of cancer cells to chemotherapy. Because autophagy is implicated in a number of diseases, a better understanding of the molecular mechanisms of autophagy is needed for therapeutic purposes, including rational design of drugs. Autophagy is a process that occurs in several steps as follows: formation of phagophores, formation of mature autophagosomes, targeting and trafficking of autophagosomes to lysosomes, formation of autolysosomes by fusion between autophagosomes and lysosomes, and finally, degradation of the autophagic bodies within the lysosomes. It has been suggested that autophagosome formation is driven by molecular motor machineries, and, once formed, autophagosomes need to reach lysosomes, enriched perinuclearly around the microtubule-organizing centre. While it is recognized that all these steps require the cytoskeletal network, little is known about the mechanisms involved. Here we assessed the role of cytoplasmic dynein in the autophagic process of human glioma cells to determine the part played by dynein in autophagy. We observed that chemical interference with dynein function led to an accumulation of autophagosomes, suggesting impaired autophagosome-lysosome fusion. In contrast, we found that overexpression of dynamitin, which disrupts the dynein complex, reduced the number of autophagosomes, suggesting the requirement of the dynein-dynactin interaction in the early membrane trafficking step in autophagosome formation. These results suggest that dynein plays a variety of crucial roles during the autophagic process in glioma cells.

We isolated and sequenced a cDNA clone corresponding to the entire coding sequence of rat liver lysosomal cathepsin D. The deduced amino acid sequence revealed that cathepsin D consists of 407 amino acid residues (Mr 44,608) and the 20 NH2-terminal residues seem to constitute a cleavable signal peptide after which 44 amino acid residues follow as a propeptide. Two putative N-linked glycosylation sites and aspartic acid in the active site are as well conserved as those of human lysosomal cathepsin D. In the NH2-terminal sequence analysis of two isolated heavy chains of the mature enzyme, the termini were assigned as tryptophan (118th residue) and glycine (165th or 166th residue), respectively, hence demonstrates that the two heavy chains derive from a split of the single chain of cathepsin D at position between 117th and 118th or between 164th and 165th or 165th and 166th amino acids. We conclude that cathepsin D in rat liver lysosomes is a mixture of three forms composed of a single and two two-chain forms. However, the amounts of the two two-chain forms are low compared with that of the single chain form. Densidometric determination after SDS-PAGE revealed that the two two-chain forms account for less than 5% of the single chain form. There is a 82% similarity in amino acid level between rat and human liver lysosomal cathepsin D.

We used the oligonucleotide probe corresponding to the internal amino acid sequence of a lysosomal membrane glycoprotein with a molecular weight of 85 K (LGP85) and isolated and characterized cDNA clones containing the entire coding region. The isolated cDNA comprised 2065 nucleotides. The predicted amino acid sequences of LGP85 consisted of 478 amino acid residues (Mr.54,090) and the protein has 11 potential N-glycosylation sites. Since the NH2 terminal sequence determined from purified LGP85 was identical to the NH2 terminal sequence deduced from the nucleotide sequence of the cDNA, except for the lack of initiator methionine which is likely to be cleaved off posttranslationally, it is likely that LGP85 has an uncleavable signal peptide at the NH2 terminus. Hydropathy plots show that LGP85 possesses two strong hydrophobic regions at the NH2 terminus (residues 4-26) and near the COOH terminus (residues 433-457), respectively. Either one or both of the domains might be used for membrane anchoring. A comparison of the sequences of the other lysosomal membrane glycoproteins with that of LGP85 revealed no homology. Glycine-tyrosine residues (so-called GY motif) which are thought an important signal for delivery of lysosomal membrane glycoproteins to lysosomes were not contained in the cytoplasmic tail of LGP85 (residues 458-478). LGP85 appears to be an unique lysosomal membrane glycoprotein that does not require tyrosine residues for targeting to lysosomes. Tyrosine residue may not be an essential signal for delivering newly synthesized lysosomal membrane glycoproteins to lysosomes.

In foregoing studies, we reported that LGP107, a major lysosomal membrane glycoprotein in the rat liver, distributes in and circulates continuously throughout the endocytic membrane system (endosomes, lysosomes and plasma membrane), in hepatocytes (1,2). In the present study we examined whether acid phosphatase (APase), an enzyme that is transported to lysosomes as a transmembrane protein, passes through the cell surface during intracellular transport, because transport of newly synthesized APase to lysosomes involves the passage of endosomes containing a ligand which is internalized via receptors on the cell surface and is finally dispatched to lysosomes for degradation (3). When localization of APase in rat hepatocytes was investigated by immunoelectron microscopy, APase was found to be localized in lysosomes and endosomes, but not in coated pits on the cell surface, which are positive for LGP107, and from which antibodies for LGP107 are internalized. Further, unlike LGP107, newly synthesized APase was not detected in plasma membranes isolated from livers of rats given [35S]methionine, and when cultured hepatocytes were exposed to 125I-labeled anti APase IgG at 37°C, there was no transfer of the antibody to lysosomes even after 24 h incubation. Therefore, these results indicate that intracellular movement of APase does not involve cell surface passage in rat hepatocytes, and clearly differs from the recent report that human APase is transported to lysosomes via the cell surface in BHK cells transfected with its cDNA (4).

The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of endosomes in transport of newly synthesized acid phosphatase to lysosomes was investigated using the Golgi fraction (GF1+2), enriched in endosomes. The Golgi fraction (GF1+2) was prepared from the livers of rats given [ 35 S]methionine and asialofetuin conjugated-horseradish peroxidase (HRP). Newly synthesized acid phosphatase in the endosomes containing internalized asialofetuin-HRP was measured as a loss of the detectable labeled enzyme after 3,3′-diaminobenzidine (DAB) and H 2 O 2 reaction, due to formation of insoluble polymers which reduce protein antigenicity. With this procedure, acid phosphatase was all but undetectable in the Golgi fraction. Thus, newly synthesized acid phosphatase is apparently transported to lysosomes by endosomes.

LGP85 is a lysosomal membrane protein possessing a type III topology and is also known as a member of the CD36 superfamily of proteins, such as CD36 and the scavenger-receptor BI (SR-BI). We have recently demonstrated that overexpression of LGP85 in various mammalian cell lines causes the enlargement of endosomal/lysosomal compartments (ELCs). Using chimeras and deletion mutants, we show here that the lumenal region of LGP85 is necessary, but not sufficient, for the development of ELCs. Effective formation of enlarged ELC was largely dependent on the presence of a preceding NH(2)-terminal transmembrane segment. Analyses of deletion mutants within the lumenal domain further revealed a requirement of the NH(2)-terminal transmembrane proximal lumenal region, with high sequence similarity with SR-BI for the enlargement of ELC. These results suggest that an interaction of the NH(2)-terminal transmembrane proximal lumenal domain of LGP85 with the inner leaflet of endosomal/lysosomal membranes through the connection with the transmembrane domain is an essential determinant for the regulation of endosomal/lysosomal membrane traffic. Interestingly, although the NH(2)-terminal transmembrane domain itself was not sufficient for the enlargement of ELCs, it appeared to be required for direct targeting of LGP85 from the trans-Golgi network to late endosomes/lysosomes. Taken together, these results indicate the involvement of distinct domain of LGP85 in the targeting to, and biogenesis and maintenance of, ELC.

Lysosomes are acidic intracellular compartments and are regarded as degradative and the end point, of the endocytic pathway. Here we provide evidence for the generation of acid hydrolase poor and non-acidic post-lysosomal compartments in NRK cells that have accumulated non-digestible macromolecules, Texas red-dextran (TR-Dex), within lysosomes. When TR-Dex was fed to the cells for 6h, most of the internalized TR-Dex colocalized with a lysosomal enzyme, cathepsin D. With an increase in the chase period, however, the internalized TR-Dex gradually accumulated in cathepsin D-negative vesicles. These vesicles were positive for a lysosomal membrane protein, LGP85, and their formation was inhibited by treatment of the cells with U18666A, which impairs membrane transport out of late endosomal/lysosomal compartments, thereby suggesting that the vesicles are derived from lysosomes. Interestingly, these compartments are non-acidic as judged for the DAMP staining. The results, therefore, suggest that the excess accumulation of non-digestible macromolecules within lysosomes induces the formation of acid hydrolase poor and non-acidic post-lysosomal compartments. The fact that treatment of the cells with lysosomotropic amines or a microtubule-depolymerization agent resulted in extensive colocalization of TR-Dex with cathepsin D further indicates that the formation of the post-lysosomal compartments depends on the lysosomal acidification and microtubule organization. Furthermore, these results suggest bi-directional membrane transport between lysosomes and the post-lysosomal compartments, which implies that the latter are not resting compartments.

3-Methyladenine (3-MA), a well-known inhibitor of autophagic sequestration, can also prevent class III phosphatidylinositide (PI) 3-kinase activity, which is required for many processes in endosomal membrane trafficking. Although much is known about the effects of other PI 3-kinase inhibitors, such as wortmannin and LY294002, on endosomal membrane trafficking, little is known about those of 3-MA. Here we show that the treatment of cells with 3-MA results in a specific redistribution of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR300) from the trans-Golgi network (TGN) to early/recycling endosomal compartments containing internalized transferrin. Importantly, in contrast to wortmannin and LY294002, 3-MA did not cause the enlargement of late endosomal/lysosomal compartments. The results suggest that the effect of 3-MA is restricted to the retrieval of MPR300 from early/recycling endosomes.

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.

Small GTPase RhoA regulates signal transduction from receptors in the membrane to a variety of cellular events related to cell morphology, motility, cytoskeletal dynamics, cytokinesis, and tumour progression, but it is unclear how RhoA regulates intracellular membrane dynamics of lysosomes. We showed previously by confocal immunofluorescence microscopy that the transfection of dominant active RhoA in MM1 cells causes the dispersal translocation of lysosomes stained for cathepsin D throughout the cytoplasm. Y-27632, a selective inhibitor of p160ROCK, impeded the cellular redistribution of lysosomes and promoted reclustering of lysosomes toward the perinuclear region. Here we have further investigated whether the acidic lysosomal vesicles dispersed throughout the cytoplasm are applied to the early endosomes in the endocytic pathway, and we demonstrate that the dispersed lysosomes were accessible to endocytosed molecule such as dextran, and their acidity was not changed, as determined by increased accumulation of the acidotropic probe LysoTracker Red. Brefeldin A did not induce the tabulation of these dispersed lysosomes, but it caused early endosomes to form an extensive tubular network. The dispersed lysosomes associated with cathepsin D and LIMPII were not colocalized with early endosomes, and these vesicles were not inaccessible to the endocytosed anti-transferrin receptor antibody. Moreover, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, induced a dramatic change in LIMPII-containing structures in which LIMPII-positive swollen large vacuoles were increased and small punctate structures disappeared in the cytoplasm. These swollen vacuoles were not doubly positive for LIMPII and transferrin receptor, and were not inaccessible to the internalized anti-transferrin receptor antibody. Therefore, our novel findings presented in this paper indicate that RhoA activity causes a selective translocation of lysosomes without perturbing the machinery of endocytic pathway.