Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. Despite considerable efforts, no essential cellular receptors for filovirus entry have been identified. We showed previously that Niemann-Pick C1 (NPC1), a lysosomal cholesterol transporter, is required for filovirus entry. Here, we demonstrate that NPC1 is a critical filovirus receptor. Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. The second luminal domain of NPC1 binds directly and specifically to the viral glycoprotein, GP, and a synthetic single-pass membrane protein containing this domain has viral receptor activity. Purified NPC1 binds only to a cleaved form of GP that is generated within cells during entry, and only viruses containing cleaved GP can utilize a receptor retargeted to the cell surface. Our findings support a model in which GP cleavage by endosomal cysteine proteases unmasks the binding site for NPC1, and GP-NPC1 engagement within lysosomes promotes a late step in entry proximal to viral escape into the host cytoplasm. NPC1 is the first known viral receptor that recognizes its ligand within an intracellular compartment and not at the plasma membrane.

The absence of the outer mitochondrial membrane protein Uth1p was found to induce resistance to rapamycin treatment and starvation, two conditions that induce the autophagic process. Biochemical studies showed the onset of a fully active autophagic activity both in wild-type and ⌬uth1 strains. On the other hand, the disorganization of the mitochondrial network induced by rapamycin treatment or 15 h of nitrogen starvation was followed in cells expressing mitochondria-targeted green fluorescent protein; a rapid colocalization of green fluorescent protein fluorescence with vacuole-selective FM4-64 labeling was observed in the wild-type but not in the ⌬uth1 strain. Degradation of mitochondrial proteins, followed by Western blot analysis, did not occur in mutant strains carrying null mutations of the vacuolar protease Pep4p, the autophagyspecific protein Atg5p, and Uth1p. These data show that, although the autophagic machinery was fully functional in the absence of Uth1p, this protein is involved in the autophagic degradation of mitochondria.The major cellular degradation pathways involved in protein and organelle turnover are autophagy and proteasome-mediated proteolysis. These processes are important for maintaining a controlled balance between anabolism and catabolism in order to have normal cell growth and development. These degradation pathways permit the cell to eliminate unwanted or unnecessary organelles and recycle the components for reuse. In eukaryotic cells, the lysosomes or the vacuole are major degradative organelles that contain a range of hydrolases able of degrading all the cellular constituents. During the last decade, autophagy has emerged as a crucial membrane trafficking process that transports bulk cytoplasm and sometimes entire organelles to the lysosome/vacuole for recycling in response to nutrient starvation or under specific physiological conditions (see Refs. 1 and 2 for reviews). Autophagy has two major forms, microautophagy and macroautophagy. Microautophagy operates by protruding or invaginating a portion of the vacuolar membrane to engulf cytosol or organelles. Only limited knowledge is available about microautophagy, which has been best characterized for the degradation of peroxisomes (3, 4) and selective portions of the nucleus (5). Macroautophagy, of which the molecular aspects are better characterized, involves the non-selective sequestration of large portions of the cytoplasm into double membrane structures termed autophagosomes and their delivery to the vacuole for degradation.Although the normal function of autophagy is thought to provide amino acids to starved cells, a fair amount of evidence suggests that it could also be required for the elimination of selected organelles, namely mitochondria, under peculiar conditions.

Niemann-Pick type C1 (NPC1) protein is needed for cellular utilization of low-density lipoprotein-derived cholesterol that has been delivered to lysosomes. The protein has 13 transmembrane domains, three large lumenal domains, and a cytoplasmic tail. NPC1's lumenally oriented, N-terminal domain binds cholesterol and has been proposed to receive cholesterol from NPC2 protein as part of the process by which cholesterol is exported from lysosomes into the cytosol. Using surface plasmon resonance and affinity chromatography, we show here that the second lumenal domain of NPC1 binds directly to NPC2 protein. For these experiments, a soluble NPC1 lumenal domain 2 was engineered by replacing adjacent transmembrane domains with antiparallel coiled-coil sequences. Interaction of NPC2 with NPC1 lumenal domain 2 is only detected at acidic pH, conditions that are optimal for cholesterol binding to NPC2 and transfer to NPC1; the pH is also appropriate for the acidic environment where binding would take place. Binding to NPC1 domain 2 requires the presence of cholesterol on NPC2 protein, a finding that supports directional transfer of cholesterol from NPC2 onto NPC1's N-terminal domain. Finally, human disease-causing mutations in NPC1 domain 2 decrease NPC2 binding, suggesting that NPC2 binding is necessary for NPC1 function in humans. These data support a model in which NPC1 domain 2 holds NPC2 in position to facilitate directional cholesterol transfer from NPC2 onto NPC1 protein for export from lysosomes.cholesterol trafficking | Niemann-Pick type C disease A major source of cellular cholesterol is endocytosed as lowdensity lipoprotein, which is delivered to late endosomes and lysosomes where cholesterol is released (1). Within late endosomes and lysosomes, Niemann-Pick type C1 (NPC1) and NPC2 proteins are required for the subsequent delivery of cholesterol to other intracellular compartments (2). NPC1 is a large, 1,254 residue, integral membrane protein that is predicted to span the bilayer 13 times (3, 4); it contains a lumenally oriented, N-terminal cholesterol binding site that interacts with the 3β-hydroxyl end of the cholesterol molecule (5-7). NPC2 is a much smaller, soluble lysosomal protein of 132 amino acid residues (8) that binds cholesterol in an opposite orientation via cholesterol's isooctyl side chain (9-11). The importance of NPC1 and NPC2 for cholesterol and glycosphingolipid homeostasis is demonstrated in Niemann-Pick type C disease: patients carrying homozygous mutations in either of these proteins suffer neurodegeneration and die in childhood due to cholesterol and glycosphingolipid accumulation in the brain, liver, and lungs (12, 13).NPC2 is thought to play an important role in extracting cholesterol from intralysosomal membranes that are rich in cholesterol and bis(monoacylglycerol) phosphate. Transfer of that cholesterol to NPC1 is proposed to facilitate passage of this hydrophobic sterol across the glycocalyx that lines the inner lysosomal membrane.Recent studies using purified NPC2 and a soluble con...

The antioxidant N-acetyl-L-cysteine prevented the autophagydependent delivery of mitochondria to the vacuoles, as examined by fluorescence microscopy of mitochondria-targeted green fluorescent protein, transmission electron microscopy, and Western blot analysis of mitochondrial proteins. The effect of N-acetyl-L-cysteine was specific to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation of alkaline phosphatase and the presence of hallmarks of non-selective microautophagy were not altered by N-acetyl-L-cysteine. The effect of N-acetyl-L-cysteine was not related to its scavenging properties, but rather to its fueling effect of the glutathione pool. As a matter of fact, the decrease of the glutathione pool induced by chemical or genetical manipulation did stimulate mitophagy but not general autophagy. Conversely, the addition of a cellpermeable form of glutathione inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the strain ⌬uth1, which is deficient in selective mitochondrial degradation. These data show that mitophagy can be regulated independently of general autophagy, and that its implementation may depend on the cellular redox status.Autophagy is a major pathway for the lysosomal/vacuolar delivery of long-lived proteins and organelles, where they are degraded and recycled. Autophagy plays a crucial role in differentiation and cellular response to stress and is conserved in eukaryotic cells from yeast to mammals (1, 2). The main form of autophagy, macroautophagy, involves the non-selective sequestration of large portions of the cytoplasm into doublemembrane structures termed autophagosomes, and their delivery to the vacuole/lysosome for degradation. Another process, microautophagy, involves the direct sequestration of parts of the cytoplasm by vacuole/lysosomes. The two processes coexist in yeast cells but their extent may depend on different factors including metabolic state: for example, we have observed that nitrogen-starved lactate-grown yeast cells develop microautophagy, whereas nitrogen-starved glucosegrown cells preferentially develop macroautophagy (3).Both macroautophagy and microautophagy are essentially non-selective, in the way that autophagosomes and vacuole invaginations do not appear to discriminate the sequestered material.