Endosomal-lysosomal Compartments Research Articles

Synthesis and Detection of BODIPY-, Biotin-, and 19 F- Labeled Single-Entity Dendritic Heparan Sulfate Mimetics.

Heparin and heparan sulfate (HS) are naturally occurring mammalian glycosaminoglycans, and their synthetic and semi-synthetic mimetics have attracted significant interest as potential therapeutics. However, understanding the mechanism of action by which HS, heparin, and HS mimetics have a biological effect is difficult due to their highly charged nature, broad protein interactomes, and variable structures. To address this, a library of novel single-entity dendritic mimetics conjugated to BODIPY, Fluorine-19 (19 F), and biotin was synthesized for imaging and localization studies. The novel dendritic scaffold allowed for the conjugation of labeling moieties without reducing the number of sulfated capping groups, thereby better mimicking the multivalent nature of HS-protein interactions. The 19 F labeled mimetics were assessed in phantom studies and were detected at concentrations as low as 5 mM. Flow cytometric studies using a fluorescently labeled mimetic showed that the compound associated with immune cells from tumors more readily than splenic counterparts and was directed to endosomal-lysosomal compartments within immune cells and cancer cells. Furthermore, the fluorescently labeled mimetic entered the central nervous system and was detectable in brain-infiltrating immune cells 24 hours after treatment. Here, we report the enabling methodology for rapidly preparing various labeled HS mimetics and molecular probes with diverse potential therapeutic applications.

Lysosomal integral membrane protein LGP85 (LIMP-2) is ubiquitinated at the N-terminal cytoplasmic domain

LGP85/LIMP-2 is a type III transmembrane glycoprotein of lysosomes, which traverses the membrane twice with an N-terminal uncleaved signal sequence and C-terminal hydrophobic domain. In addition to functioning as a receptor for a lysosomal enzyme β-glucocerebrosidase and for several enteroviruses, LGP85 plays a key role in the biogenesis and maintenance of endosomal/lysosomal compartments (ELCs). Our previous studies have demonstrated that overexpression of rat LGP85 into COS cells results in the enlarged ELCs, from where membrane trafficking is impaired. We show here that rat LGP85 is polyubiquitinated at the N-terminal short cytoplasmic domain that comprises of only three amino acid residues, alanine, arginine, and cysteine. Replacement of either arginine or cysteine with alanine within the N-terminal cytoplasmic domain did not influence the ubiquitination of LGP85, thereby indicating that ubiquitin (Ub) is conjugated to the α-NH2 group of the N-terminal alanine residue. Furthermore, we were able to define a domain necessary for ubiquitination in a region ranging from the amino acids 156 to 255 within the lumenal domain of LGP85. This is the first report showing that the integral lysosomal membrane protein LGP85 is ubiquitinated.

Theranostic iRGD peptide containing cisplatin prodrug: Dual-cargo tumor penetration for improved imaging and therapy

In theranostics, peptide-based platforms have widely been exploited owing to their unique biological functions and chemical versatilities. As a tumor-homing ligand, internalizing RGD peptide (iRGD), composed of a tumor-targeting sequence (RGD) and a cell-penetrating C-end Rule (CendR) motif, is known to facilitate the tumor-specific delivery of payloads that are covalently conjugated on its N-terminal fragment or co-administered without any covalent linkages. However, theranostic uses of the iRGD-based platform remain in its infancy with its full potential unexplored; for instance, detailed mechanism of iRGD fragmentation during internalization, strategies for the tumor-specific release of payloads from iRGD and the role of the C-terminal iRGD fragment in delivery have yet to be revealed. In this study, we designed a dual-channel fluorescent cyclic iRGD (TAMRA-iRGDC-Cy5.5) to track each of the N- and C-terminal fragments separately during the tumor internalization process. It turned out that both fragments undergo translocation into cancer cells together and are localized within endosomal-lysosomal compartments. The resulting co-internalization of both iRGD fragments allowed us to develop a new theranostic peptide platform (Cy5.5-iRGDC-Pt(IV)) by conjugating a fluorescent dye and a cisplatin prodrug on each terminus of cyclic iRGD for simultaneous cancer-targeted imaging and therapy. Compared to a control peptide having a non-iRGD sequence, the Cy5.5-iRGDC-Pt(IV) did not only provide a better tumor imaging contrast but also induced tumor-specific apoptosis leading to efficacious tumor suppression. Besides the outstanding cancer imaging and therapeutic performance, the Cy5.5-iRGDC-Pt(IV) revealed negligible systemic toxicity, holding potential to be applied for theranostic uses.

Fluorescent probes towards selective cathepsin B detection and visualization in cancer cells and patient samples.

Human cysteine cathepsins constitute an 11-membered family of proteases responsible for degradation of proteins in cellular endosomal-lysosomal compartments as such, they play important roles in antigen processing, cellular stress signaling, autophagy, and senescence. Moreover, for many years these enzymes were also linked to tumor growth, invasion, angiogenesis and metastasis when upregulated. Individual biological roles of each cathepsin are difficult to establish, because of their redundancy and similar substrate specificities. Selective chemical tools that enable imaging of individual cathepsin activities in living cells, tumors, and the tumor microenvironment may provide a better insight into their functions. In this work, we used HyCoSuL technology to profile the substrate specificity of human cathepsin B. The use of unnatural amino acids in the substrate library enabled us to uncover the broad cathepsin B preferences that we utilized to design highly-selective substrates and fluorescent activity-based probes (ABPs). We further demonstrated that Cy5-labeled MP-CB-2 probe can selectively label cathepsin B in eighteen cancer cell lines tested, making this ABP highly suitable for other biological setups. Moreover, using Cy5-labelled MP-CB-2 we were able to demonstrate by fluorescence microscopy that in cancer cells cathepsins B and L share overlapping, but not identical subcellular localization.

A potential gain-of-function variant of SLC9A6 leads to endosomal alkalinization and neuronal atrophy associated with Christianson Syndrome

Loss-of-function mutations in the recycling endosomal (Na+,K+)/H+ exchanger gene SLC9A6/NHE6 result in overacidification and dysfunction of endosomal-lysosomal compartments, and cause a neurodevelopmental and degenerative form of X-linked intellectual disability called Christianson Syndrome (CS). However, knowledge of the disease heterogeneity of CS is limited. Here, we describe the clinical features and underlying molecular and cellular mechanisms associated with a CS patient carrying a de novo missense variant (p.Gly218Arg; G218R) of a conserved residue in its ion translocation domain that results in a potential gain-of-function. The patient manifested several core symptoms typical of CS, including pronounced cognitive impairment, mutism, epilepsy, ataxia and microcephaly; however, deterioration of motor function often observed after the first decade of life in CS children with total loss of SLC9A6/NHE6 function was not evident. In transfected non-neuronal cells, complex glycosylation and half-life of the G218R were significantly decreased compared to the wild-type transporter. This correlated with elevated ubiquitination and partial proteasomal-mediated proteolysis of G218R. However, a major fraction was delivered to the plasma membrane and endocytic pathways. Compared to wild-type, G218R-containing endosomes were atypically alkaline and showed impaired uptake of recycling endosomal cargo. Moreover, instead of accumulating in recycling endosomes, G218R was redirected to multivesicular bodies/late endosomes and ejected extracellularly in exosomes rather than progressing to lysosomes for degradation. Attenuated acidification and trafficking of G218R-containing endosomes were also observed in transfected hippocampal neurons, and correlated with diminished dendritic branching and density of mature mushroom-shaped spines and increased appearance of filopodia-like protrusions. Collectively, these findings expand our understanding of the genetic diversity of CS and further elucidate a critical role for SLC9A6/NHE6 in fine-tuning recycling endosomal pH and cargo trafficking, processes crucial for the maintenance of neuronal polarity and mature synaptic structures.

The Endosomal-Lysosomal Pathway Is Dysregulated by APOE4 Expression in Vivo.

Possession of the ε4 allele of apolipoprotein E (APOE) is the major genetic risk factor for late-onset Alzheimer's disease (AD). Although numerous hypotheses have been proposed, the precise cause of this increased AD risk is not yet known. In order to gain a more comprehensive understanding of APOE4's role in AD, we performed RNA-sequencing on an AD-vulnerable vs. an AD-resistant brain region from aged APOE targeted replacement mice. This transcriptomics analysis revealed a significant enrichment of genes involved in endosomal–lysosomal processing, suggesting an APOE4-specific endosomal–lysosomal pathway dysregulation in the brains of APOE4 mice. Further analysis revealed clear differences in the morphology of endosomal–lysosomal compartments, including an age-dependent increase in the number and size of early endosomes in APOE4 mice. These findings directly link the APOE4 genotype to endosomal–lysosomal dysregulation in an in vivo, AD pathology-free setting, which may play a causative role in the increased incidence of AD among APOE4 carriers.

Apolipoprotein E3-mediated cellular uptake of reconstituted high-density lipoprotein bearing core 3, 10, or 17 nm hydrophobic gold nanoparticles

We have developed a high-density lipoprotein (HDL)-based platform for transport and delivery of hydrophobic gold nanoparticles (AuNPs). The ability of apolipoprotein E3 (apoE3) to act as a high-affinity ligand for the low-density lipoprotein receptor (LDLr) was exploited to gain entry of HDL with AuNPs into glioblastoma cells. AuNPs of 3, 10, and 17 nm diameter, the latter two synthesized by phase transfer process, were solubilized by integration with phospholipids and apoE3, yielding reconstituted HDL (rHDL) bearing AuNPs. Ultraviolet–visible spectra of rHDL-AuNP indicated the presence of stable particles with surface plasmon band at ~530 nm. Transmission electron microscopy (TEM) of rHDL-AuNP revealed roughly spherical particles with AuNPs embedded in the core. The rHDL-AuNP particles displayed robust binding to the LDLr and were internalized by receptor-mediated endocytosis in glioblastoma cells. Confocal microscopy confirmed cellular uptake of AuNPs in the endosomal–lysosomal compartments, while TEM revealed intracellular aggregated AuNPs. Cell viability assay demonstrated that >85% of cells were viable with rHDL-AuNP treatment of 0.1–100 μg/mL for 24 hours. These findings are significant since they offer an effective means of delivering AuNPs across the cell membrane, which is particularly relevant in tumor cells that overexpress LDLr.

IRAP+ endosomes restrict TLR9 activation and signaling.

The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP+ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP+ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.

Revisiting the neuronal localization and trafficking of CLN3 in juvenile neuronal ceroid lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis, the most common neurodegenerative disease affecting children, is caused by mutations of the CLN3 gene encoding CLN3, a transmembrane protein with so far undefined function. The embryonic expression of the gene has not been studied in detail before. Moreover, the protein CLN3 was mostly localized on the subcellular level to lysosomes but the exclusiveness is still under debate. Here, we analyze the expression pattern of murine CLN3 at different developmental stages by insitu hybridizations. We observe expression maxima in the developing thalamus and cerebral cortex and outside of the central nervous system in the gastrointestinal tract and other peripheral organs. In differentiated primary neurons, the protein CLN3 shows mainly a somatodendritic localization. In primary neurons, we thoroughly revisit the subcellular localization of CLN3 and find a predominant localization in late endosomal-lysosomal compartments. Moreover, we expressed the major mutant form of CLN3 - CLN3deltaExon7/8 - in neurons and demonstrate that it is retained in the endoplasmatic reticulum. Time-lapse microscopy analysis of neurons revealed co-trafficking of CLN3 with the late endosomal marker Rab7, but not with the early endosomal marker Rab5. Furthermore, a constitutive active mutant of Rab7 traps CLN3 in enlarged endosomes. Our subcellular localization study in neurons refines the localization and subcellular targeting of CLN3 to late endosomal-lysosomal compartments and provides information on the velocity of CLN3 in living neurons which has not been investigated before.

Abstract 394: ApoE3-containing High Density Lipoprotein Serves as a Multifunctional Platform for Delivery of Gold Nanoparticles for Hyperthermic Treatment

We have developed a novel HDL-based multifunctional platform for transport and delivery of highly hydrophobic gold nanoparticles (AuNP) bearing photothermic properties. We exploit the ability of apolipoprotein E3 (apoE3) to act as a high affinity ligand for the low-density lipoprotein receptor (LDLr) to gain entry into glioblastoma cells. The issue of poor aqueous solubility of AuNP was overcome by integrating them with phospholipids and apoE3, yielding reconstituted rHDL bearing 3, 10, or 10 nm AuNP. UV-Vis spectra of rHDL-AuNP indicated the presence of stable particles with surface plasmon band at ~530 nm, a signature feature of AuNP. Transmission electron microscopy (TEM) revealed discoidal geometry for rHDL with 3, 10 and 17 nm particles. Co-immunoprecipitation assay using a soluble form of the LDLr indicated robust binding of rHDL-AuNP to the receptor. Immunofluorescence analysis indicated that all 3 rHDL particles were internalized by glioblastoma cells, as revealed by the presence of punctate, peri-nuclear endocytic/lysosomal vesicles; this suggests cellular uptake of rHDL-AuNP by receptor-mediated endocytosis. Cellular uptake was further confirmed by TEM, in which aggregated AuNP were found in the endosomal-lysosomal compartments. Finally, cytotoxicity studies demonstrated that more than 50% of cells were viable with rHDL-AuNP treatment of up to 0.1 mg/ml for 24 h. The findings that apoE3: (i) acts as a detergent in solubilizing and dramatically improving the aqueous solubility of AuNP, and, (ii) facilitates cellular uptake of rHDL-AuNP by the LDLr pathway, are significant since they offer an effective means of delivering AuNP across the cell membrane. This is particularly relevant in tumor cells since they overexpress LDLr to meet the high demand for cholesterol that is required for rapid proliferation and membrane biogenesis.

Overexpression of the Insulin-Like Growth Factor II Receptor Increases β-Amyloid Production and Affects Cell Viability.

Amyloid β (Aβ) peptides originating from amyloid precursor protein (APP) in the endosomal-lysosomal compartments play a critical role in the development of Alzheimer's disease (AD), the most common type of senile dementia affecting the elderly. Since insulin-like growth factor II (IGF-II) receptors facilitate the delivery of nascent lysosomal enzymes from the trans-Golgi network to endosomes, we evaluated their role in APP metabolism and cell viability using mouse fibroblast MS cells deficient in the murine IGF-II receptor and corresponding MS9II cells overexpressing the human IGF-II receptors. Our results show that IGF-II receptor overexpression increases the protein levels of APP. This is accompanied by an increase of β-site APP-cleaving enzyme 1 levels and an increase of β- and γ-secretase enzyme activities, leading to enhanced Aβ production. At the cellular level, IGF-II receptor overexpression causes localization of APP in perinuclear tubular structures, an increase of lipid raft components, and increased lipid raft partitioning of APP. Finally, MS9II cells are more susceptible to staurosporine-induced cytotoxicity, which can be attenuated by β-secretase inhibitor. Together, these results highlight the potential contribution of IGF-II receptor to AD pathology not only by regulating expression/processing of APP but also by its role in cellular vulnerability.

Fusion to the Lysosome Targeting Signal of the Invariant Chain Alters the Processing and Enhances the Immunogenicity of HIV-1 Reverse Transcriptase

Intracellular processing of the antigen encoded by a DNA vaccine is one of the key steps in generating an immune response. Immunization with DNA constructs targeted to the endosomal-lysosomal compartments and to the MHC class II pathway can elicit a strong immune response. Herein, the weakly immunogenic reverse transcriptase of HIV-1 was fused to the minimal lysosomal targeting motif of the human MHC class II invariant chain. The motif fused to the N-terminus shifted the enzyme intracellular localization and accelerated its degradation. Degradation of the chimeric protein occurred predominantly in the lysosomal compartment. BALB/c mice immunized with the plasmid encoding the chimeric protein demonstrated an enhanced immune response, in the form of an increased antigen-specific production of Th1 cytokines, INF-γ and IL-2, by mouse splenocytes. Moreover, the majority of the splenocytes secreted both cytokines; i.e., were polyfunctional. These findings suggest that retargeting of the antigen to the lysosomes enhances the immune response to DNA vaccine candidates with low intrinsic immunogenicity.

Dopamine induces apoptosis in APPswe-expressing Neuro2A cells following Pepstatin-sensitive proteolysis of APP in acid compartments

A pathological hallmark of Alzheimer's disease (AD) is the presence within neurons and the interneuronal space of aggregates of β-amyloid (Aβ) peptides that originate from an abnormal proteolytic processing of the amyloid precursor protein (APP). The aspartyl proteases that initiate this processing act in the Golgi and endosomal compartments. Here, we show that the neurotransmitter dopamine stimulates the rapid endocytosis and processing of APP and induces apoptosis in neuroblastoma Neuro2A cells over-expressing transgenic human APP (Swedish mutant). Apoptosis could be prevented by impairing Pepstatin-sensitive and acid-dependent proteolysis of APP within endosomal–lysosomal compartments. The γ-secretase inhibitor L685,458 and the α-secretase stimulator phorbol ester elicited protection from dopamine-induced proteolysis of APP and cell toxicity. Our data shed lights on the mechanistic link between dopamine excitotoxicity, processing of APP and neuronal cell death. Since AD often associates with parkinsonian symptoms, which is suggestive of dopaminergic neurodegeneration, the present data provide the rationale for the therapeutic use of lysosomal activity inhibitors such as chloroquine or Pepstatin A to alleviate the progression of AD leading to onset of parkinsonism.

Salmonella infects B cells by macropinocytosis and formation of spacious phagosomes but does not induce pyroptosis in favor of its survival

We have previously reported that Salmonella infects B cells and survives within endosomal-lysosomal compartments. However, the mechanisms used by Salmonella to enter B cells remain unknown. In this study, we have shown that Salmonella induces its own entry by the induction of localized ruffling, macropinocytosis, and spacious phagosome formation. These events were associated with the rearrangement of actin and microtubule networks. The Salmonella pathogenesis island 1 (SPI-1) was necessary to invade B cells. In contrast to macrophages, B cells were highly resistant to cell death induced by Salmonella. These data demonstrate the ability of Salmonella to infect these non-professional phagocytic cells, where the bacterium can find an ideal intracellular niche to support persistence and the possible dissemination of infection.

PH-sensitive siRNA delivery systems

RNA interference is a promising therapeutic modality to treat human diseases by specifically silencing the expression of disease-related genes. Effective gene silencing requires specific delivery of small interfering RNA (siRNA) into target tissue and cytosolic release in target cells. The cytoplasmic membrane of endosomal-lysosomal compartments is the final barrier for effective cytosolic siRNA delivery. pH-sensitive siRNA delivery systems have been designed to overcome this barrier and to facilitate cytosolic siRNA release utilizing the pH difference between plasma and endosomal-lysosomal compartments. The pH-sensitive delivery systems have a common feature of using materials with pH-sensitive amphiphilicity. The pH-sensitive materials, both lipids and polymers, become more amphiphilic at decreased pH in endosomes and destabilize the cytosolic membrane to facilitate cytosolic siRNA release. Significant progress has been made on the design and development of both lipid and polymer based pH-sensitive siRNA delivery systems. These systems exhibit pH-sensitive cell membrane destabilization and enhanced intracellular gene silencing efficiency. The pH-sensitive siRNA delivery systems have shown a great promise for efficient delivery of therapeutic siRNA. This review summarizes the concept, design and recent development on pH-sensitive siRNA delivery systems.

Lipoprotein Lipase Is a Novel Amyloid β (Aβ)-binding Protein That Promotes Glycosaminoglycan-dependent Cellular Uptake of Aβ in Astrocytes

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules in plasma lipoprotein particles. LPL also plays a role in the binding of lipoprotein particles to cell-surface molecules, including sulfated glycosaminoglycans (GAGs). LPL is predominantly expressed in adipose and muscle but is also highly expressed in the brain where its specific roles are unknown. It has been shown that LPL is colocalized with senile plaques in Alzheimer disease (AD) brains, and its mutations are associated with the severity of AD pathophysiological features. In this study, we identified a novel function of LPL; that is, LPL binds to amyloid β protein (Aβ) and promotes cell-surface association and uptake of Aβ in mouse primary astrocytes. The internalized Aβ was degraded within 12 h, mainly in a lysosomal pathway. We also found that sulfated GAGs were involved in the LPL-mediated cellular uptake of Aβ. Apolipoprotein E was dispensable in the LPL-mediated uptake of Aβ. Our findings indicate that LPL is a novel Aβ-binding protein promoting cellular uptake and subsequent degradation of Aβ.

CD40, autophagy and Toxoplasma gondii

Toxoplasma gondii represents a pathogen that survives within host cells by preventing the endosomal-lysosomal compartments from fusing with the parasitophorous vacuoles. The dogma had been that the non-fusogenic nature of these vacuoles is irreversible. Recent studies revealed that this dogma is not correct. Cell-mediated immunity through CD40 re-routes the parasitophorous vacuoles to the lysosomal compartment by a process called autophagy. Autophagosome formation around the parasitophorous vacuole results in killing of the T. gondii. CD40-induced autophagy likely contributes to resistance against T. gondii particularly in neural tissue.

Reduced brain volumes in mice expressing APP-Austrian mutation but not in mice expressing APP-Swedish–Austrian mutations

We previously described two transgenic mouse lines expressing sub-endogenous levels of the ‘Austrian’ APP-T714I mutation (driven by the prenatally active PDGF-β promoter; APP-Au mice) and showing intraneuronal Aβ pathology and reduced brain volumes on MRI at 12 and 20 months of age. To further investigate whether reduced brain sizes were caused by neurodegeneration or a neurodevelopmental defect, we now measured brain volumes as early as postnatal day 10. At this age, a distinguishable reduction in brain volumes was absent, indicating that brain volume deficits in APP-Au mice are not caused by a neurodevelopmental defect. To further study the association between intraneuronal Aβ and reduced brain volumes, we further generated and analyzed an APP transgenic mouse model expressing both Austrian and Swedish (K670N/M671L) mutations (APP-SwAu mice). APP-Swedish mutation is known to lead to altered APP processing in the secretory pathway, precluding its later processing in endosomal–lysosomal compartments, the site of intraneuronal Aβ accumulation. Also, to have higher levels of transgene expression only after birth, a murine Thy-1 promoter was utilized for APP-SwAu mouse lines. Despite having five times higher transgene APP levels compared to APP-Au mice, APP-SwAu mice showed significantly lower intraneuronal Aβ levels in the absence of reduced brain volumes, suggesting that intraneuronal Aβ accumulation is related to reduced brain volumes in APP-Au mice. These data also provide a first in vivo indication of altered processing of APP-Swedish at sub-endogenous levels, an effect not observed in mouse models expressing the APP-Swedish mutation in high amounts.

The Single Dynamin Family Protein in the Primitive Protozoan Giardia lamblia Is Essential for Stage Conversion and Endocytic Transport

Dynamins are universally conserved large guanosine triphosphatases, which function as mechanoenzymes in membrane scission. The primitive protozoan Giardia lamblia has a single dynamin-related protein (GlDRP) with an unusual domain structure. Giardia lacks a Golgi apparatus but generates transient Golgi-like delay compartments dubbed encystation-specific vesicles (ESVs), which serve to accumulate and mature cyst wall proteins during differentiation to infectious cyst forms. Here, we analyze the function of GlDRP during growth and encystation and demonstrate that it relocalizes from peripheral endosomal-lysosomal compartments to nascent ESVs. We show that GlDRP is necessary for secretion of the cyst wall material and ESV homeostasis. Expression of a dominant-negative GlDRP variant does not interfere with ESV formation but blocks cyst formation completely prior to regulated exocytosis. GlDRP colocalizes with clathrin at the cell periphery and is necessary for endocytosis of surface proteins to endosomal-lysosomal organelles in trophozoites. Electron microscopy and live cell imaging reveal gross morphological changes as well as functional impairment of the endocytic system in cells expressing the dominant-negative GlDRP. Thus, giardial DRP plays a key role in two distinct trafficking pathways and in organelle homeostasis, both essential functions for the proliferation of the parasite in the gut and its transmission to a new host.

The NH2‐Terminal Transmembrane and Lumenal Domains of LGP85 are Needed for the Formation of Enlarged Endosomes/Lysosomes

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.