Melanosomes--dark organelles enlighten endosomal membrane transport

Melanosomes are tissue-specific lysosome-related organelles of pigment cells in which melanins are synthesized and stored. Analyses of the trafficking and fate of melanosomal components are beginning to reveal how melanosomes are formed through novel pathways from early endosomal intermediates. These studies unveil generalized structural and functional modifications of the endosomal system in specialized cells, and provide unexpected insights into the biogenesis of multivesicular bodies and how compartmentalization regulates protein refolding. Moreover, genetic disorders that affect the biogenesis of melanosomes and other lysosome-related organelles have shed light onto the molecular machinery that controls specialized endosomal sorting events.     

Sterol-binding proteins and endosomal cholesterol transport

Endosomal compartments sort and deliver exogenous lipoprotein-derived cholesterol to the endoplasmic reticulum for regulating cellular cholesterol homeostasis. A large number of studies have focused on the removal of endosomal cholesterol, since its accumulation leads to devastating human diseases. Recent studies suggest that cytoplasmic sterol-binding proteins may be involved in endosomal cholesterol transport. In particular, endosome/lysosome-localized or -associated cholesterol-binding proteins may serve as key mediators of cholesterol removal in a non-vesicular manner. Further characterization of these cholesterol-binding proteins will shed light on the molecular mechanisms that regulate endosomal cholesterol sorting.     

Membrane transport: ubiquitylation in endosomal sorting.

In yeast, membrane proteins from the biosynthetic and endocytic pathways must be ubiquitylated for sorting to inward-budding vesicles in late endosomes, which give rise to multivesicular bodies. A conserved protein complex containing the yeast Vps23p or its mammalian counterpart Tsg101 may act as the ubiquitin receptor.     

Separation and characterization of late endosomal membrane domains

Very little is known about the biophysical properties and the lipid or protein composition of membrane domains presumably present in endocytic and biosynthetic organelles. Here we analyzed the membrane composition of late endosomes by suborganellar fractionation in the absence of detergent. We found that the internal membranes of this multivesicular organelle can be separated from the limiting membrane and that each membrane population exhibited a defined composition. Our data also indicated that internal membranes may consist of at least two populations, containing primarily phosphatidylcholine or lysobisphosphatidic acid as major phospholipid, arguing for the existence of significant microheterogeneity within late endosomal membranes. We also found that lysobisphosphatidic acid exhibited unique pH-dependent fusogenic properties, and we speculated that this lipid is an ideal candidate to regulate the dynamic properties of this internal membrane mosaic.     

Snc1p v-SNARE transport to the prospore membrane during yeast sporulation is dependent on endosomal retrieval pathways

Vesicular traffic is essential for sporulation in Saccharomyces cerevisiae. The Golgi-associated retrograde protein (GARP) tethering complex is required for retrograde traffic from both the early and late endosomes to the Golgi. Analyses of GARP complex mutants in sporulation reveal defects in meiotic progression and spore formation. In contrast, inactivation of the retromer complex, which mediates vesicle budding and cargo selection from the late endosome, or Snx4p, which is involved in retrieval of proteins from the early endosome, has little effect on sporulation. A retromer GARP double mutant is defective in the formation of the prospore membrane (PSM) that surrounds the haploid nuclei. In the retromer GARP double mutant, PSM precursor vesicles carrying the cargo, Dtr1p, are transported to the spindle pole body (SPB), where PSM formation is initiated. However, the v-SNARE Snc1p is not transported to the SPB in the double mutant, suggesting that the defect in PSM formation is because of the failure to retrieve Snc1p, and perhaps other proteins, from the endosomal pathway. Taken together, these results indicate that retrograde trafficking from the endosome is essential for sporulation by retrieving molecules important for PSM and spore wall formation.     

Modulation of endosomal cholesteryl ester metabolism by membrane cholesterol

Cells acquire cholesterol in part by endocytosis of cholesteryl ester containing lipoproteins. In endosomes and lysosomes cholesteryl ester is hydrolyzed by acidic cholesteryl ester hydrolase producing cholesterol and fatty acids. Under certain pathological conditions, however, such as in atherosclerosis, excessive levels of cholesteryl ester accumulate in lysosomes for reasons that are poorly understood. Here, we have studied endosomal and lysosomal cholesteryl ester metabolism in cultured mouse macrophages and with cell-free extracts. We show that net hydrolysis of cholesteryl ester is coupled to the transfer of cholesterol to membranes. When membrane cholesterol levels are low, absorption of cholesterol effectively drives cholesteryl ester hydrolysis. When cholesterol levels in acceptor membranes approach saturation or when cholesterol export is blocked, cholesterol is re-esterified in endosomes. These results reveal a new facet of cellular cholesterol homeostasis and provide a potential explanation for cholesteryl ester accumulation in lysosomes of atherosclerotic cells.     

Spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape

A key step in adenovirus cell entry is viral penetration of cellular membranes to gain access to the cytoplasm and deliver the genome to the nucleus. Yet little is known about this important event in the adenoviral life cycle. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture with both live- and fixed-cell imaging, we demonstrate that in the majority of instances, exposure of pVI and recruitment of gal3 to ruptured membranes occur early at or near the cell surface and occur minimally in EEA-1-positive (EEA-1(+)) early endosomes or LAMP-1(+) late endosomes/lysosomes. Live-cell imaging of Ad5 egress from gal3(+) endosomes occurs most frequently from perinuclear locations. While the Ad5 capsid is observed escaping from gal3(+) endosomes, pVI appears to remain associated with the gal3(+) ruptured endosomes. Thus, Ad5 membrane rupture and endosomal escape appear to be both spatially and temporally distinct events.     

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.     

Bidirectional transport between the trans-Golgi network and the endosomal system

Abstract

The exchange of proteins and lipids between the trans-Golgi network (TGN) and the endosomal system requires multiple cellular machines, whose activities are coordinated in space and time to generate pleomorphic, tubulo-vesicular carriers that deliver their content to their target compartments. These machines and their associated protein networks are recruited and/or activated on specific membrane domains where they select proteins and lipids into carriers, contribute to deform/elongate and partition membrane domains using the mechanical forces generated by actin polymerization or movement along microtubules. The coordinated action of these protein networks contributes to regulate the dynamic state of multiple receptors recycling between the cell surface, endosomes and the TGN, to maintain cell homeostasis as exemplified by the biogenesis of lysosomes and related organelles, and to establish/maintain cell polarity. The dynamic assembly and disassembly of these protein networks mediating the exchange of membrane domains between the TGN and endosomes regulates cell-cell signalling and thus the development of multi-cellular organisms. Somatic mutations in single network components lead to changes in transport dynamics that may contribute to pathological modifications underlying several human diseases such as mental retardation.     

Accumulation of glycosphingolipids in Niemann-Pick C disease disrupts endosomal transport

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1(-/-) mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.     

Role for LAMP‐2 in endosomal cholesterol transport

The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein-2 (LAMP-2) and LAMP-1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP-1 and LAMP-2 (LAMP^−/−), low-density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild-type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP^−/− cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP-2 deficient liver and that overexpression of LAMP-2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP-2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP^−/− cells was diminished by overexpression of any of the three isoforms of LAMP-2, but not by LAMP-1. The LAMP-2 luminal domain, the membrane-proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP-2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone-mediated autophagy function of LAMP-2.     

Bidirectional transport between the trans-Golgi network and the endosomal system

The exchange of proteins and lipids between the trans-Golgi network (TGN) and the endosomal system requires multiple cellular machines, whose activities are coordinated in space and time to generate pleomorphic, tubulo-vesicular carriers that deliver their content to their target compartments. These machines and their associated protein networks are recruited and/or activated on specific membrane domains where they select proteins and lipids into carriers, contribute to deform/elongate and partition membrane domains using the mechanical forces generated by actin polymerization or movement along microtubules. The coordinated action of these protein networks contributes to regulate the dynamic state of multiple receptors recycling between the cell surface, endosomes and the TGN, to maintain cell homeostasis as exemplified by the biogenesis of lysosomes and related organelles, and to establish/maintain cell polarity. The dynamic assembly and disassembly of these protein networks mediating the exchange of membrane domains between the TGN and endosomes regulates cell-cell signalling and thus the development of multi-cellular organisms. Somatic mutations in single network components lead to changes in transport dynamics that may contribute to pathological modifications underlying several human diseases such as mental retardation.     

Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion

We examined the effect of Niemann-Pick disease type 2 (NPC2) protein and some late endosomal lipids [sphingomyelin, ceramide and bis(monoacylglycero)phosphate (BMP)] on cholesterol transfer and membrane fusion. Of all lipid-binding proteins tested, only NPC2 transferred cholesterol at a substantial rate, with no transfer of ceramide, GM3, galactosylceramide, sulfatide, phosphatidylethanolamine, or phosphatidylserine. Cholesterol transfer was greatly stimulated by BMP, little by ceramide, and strongly inhibited by sphingomyelin. Cholesterol and ceramide were also significantly transferred in the absence of protein. This spontaneous transfer of cholesterol was greatly enhanced by ceramide, slightly by BMP, and strongly inhibited by sphingomyelin. In our transfer assay, biotinylated donor liposomes were separated from fluorescent acceptor liposomes by streptavidin-coated magnetic beads. Thus, the loss of fluorescence indicated membrane fusion. Ceramide induced spontaneous fusion of lipid vesicles even at very low concentrations, while BMP and sphingomyelin did so at about 20 mol% and 10 mol% concentrations, respectively. In addition to transfer of cholesterol, NPC2 induced membrane fusion, although less than saposin-C. In this process, BMP and ceramide had a strong and mild stimulating effect, and sphingomyelin an inhibiting effect, respectively. Note that the effects of the lipids on cholesterol transfer mediated by NPC2 were similar to their effect on membrane fusion induced by NPC2 and saposin-C.     

Securin and separase modulate membrane traffic by affecting endosomal acidification

Securin and separase play a key role in sister chromatid separation during anaphase. However, a growing body of evidence suggests that in addition to regulating chromosome segregation, securin and separase display functions implicated in membrane traffic in Caenorhabditis elegans and Drosophila. Here we show that in mammalian cells both securin and separase associate with membranes and that depletion of either protein causes robust swelling of the trans-Golgi network (TGN) along with the appearance of large endocytic vesicles in the perinuclear region. These changes are accompanied by diminished constitutive protein secretion as well as impaired receptor recycling and degradation. Unexpectedly, cells depleted of securin or separase display defective acidification of early endosomes and increased membrane recruitment of vacuolar (V-) ATPase complexes, mimicking the effect of the specific V-ATPase inhibitor Bafilomycin A1. Taken together, our findings identify a new functional role of securin and separase in the modulation of membrane traffic and protein secretion that implicates regulation of V-ATPase assembly and function.     

SKD1 AAA ATPase-dependent endosomal transport is involved in autolysosome formation

Mouse SKD1 AAA ATPase is involved in the sorting and transport from endosomes; cells overexpressing a dominant-negative mutant, SKD1(E235Q) were defective in endosomal transport to both the plasma membranes and lysosomes (Yoshimori et al., 2000). In the present study, we demonstrated that overexpression of SKD1(E235Q) using an adenovirus delivery system caused a defect in autophagy-dependent bulk protein degradation. Morphological observations suggested that this inhibition of autophagy results from an impairment of autolysosome formation. SKD1(E235Q) overexpression also inhibited transport from endosomes to autophagosomes, an event normally occurring prior to fusion with lysosomes. These results indicate that SKD1-dependent endosomal membrane trafficking is required for formation of autolysosomes.     

ARF6-mediated endosomal transport of Telencephalin affects dendritic filopodia-to-spine maturation

Dendritic filopodia are dynamic structures thought to be the precursors of spines during synapse development. Morphological maturation to spines is associated with the stabilization and strengthening of synapses, and can be altered in various neurological disorders. Telencephalin (TLN/intercellular adhesion molecule-5 (ICAM5)) localizes to dendritic filopodia, where it facilitates their formation/maintenance, thereby slowing spine morphogenesis. As spines are largely devoid of TLN, its exclusion from the filopodia surface appears to be required in this maturation process. Using HeLa cells and primary hippocampal neurons, we demonstrate that surface removal of TLN involves internalization events mediated by the small GTPase ADP-ribosylation factor 6 (ARF6), and its activator EFA6A. This endocytosis of TLN affects filopodia-to-spine transition, and requires Rac1-mediated dephosphorylation/release of actin-binding ERM proteins from TLN. At the somato-dendritic surface, TLN and EFA6A are confined to distinct, flotillin-positive membrane subdomains. The co-distribution of TLN with this lipid raft marker also persists during its endosomal targeting to CD63-positive late endosomes. This suggests a specific microenvironment facilitating ARF6-mediated mobilization of TLN that contributes to promotion of dendritic spine development.     

A role for EHD4 in the regulation of early endosomal transport

All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway.