Ncr1p, the yeast ortholog of mammalian Niemann Pick C1 protein, is dispensable for endocytic transport

The Niemann Pick C1 protein localizes to late endosomes and plays a key role in the intracellular transport of cholesterol in mammalian cells. Cholesterol and other lipids accumulate in a lysosomal or late endosomal compartment in cells lacking normal NPC1 function. Other than accumulation of lipids, defects in lysosomal retroendocytosis, sorting of a multifunctional receptor and endosomal movement have also been detected in NPC1 mutant cells. Ncr1p is an ortholog of NPC1 in the budding yeast Saccharomyces cerevisiae. In this study, we show that Ncr1p is a vacuolar membrane protein that transits through the biosynthetic vacuolar protein sorting pathway, and that it can be solubilized by Triton X-100 at 4 degrees C. Using well-established assays, we demonstrate that the absence of Ncr1p had no effect on fluid phase and receptor- mediated endocytosis, biosynthetic delivery to the vacuole, retrograde transport from endosome to Golgi and ubiquitin- and nonubiquitin-dependent multivesicular body sorting. We conclude that Ncr1p does not have an essential role in known endocytic transport pathways in yeast.     

Transport between the cytoplasm and the nucleus

Summary Active transport of proteins and RNAs across the nuclear-pore complex (NPC) is mediated by a family of related transport receptors which shuttle between the cytoplasm and the nucleoplasm. A number of import and export pathways have been described. Some transport substrates require adapters which mediate association with certain transporters. The transport receptors specifically bind to a recognition signal within the transport substrate or adapter, pass the NPC in one direction, and deliver their cargo to the other side of the nuclear envelope. The Ran GTPase is the crucial regulator of bidirectional transport. Ran-modulating proteins establish an asymmetric intracellular distribution of Ran. As a result, Ran is mainly bound to GTP in the nucleus and to GDP in the cytoplasm. Evidently, RanGTP regulates binding and release of the transport substrates by binding to the transport receptors in the nucleus as well as the transport direction across the NPC. However, little is known about the molecular mechanism of translocation through the NPC.     

The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm

Compositional analysis of nuclear pore complexes (NPCs) is nearing completion, and efforts are now focused on understanding how these protein machines work. Recent analysis of soluble transport factor interactions with NPC proteins reveals distinct and overlapping pathways for movement between the nucleus and cytoplasm. New fluorescence- and microscopy-based strategies have been used to monitor the pathway of NPC assembly and to reveal the dynamics of the NPC during transport.     

Nucleoporin's Like Charge Regions Are Major Regulators of FG Coverage and Dynamics Inside the Nuclear Pore Complex

Nucleocytoplasmic transport has been the subject of a large body of research in the past few decades. Recently, the focus of investigations in this field has shifted from studies of the overall function of the nuclear pore complex (NPC) to the examination of the role of different domains of phenylalanine-glycine nucleoporin (FG Nup) sequences on the NPC function. In our recent bioinformatics study, we showed that FG Nups have some evolutionarily conserved sequence-based features that might govern their physical behavior inside the NPC. We proposed the ‘like charge regions’ (LCRs), sequences of charged residues with only one type of charge, as one of the features that play a significant role in the formation of FG network inside the central channel. In this study, we further explore the role of LCRs in the distribution of FG Nups, using a recently developed coarse-grained molecular dynamics model. Our results demonstrate how LCRs affect the formation of two transport pathways. While some FG Nups locate their FG network at the center of the NPC forming a homogeneous meshwork of FG repeats, other FG Nups cover the space adjacent to the NPC wall. LCRs in the former group, i.e. FG Nups that form an FG domain at the center, tend to regulate the size of the highly dense, doughnut-shaped FG meshwork and leave a small low FG density area at the center of the pore for passive diffusion. On the other hand, LCRs in the latter group of FG Nups enable them to maximize their interactions and cover a larger space inside the NPC to increase its capability to transport numerous cargos at the same time. Finally, a new viewpoint is proposed that reconciles different models for the nuclear pore selective barrier function.     

Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

Niemann–Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.     

Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex

The interactions between transport receptors and proteins of the nuclear pore complex (NPC) are fundamental to understanding nucleocytoplasmic transport. In order to delineate the path that a particular transport receptor takes through the NPC, we have employed fluorescence resonance energy transfer (FRET) between enhanced cyan and yellow fluorescent proteins (ECFP, EYFP) in living cells. A panel of yeast strains expressing functional receptor--ECFP and nucleoporin--EYFP fusions has been analyzed with a FRET assay. With this approach, we define points of contact in the NPC for the related importin Pse1/Kap121 and exportin Msn5. These data demonstrate the utility of FRET in mapping dynamic protein interactions in a genetic system. Furthermore, the data indicate that an importin and exportin have overlapping pathways through the NPC.     

The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function

The nuclear pore complex (NPC) is a large channel that spans the two lipid bilayers of the nuclear envelope and mediates transport events between the cytoplasm and the nucleus. Only a few NPC components are transmembrane proteins, and the role of these proteins in NPC function and assembly remains poorly understood. We investigate the function of the three integral membrane nucleoporins, which are Ndc1p, Pom152p, and Pom34p, in NPC assembly and transport in Saccharomyces cerevisiae. We find that Ndc1p is important for the correct localization of nuclear transport cargoes and of components of the NPC. However, the role of Ndc1p in NPC assembly is partially redundant with Pom152p, as cells lacking both of these proteins show enhanced NPC disruption. Electron microscopy studies reveal that the absence of Ndc1p and Pom152p results in aberrant pores that have enlarged diameters and lack proteinaceous material, leading to an increased diffusion between the cytoplasm and the nucleus.     

Niemann-Pick Type C2 protein contributes to the transport of endosomal cholesterol to mitochondria without interacting with NPC1

Mitochondrial cholesterol is maintained within a narrow range to regulate steroid and oxysterol synthesis and to ensure mitochondrial function. Mitochondria acquire cholesterol through several pathways from different cellular pools. Here we have characterized mitochondrial import of endosomal cholesterol using Chinese hamster ovary cells expressing a CYP11A1 fusion protein that converts cholesterol to pregnenolone at the mitochondrial inner membrane. RNA interference-mediated depletion of the voltage-dependent anion channel 1 in the mitochondrial outer membrane or of Niemann-Pick Type C2 (NPC2) in the endosome lumen decreased arrival of cholesterol at the mitochondrial inner membrane. Expression of NPC2 mutants unable to transfer cholesterol to NPC1 still restored mitochondrial cholesterol import in NPC2-depleted cells. Transport assays in semi-permeabilized cells showed nonvesicular cholesterol trafficking directly from endosomes to mitochondria that did not require cytosolic transport proteins but that was reduced in the absence of NPC2. Our findings indicate that NPC2 delivers cholesterol to the perimeter membrane of late endosomes, where it becomes available for transport to mitochondria without requiring NPC1.     

Identification of a pharmaceutical compound that partially corrects the Niemann-Pick C phenotype in cultured cells

Niemann-Pick C (NPC) is an autosomal recessive lysosomal lipid storage disease characterized by progressive central nervous system degeneration. In cultured human NPC fibroblasts, LDL-derived cholesterol accumulates in lysosomes and endosomes, LDL-cholesterol transport from endocytic compartments to other cellular compartments is delayed, and LDL does not elicit normal homeostatic responses. Currently, there is no therapy that delays the onset of neurological symptoms or prolongs the life span of NPC children. We have developed and implemented an amphotericin B-mediated cytotoxicity assay to screen for potential therapeutic drugs that induce cholesterol movement in cultured NPC cells. NPC cells are relatively resistant to amphotericin B killing due to intracellular sequestration of cellular cholesterol. The screen was carried out using simian virus 40-transformed ovarian granulosa cells from the npc (nih) mouse model of NPC disease. A library of 44240 compounds was screened and 55 compounds were identified that promote amphotericin B-mediated killing of NPC cells. One compound, NP-27, corrected the NPC phenotype by four different measures of cholesterol homeostasis. In addition to making NPC cells more sensitive to amphotericin B, NP-27 stimulated two separate cholesterol transport pathways and restored LDL stimulation of cholesterol esterification to near normal levels.     

The Role of Cohesiveness in the Permeability of the Spatial Assemblies of FG Nucleoporins

Nuclear pore complexes (NPCs) conduct selective, bidirectional transport across the nuclear envelope. The NPC passageway is lined by intrinsically disordered proteins that contain hydrophobic phenylalanine-glycine (FG) motifs, known as FG nucleoporins (FG nups), that play the key role in the NPC transport mechanism. Cohesive interactions among the FG nups, which arise from the combination of hydrophobic, electrostatic, and other forces, have been hypothesized to control the morphology of the assemblies of FG nups in the NPC, as well as their permeability with respect to the transport proteins. However, the role of FG nup cohesiveness is still vigorously debated. Using coarse-grained polymer theory and numerical simulations, we study the effects of cohesiveness on the selective permeability of in vitro FG nup assemblies in different geometries that have served as proxies for the morphological and transport properties of the NPC. We show that in high-density FG nup assemblies, increase in cohesiveness leads to the decrease in their permeability, in accordance with the accepted view. On the other hand, the permeability of low-density assemblies is a nonmonotonic function of the cohesiveness, and a moderate increase in cohesiveness can enhance permeability. The density- and cohesiveness-dependent effects on permeability are explained by considering the free-energy cost associated with penetrating the FG nup assemblies. We discuss the implications of these findings for the organization and function of the NPC.     

Influence of cargo size on Ran and energy requirements for nuclear protein import

Previous work has shown that the transport of some small protein cargoes through the nuclear pore complex (NPC) can occur in vitro in the absence of nucleoside triphosphate hydrolysis. We now demonstrate that in the importin alpha/beta and transportin import pathways, efficient in vitro transport of large proteins, in contrast to smaller proteins, requires hydrolyzable GTP and the small GTPase Ran. Morphological and biochemical analysis indicates that the presence of Ran and GTP allows large cargo to efficiently cross central regions of the NPC. We further demonstrate that this function of RanGTP at least partly involves its direct binding to importin beta and transportin. We suggest that RanGTP functions in these pathways to promote the transport of large cargo by enhancing the ability of import complexes to traverse diffusionally restricted areas of the NPC.     

Karyopherins: from nuclear-transport mediators to nuclear-function regulators

The karyopherin beta (or importin beta) family comprises soluble transport factors that mediate the movement of proteins and RNAs between the nucleus and cytoplasm. Recent studies have extended the role of karyopherins to regulating assembly of the nuclear pore complex (NPC), assembly of the nuclear envelope, mitosis and replication. New data also address how karyopherins specifically recognize and transport many distinct cargoes and traverse the NPC. These data raise the possibility that, although there might be a universal mechanism for nuclear transport, specific interactions between karyopherins and components of the NPC might function to regulate differentially the ability of the different karyopherins to cross the NPC.     

Nuclear transport of single molecules: dwell times at the nuclear pore complex

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 +/- 0.2 and 7.1 +/- 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.     

The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo

Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs). Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats. Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC. We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry. The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1. Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups. These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC.     

Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport

The fate of free cholesterol released after endocytosis of low-density lipoproteins remains obscure. Here we report that late endosomes have a pivotal role in intracellular cholesterol transport. We find that in the genetic disease Niemann-Pick type C (NPC), and in drug-treated cells that mimic NPC, cholesterol accumulates in late endosomes and sorting of the lysosomal enzyme receptor is impaired. Our results show that the characteristic network of lysobisphosphatidic acid-rich membranes contained within multivesicular late endosomes regulates cholesterol transport, presumably by acting as a collection and distribution device. The results also suggest that similar endosomal defects accompany the anti-phospholipid syndrome and NPC.     

Genetic Analysis of Macromolecular Transport across the Nuclear Envelope

Numerous factors that promote movement of macromolecules in and out of the nucleus have now been identified. These include both soluble cytoplasmic and nucleoplasmic proteins and proteins of the nuclear pore complex (NPC). Genetic analyses of the nuclear transport process in the model organism, the budding yeastSaccharomyces cerevisiae,have revealed remarkable conservation of all of these factors. In addition, important clues as to how these factors promote the unique bidirectional movement across the NPC have emerged from studies of yeast. We summarize the characterization and genetic interactions of the soluble transport factors and present data to illustrate how genetic experiments can be used to further define the import and export pathways.     

The NPC1 protein: structure implies function

Niemann-Pick type C (NPC) is a lysosomal storage disorder, characterized by intracellular accumulation of low-density lipoprotein (LDL)-derived cholesterol and neurodegeneration leading to premature death. The most common form of the disease, NPC1, results from mutations in the NPC1 gene. Thus, the NPC1 protein is the focus of intense investigation to elucidate the function of this protein and its role in the disease pathogenesis. Recent studies have revealed the NPC1 subcellular location, topology and potential functions of the NPC1 protein. In lieu of direct experimental evidence, certain hypotheses about the function of NPC1 can be inferred by analyzing disease-causing mutations, NPC1 protein sequence homology to other related proteins, and the potential tertiary structure similarity between NPC1 and its prokaryotic ancestors, such as the E. coli RND permease AcrB. This review will discuss recent work on the characterization and function of the NPC1 protein and highlight structural features that may be important in assisting in the elucidation of NPC1 function and role in subcellular lipid transport and homeostasis.     

Regulation of sterol transport between membranes and NPC2

Niemann-Pick disease type C (NPC) is caused by defects in either the NPC1 or NPC2 gene and is characterized by accumulation of cholesterol and glycolipids in the late endosome/lysosome compartment. NPC2 is an intralysosomal protein that binds cholesterol in vitro. Previous studies demonstrated rapid rates of cholesterol transfer from NPC2 to model membranes [Cheruku, S. R., et al. (2006) J. Biol. Chem. 281, 31594-31604]. To model the potential role of NPC2 as a lysosomal cholesterol export protein, in this study we used fluorescence spectroscopic approaches to examine cholesterol transfer from membranes to NPC2, assessing the rate, mechanism, and regulation of this transport step. In addition, we examined the effect of NPC2 on the rate and kinetic mechanism of intermembrane sterol transport, to model the movement of cholesterol from internal lysosomal membranes to the limiting lysosomal membrane. The results support the hypothesis that NPC2 plays an important role in endo/lysosomal cholesterol trafficking by markedly accelerating the rates of cholesterol transport. Rates of sterol transfer from and between membranes were increased by as much as 2 orders of magnitude by NPC2. The transfer studies indicate that the mechanism of NPC2 action involves direct interaction of the protein with membranes. Such interactions were observed directly using FTIR spectroscopy and protein tryptophan spectral shifts. Additionally, cholesterol transfer by NPC2 was found to be greatly enhanced by the unique lysosomal phospholipid lyso-bisphosphatidic acid (LBPA), suggesting an important role for LBPA in NPC2-mediated cholesterol trafficking.     

Ran alters nuclear pore complex conformation

Transport across the nuclear membranes occurs through the nuclear pore complex (NPC), and is mediated by soluble transport factors including Ran, a small GTPase that is generally GDP-bound during import and GTP-bound for export. The dynamic nature of the NPC structure suggests a possible active role for it in driving translocation. Here we show that RanGTP but not RanGDP causes alterations of NPC structure when injected into the cytoplasm of Xenopus oocytes, including compaction of the NPC and extension of the cytoplasmic filaments. RanGTP caused accumulation of nucleoplasmin-gold along the length of extended cytoplasmic filaments, whereas RanGDP caused accumulation around the cytoplasmic rim of the NPC. This suggests a possible role for Ran in altering the conformation of the cytoplasmic filaments during transport.