Differential trafficking of the Niemann-Pick C1 and 2 proteins highlights distinct roles in late endocytic lipid trafficking.

UNLABELLED: The cellular location of Niemann-Pick C2 protein (NPC2) in cultured human fibroblasts and Chinese hamster ovary cells was examined immunocytochemically and in living cells by expression of a functional red fluorescent protein chimeric analogue. RESULTS: NPC2 is present in the lysosomes of both cholesterol-depleted and -replenished cells, unlike Niemann-Pick C1 protein (NPC1) which is recruited to late endosomes only upon uptake of low-density lipoprotein. With mobilization of cholesterol from lysosomes, immunocytochemical detection of NPC2 in lysosomes is greatly diminished, whereas NPC1 remains in the late endosomal compartment. We found a partial overlap in the trafficking and organellar sites of accumulation of NPC2 and NPC1. In living cells, NPC2 traffics with NPC1 in late endosomal tubules. However, in contrast to NPC1, which remains either in late endosomal vesicles and tubules or at the peripheries of cholesterol-laden lysosomes, NPC2 moves into the central core of lysosomes. Glycolipid analysis reveals that, in contrast to null mutant NPC1 cells, which accumulate GM2 ganglioside only at the plasma membrane, with no endocytic storage, absence of NPC2 protein in null mutant NPC2 cells does not block internalization of GM2 into endocytic vesicles. This difference in the cellular distribution of GM2 in NPC1 and NPC2 null mutants is the first report of a variation in the phenotypic expression of these genotypically distinct lesions. CONCLUSION: We speculate that while NPC1 may play a major role in the sorting of glycolipids as well as cholesterol within the late endosomes, NPC2 primarily plays a role in the egress of cholesterol and, potentially, glycolipids from lysosomes. These proteins appear not to be integrated into a tightly bound biological complex, but rather represent separate functional entities that complement each other.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease.

Niemann-Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes.

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.

Determinants of NPC1 expression and action: key promoter regions, posttranscriptional control, and the importance of a "cysteine-rich" loop.

Mutations in the NPC1 gene cause Niemann-Pick type C disease, which is characterized by the accumulation of free cholesterol and other lipids in lysosomes. The NPC1 glycoprotein is located in a late endosomal compartment that transiently interacts with lysosomes. To identify factors regulating NPC1 expression and action, we analyzed the function of the human NPC1 promoter in human-derived ovarian, hepatic, and neuronal cells. A fragment containing the first 208 base pairs upstream from the major transcription initiation site was sufficient to drive near maximal NPC1 promoter activity. Deletion analysis revealed that sequences between base pairs -111 and -37 play an important role in controlling NPC1 transcription. Treatment of proliferating granulosa cells with 30 microM progesterone, which induces a reversible phenocopy of the cholesterol trafficking defect of Niemann-Pick type C disease, increased NPC1 mRNA levels threefold. The protein synthesis inhibitor, cycloheximide, also increased NPC1 mRNA levels, augmenting the progesterone-induced increase in NPC1 mRNA abundance. Progesterone treatment was shown to increase the mRNA half-life, but did not affect NPC1 promoter activity. Cysteine residues in a "cysteine-rich" loop predicted to reside in the intralumenal compartment of vesicles containing NPC1 were mutated, resulting in proteins that were incapable of correcting the cholesterol trafficking defect in CT60 cells, a Chinese hamster cell line in which the endogenous NPC1 gene is inactivated. Converting isoleucine 1061, also predicted to lie within the cysteine-rich loop, to a threonine residue inactivated the protein as well. The I1061T mutation is one of the most common mutations in Niemann-Pick type C disease. All of the cysteine-rich loop mutants were localized to cholesterol-engorged lysosomes in a pattern mimicking the distribution of NPC1 in progesterone-treated cells. A recombinant protein representing the cysteine-rich loop was shown to bind to a zinc-NTA agarose column. We conclude: (1) that cis elements residing in the first 111 base pairs upstream from the transcription start site are critical for transcription of the NPC1 gene; (2) that NPC1 expression is subject to posttranscriptional regulation in response to treatments that disrupt NPC1 function; and (3) that an intralumenal cysteine-rich loop with zinc-binding activity is critical to NPC1's ability to unload lysosomal cargo.

NPC1-containing compartment of human granulosa-lutein cells: a role in the intracellular trafficking of cholesterol supporting steroidogenesis.

Steroidogenic cells represent unique systems for the exploration of intracellular cholesterol trafficking. We employed cytochemical and biochemical methods to explore the expression, regulation, and function of the Niemann-Pick C1 protein (NPC1) in human granulosa-lutein cells. NPC1 was localized in a subset of lysosome-associated membrane glycoprotein 2 (LAMP-2)-positive vesicles. By analyzing the sensitivity of NPC1 N-linked oligosaccharide chains to glycosidases and neuraminidase, evidence was obtained for movement of nascent NPC1 from the endoplasmic reticulum through the medial and trans compartments of the Golgi apparatus prior to its appearance in cytoplasmic vesicles. NPC1 protein content and the morphology and cellular distribution of NPC1-containing vesicles were not affected by treatment of the granulosa-lutein cells with 8-Br-cAMP, which stimulates cholesterol metabolism into progesterone. In contrast, steroidogenic acute regulatory (StAR) protein levels were increased by 8-Br-cAMP. Incubation of granulosa-lutein cells with low-density lipoprotein (LDL) in the presence of the hydrophobic amine, U18666A, caused accumulation of free cholesterol in granules, identified by filipin staining, that contained LAMP-2 and NPC1. These granules also stained for neutral lipid with Nile red, reflecting accumulation of LDL-derived cholesterol esters. LDL-stimulated progesterone synthesis was completely blocked by U18666A, leaving steroid output at levels similar to those of cells incubated in the absence of LDL. The hydrophobic amine also blocked the LDL augmentation of 8-Br-cAMP-stimulated progesterone synthesis, reducing steroid production to levels seen in cells stimulated with 8-Br-cAMP in the absence of LDL. Steroidogenesis recovered after U18666A was removed from the culture medium. U18666A treatment caused a 2-fold or more increase in NPC1 protein and mRNA levels, suggesting that disruption of NPC1's function activates a compensatory mechanism resulting in increased NPC1 synthesis. We conclude that the NPC1 compartment plays an important role in the trafficking of LDL-derived substrate in steroidogenic cells; that NPC1 expression is up-regulated when NPC1 action is blocked; and that the NPC1 compartment can be functionally separated from other intracellular pathways contributing substrate for steroidogenesis.

Adrenal and liver in normal and cld/cld mice synthesize and secrete hepatic lipase, but the lipase is inactive in cld/cld mice.

Combined lipase deficiency (cld) is a recessive mutation in mice that causes a severe lack of lipoprotein lipase (LPL) and hepatic lipase (HL) activities, hyperlipemia, and death within 3 days after birth. Earlier studies showed that inactive LPL and HL were synthesized by cld/cld tissues and that LPL synthesized by cld/cld brown adipocytes was retained in their ER. We report here a study of HL in liver, adrenal, and plasma of normal newborn and cld/cld mice. Immunofluorescence studies showed HL was present in extracellular space, but not in cells, in liver and adrenal of both normal and cld/cld mice. When protein secretion was blocked with monensin, HL was retained intracellularly in liver cell cultures and in incubated adrenal tissues of both groups of mice. These findings demonstrated that HL was synthesized and secreted by liver and adrenal cells in normal newborn and cld/cld mice. HL activities in liver, adrenal, and plasma in cld/cld mice were very low, <8% of that in normal newborn mice, indicating that HL synthesized and secreted by cld/cld cells was inactive. Livers of both normal newborn and cld/cld mice synthesized LPL, but the level of LPL activity in cld/cld liver was very low, <9% of that in normal liver. Immunofluorescence studies showed that LPL was present intracellularly in liver of cld/cld mice, indicating that LPL was synthesized but not secreted by cld/cld liver cells. Immunofluorescent LPL was not found in normal newborn liver cells unless the cells were treated with monensin, thus demonstrating that normal liver cells synthesized and secreted LPL. Livers of both groups of mice contained an unidentified alkaline lipase activity which accounted for 34-54% of alkaline lipase activity in normal and 65% of that in cld/cld livers. Our findings indicate that liver and adrenal cells synthesized and secreted HL in both normal newborn and cld/cld mice, but the lipase was inactive in cld/cld mice. That cld/cld liver cells secreted inactive HL while retaining inactive LPL indicates that these closely related lipases were processed differently.

Induction of endocytic vesicles by exogenous C(6)-ceramide.

Ceramide is a newly discovered second messenger that has been shown to cause cell growth arrest and apoptosis. Here, we present evidence that exogenously added C(6)-ceramide induces enlargement of late endosomes and lysosomes. 10 microM C(6)-ceramide caused the formation of numerous vesicles of varying sizes (2-10 micrometers) in fibroblasts (3T3-L1 and 3T3-F442A), without toxic effects. Vesicle formation induced by C(6)-ceramide was time- and dose-dependent, rapid, and reversible. Numerous small vesicles appeared within 8 h of treatment with 10 microM C(6)-ceramide. They enlarged with time, with large vesicles found in the perinuclear region and small ones observed at the cell periphery. Within 24 h of treatment, approximately 30% of the cells exhibited these vesicles. Removal of ceramide from the culture medium caused disappearance of the vesicles, which reappeared upon readdition of ceramide. Confocal immunofluorescence microscopic analysis using an anti-lysosome-associated membrane protein antibody identified the enlarged vesicles as late endosomes/lysosomes. The fluorescent C(6)-NBD-ceramide, a vital stain for the Golgi apparatus, did not stain these vesicles. The effect on vesicle formation was influenced by ceramide structure; D-erythro-C(6)-ceramide was the most active ceramide analogue tested. Short chain ceramide metabolites, such as sphingosine, sphingosine 1-phosphate, N-hexanoyl-sphingosylphosphorylcholine, N-acetylpsychosine, and C(2)-ceramide G(M3), (G(M3), N-acetylneuraminosyl-alpha(2, 3)-galactosyl-beta(1,4)-glucosylceramide), were inactive in causing vesicle formation when added exogenously. Together, these studies demonstrate that exogenous C(6)-ceramide induces endocytic vesicle formation and causes enlarged late endosomes and lysosomes in mouse fibroblasts.

Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein.

Niemann-Pick type C (NPC) disease, characterized by accumulation of low density lipoprotein-derived free cholesterol in lysosomes, is caused by mutations in the NPC1 gene. We examined the ability of wild-type NPC1 and NPC1 mutants to correct the NPC sterol trafficking defect and their subcellular localization in CT60 cells. Cells transfected with wild-type NPC1 expressed 170- and 190-kDa proteins. Tunicamycin treatment resulted in a 140-kDa protein, the deduced size of NPC1, suggesting that NPC1 is N-glycosylated. Mutation of all four asparagines in potential N-terminal N-glycosylation sites to glutamines resulted in a 20-kDa reduction of the expressed protein. Proteins with a single N-glycosylation site mutation localized to late endosome/lysosomal compartments, as did wild-type NPC1, and each corrected the cholesterol trafficking defect. However, mutation of all four potential N-glycosylation sites reduced ability to correct the NPC phenotype commensurate with reduced expression of the protein. Mutations in the putative sterol-sensing domain resulted in inactive proteins targeted to lysosomal membranes encircling cholesterol-laden cores. N-terminal leucine zipper motif mutants could not correct the NPC defect, although they accumulated in lysosomal membranes. We conclude that NPC1 is a glycoprotein that must have an intact sterol-sensing domain and leucine zipper motif for cholesterol-mobilizing activity.

The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo.

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.

Localization of Niemann-Pick C1 protein in astrocytes: implications for neuronal degeneration in Niemann- Pick type C disease.

Niemann-Pick type C disease (NP-C) is an inherited neurovisceral lipid storage disorder characterized by progressive neurodegeneration. Most cases of NP-C result from inactivating mutations of NPC1, a recently identified member of a family of genes encoding membrane-bound proteins containing putative sterol sensing domains. By using a specific antipeptide antibody to human NPC1, we have here investigated the cellular and subcellular localization and regulation of NPC1. By light and electron microscopic immunocytochemistry of monkey brain, NPC1 was expressed predominantly in perisynaptic astrocytic glial processes. At a subcellular level, NPC1 localized to vesicles with the morphological characteristics of lysosomes and to sites near the plasma membrane. Analysis of the temporal and spatial pattern of neurodegeneration in the NP-C mouse, a spontaneous mutant model of human NP-C, by amino-cupric-silver staining, showed that the terminal fields of axons and dendrites are the earliest sites of degeneration that occur well before the appearance of a neurological phenotype. Western blots of cultured human fibroblasts and monkey brain homogenates revealed NPC1 as a 165-kDa protein. NPC1 levels in cultured fibroblasts were unchanged by incubation with low density lipoproteins or oxysterols but were increased 2- to 3-fold by the drugs progesterone and U-18666A, which block cholesterol transport out of lysosomes, and by the lysosomotropic agent NH4Cl. These studies show that NPC1 in brain is predominantly a glial protein present in astrocytic processes closely associated with nerve terminals, the earliest site of degeneration in NP-C. Given the vesicular localization of NPC1 and its proposed role in mediating retroendocytic trafficking of cholesterol and other lysosomal cargo, these results suggest that disruption of NPC1-mediated vesicular trafficking in astrocytes may be linked to neuronal degeneration in NP-C.

Niemann-Pick C1 protein: obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization.

Niemann-Pick type C (NPC) disease is an inherited lipid storage disorder that affects the viscera and central nervous system. A characteristic feature of NPC cells is the lysosomal accumulation of low density lipoprotein-derived cholesterol. To elucidate important structural features of the recently identified NPC1 gene product defective in NPC disease, we examined the ability of wild-type NPC1 and NPC1 mutants to correct the excessive lysosomal storage of low density lipoprotein-derived cholesterol in a model cell line displaying the NPC cholesterol-trafficking defect (CT60 Chinese hamster ovary cells). CT60 cells transfected with human wild-type NPC1 contained immunoreactive proteins of 170 and 190 kDa localized to the lysosomal/endosomal compartment. Wild-type NPC1 protein corrected the NPC cholesterol-trafficking defect in the CT60 cells. Mutation of conserved cysteine residues in the NPC1 N terminus to serine residues resulted in proteins targeted to lysosomal membranes encircling cholesterol-laden cores, whereas deletion of the C-terminal 4-aa residues containing the LLNF lysosome-targeting motif resulted in the expression of protein localized to the endoplasmic reticulum. None of these mutant NPC1 proteins corrected the NPC cholesterol-trafficking defect in CT60 cells. We conclude that transport of the NPC1 protein to the cholesterol-laden lysosomal compartment is essential for expression of its biological activity and that domains in the N terminus of the NPC1 protein are critical for mobilization of cholesterol from lysosomes.

Combined lipase deficiency (cld/cld) in mice affects differently post-translational processing of lipoprotein lipase, hepatic lipase and pancreatic lipase.

Lipoprotein lipase (LPL) and hepatic lipase (HL), which act on plasma lipoproteins, belong to the same gene family as pancreatic lipase. LPL is synthesized in heart, muscle and adipose tissue, while HL is synthesized primarily in liver. LPL is also synthesized in liver of newborn rodents. The active form of LPL is a dimer, whereas that of HL has not been established. Combined lipase deficiency (CLD) is an autosomal recessive mutation (cld) in mice which impairs post-translational processing of LPL and HL. Cld/cld mice have very low LPL and HL activities (< 5% of normal), yet normal pancreatic lipase activity. They develop massive hypertriglyceridemia and die within 3 days after birth. The CLD mutation allows synthesis, glycosylation and dimerization of LPL, but blocks activation and secretion of the lipase. Thus, dimerization per se does not result in production of active LPL. Immunofluorescence studies showed that LPL is retained in endoplasmic reticulum (ER) in cld/cld cells. Translocation of Golgi components to ER by treatment with brefeldin A (BFA) enabled synthesis of active LPL in cultured cld/cld brown adipocytes. Thus, production of inactive LPL in cld/cld cells results from inability of the cells to transport LPL from ER. The CLD mutation allows synthesis and glycosylation of HL, but blocks activation of the lipase. Immunofluorescence studies located HL mostly outside of cells in liver, liver cell cultures and incubated adrenal tissue of normal and cld/cld mice and mostly inside of cells in liver cell cultures and adrenal tissues treated with monensin (to block secretion of protein). These findings demonstrate synthesis and secretion of HL by both liver and adrenal cells of normal and cld/cld mice. Thus, the CLD mutation allows secretion of inactive HL by liver and adrenals. However, it does not block synthesis or secretion of active pancreatic lipase. Our findings indicate that LPL, HL and pancreatic lipase, although closely related, are processed differently.

Sickling of nucleated erythroid precursors from patients with sickle cell anemia.

The pathophysiology of sickle cell anemia is primarily explained in terms of the oxygen-dependent polymerization of sickle hemoglobin (HbS) followed by sickling of erythrocytes. Since the rate and extent of HbS polymerization depend on its intracellular concentration, it has been generally assumed that sickling occurs primarily in mature erythrocytes with their high intracellular hemoglobin concentration. In the present study, we investigated the propensity of nucleated erythroid precursors to undergo sickling; both cultured and fresh marrow-derived erythroid precursors from patients with homozygous sickle cell anemia were studied. The results revealed that upon deoxygenation cultured erythroblasts underwent characteristic morphological deformation in the form of fine, fragile, elongated spicules. Ultrastructural analysis demonstrated highly organized and tightly aligned hemoglobin fibers in the protruded regions. Bone marrow cells examined under partial or complete deoxygenated conditions displayed similar morphological changes. When cultured SS erythroid precursors were exposed to hydroxyurea or butyrate, drugs that may increase fetal hemoglobin (HbF) and inhibit intracellular polymerization, a significant decrease was observed in the propensity of these precursors to undergo sickling, accompanied by a three- to fivefold increase in HbF. These results suggest that, in addition to mature erythrocytes, nucleated erythroid precursors in the bone marrow have the capacity to undergo characteristic sickling as a result of HbS polymerization and may be involved in several aspects of the pathophysiology of sickle cell anemia. Treatment with HbF-stimulating drugs may benefit patients with this disease by inhibiting polymerization-induced sickling of erythroid precursors in the marrow as well as mature erythrocytes in the peripheral blood.

Constitutive achlorhydria in mucolipidosis type IV.

Mucolipidosis type IV is an autosomal recessive lysosomal storage disease of unknown etiology that causes severe neurological and ophthalmological abnormalities. In an attempt to obtain insight into the nature of the metabolic abnormality in this disorder, we prospectively evaluated 15 consecutive patients, aged 2 to 23 years, over a period of 22 months. The finding of iron deficiency in some of the patients led us to the discovery that all patients but one had markedly elevated blood gastrin levels. None had vitamin B12 deficiency. Gastroscopy in three patients showed normal gross appearance of the mucosa in two patients, 4 and 7 years old, and mucosal atrophy in a 22-year-old. Parietal cells were present in normal numbers and contained large cytoplasmic inclusions that were confirmed immunohistochemically to be lysosomal in nature. Other gastric epithelial cells appeared normal. Parietal cells contained very few tubulovesicular membranes, suggesting cellular activation, whereas apical canaliculi appeared relatively nonactivated. Both subunits of the parietal cell H+/K+-ATPase were present, and both partially colocalized with f-actin at the apical membrane. We conclude that patients with mucolipidosis type IV are constitutively achlorhydric and have partially activated parietal cells. We hypothesize that the defective protein in this disease is closely associated with the final stages of parietal cell activation and is critical for a specific type of cellular vacuolar trafficking between the cytoplasm and the apical membrane domain.

Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein.

The adipose differentiation-related protein (ADRP) was first characterized as a mRNA induced early during adipocyte differentiation (Jiang, H. P., and G. Serrero. 1992. Proc. Natl. Acad. Sci. USA. 89:7856-7860). The present study demonstrates that ADRP mRNA is expressed in a variety of tissues and cultured cell lines. Immunocytochemical examination revealed that ADRP localizes to neutral lipid storage droplets in cultured murine 3T3-L1 adipocytes, murine MA-10 Leydig cells, Chinese hamster ovary (CHO) fibroblasts, and human HepG2 hepatoma cells; the association of ADRP with lipid droplets was confirmed by subcellular fractionation of MA-10 Leydig cells. In addition to ADRP, steroidogenic cells and adipocytes express the perilipins, a family of lipid droplet-associated proteins that share a highly related sequence domain with ADRP. ADRP and perilipins co-localize on lipid droplets in MA-10 Leydig cells. While ADRP was found on small lipid droplets in 3T3-L1 preadipocytes and early differentiated adipocytes, it was absent in maturing adipocytes. In contrast, perilipins were absent early during differentiation, but were found on small and large lipid droplets at later stages. The transition in surface protein composition of adipocyte lipid droplets from ADRP to perilipins occurred 3 days after the initiation of differentiation when cells displayed co-localizatioin of both proteins on the same lipid droplets. The specific localization of adipose differentiation-related protein to lipid droplets in a wide variety of cells suggests that ADRP plays a role in management of neutral lipid stores.

Immunocytochemical evidence that GLUT4 resides in a specialized translocation post-endosomal VAMP2-positive compartment in rat adipose cells in the absence of insulin.

Insulin stimulates glucose transport in rat adipose cells through the translocation of GLUT4 from a poorly defined intracellular compartment to the cell surface. We employed confocal microscopy to determine the in situ localization of GLUT4 relative to vesicle, Golgi, and endosomal proteins in these physiological insulin target cells. Three-dimensional analyses of GLUT4 immunostaining in basal cells revealed an intracellular punctate, patchy distribution both in the perinuclear region and scattered throughout the cytoplasm. VAMP2 closely associates with GLUT4 in many punctate vesicle-like structures. A small fraction of GLUT4 overlaps with TGN38-mannosidase II, gamma-adaptin, and mannose-6-phosphate receptors in the perinuclear region, presumably corresponding to late endosome and trans-Golgi network structures. GLUT4 does not co-localize with transferrin receptors, clathrin, and Igp-120. After insulin treatment, GLUT4 partially redistributes to the cell surface and decreases in the perinuclear area. However, GLUT4 remains co-localized with TGN38-mannosidase II and gamma-adaptin. Therefore, the basal compartment from which GLUT4 is translocated in response to insulin comprises specialized post-endosomal VAMP2-positive vesicles, distinct from the constitutively recycling endosomes. These results are consistent with a kinetic model in which GLUT4 is sequestered through two or more intracellular pools in series.

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Intracellular trafficking of cholesterol monitored with a cyclodextrin.

The sterol binding agent 2-hydroxypropyl-beta-cyclodextrin is shown to be a convenient and useful experimental tool to probe intracellular pathways of cholesterol transport. Biochemical and cytochemical studies reveal that cyclodextrin specifically removes plasma membrane cholesterol. Depletion of plasma membrane sphingomyelin greatly accelerated cyclodextrin-mediated cholesterol removal. Cholesterol arriving at the plasma membrane from lysosomes and the endoplasmic reticulum was also removed by cyclodextrin. Cellular cholesterol esterification linked to the mobilization of cholesterol from lysosomes was strongly attenuated by cyclodextrin, suggesting that the major portion of endocytosed cholesterol is delivered from lysosomes to the endoplasmic reticulum via the plasma membrane. Evidence for translocation of lysosomal cholesterol to the endoplasmic reticulum by a plasma membrane-independent pathway is provided by the finding that cyclodextrin loses its ability to suppress esterification when plasma membrane sphingomyelin is depleted. The Golgi apparatus appears to play an active role in directing the relocation of lysosomal cholesterol to the plasma membrane since brefeldin A also abrogated cyclodextrin-mediated suppression of cholesterol esterification. Using cyclodextrin we further show that attenuated esterification of lysosomal cholesterol in Niemann-Pick C cells reflects defective translocation of cholesterol to the plasma membrane that may be linked to abnormal Golgi trafficking.