The yeast vps class E mutants: the beginning of the molecular genetic analysis of multivesicular body biogenesis

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this "class E compartment" contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.     

Cellular vacuolation induced by Clostridium perfringens epsilon-toxin

The epsilon-toxin of Clostridium perfringens forms a heptamer in the membranes of Madin-Darby canine kidney cells, leading to cell death. Here, we report that it caused the vacuolation of Madin-Darby canine kidney cells. The toxin induced vacuolation in a dose-dependent and time-dependent manner. The monomer of the toxin formed oligomers on lipid rafts in membranes of the cells. Methyl-β-cyclodextrin and poly(ethylene glycol) 4000 inhibited the vacuolation. Epsilon-toxin was internalized into the cells. Confocal microscopy revealed that the internalized toxin was transported from early endosomes (early endosome antigen 1 staining) to late endosomes and lysosomes (lysosomal-associated membrane protein 2 staining) and then distributed to the membranes of vacuoles. Furthermore, the vacuolation was inhibited by bafilomycin A1, a V-type ATPase inhibitor, and colchicine and nocodazole, microtubule-depolymerizing agents. The early endosomal marker green fluorescent protein-Rab5 and early endosome antigen 1 did not localize to vacuolar membranes. In contrast, the vacuolar membranes were specifically stained by the late endosomal and lysosomal marker green fluorescent protein-Rab7 and lysosomal-associated membrane protein 2. The vacuoles in the toxin-treated cells were stained with LysoTracker Red DND-99, a marker for late endosomes and lysosomes. A dominant negative mutant of Rab7 prevented the vacuolization, whereas a mutant form of Rab5 was less effective. These results demonstrate, for the first time, that: (a) oligomers of epsilon-toxin formed in lipid rafts are endocytosed; and (b) the vacuoles originating from late endosomes and lysosomes are formed by an oligomer of epsilon-toxin.     

Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments

Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS) toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN) Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.     

Infectious HIV-1 assembles in late endosomes in primary macrophages

Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.     

ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking

The yeast vacuolar sorting protein Vps4p is an ATPase required for endosomal trafficking that couples membrane association to its ATPase cycle. To investigate the function of mammalian VPS4 in endosomal trafficking, we have transiently expressed wild-type or ATPase-defective human VPS4 (hVPS4) in cultured cells. Wild-type hVPS4 was cytosolic, whereas a substantial fraction of hVPS4 that was unable to either bind or hydrolyze ATP was localized to membranes, including those of specifically induced vacuoles. Vacuoles were exclusively endocytic in origin, and subsets of enlarged vacuoles stained with markers for each stage of the endocytic pathway. Sorting of receptors from the early endosome to the recycling compartment or to the trans-Golgi network was not significantly affected, and no mutant hVPS4 associated with these compartments. However, many hVPS4-induced vacuoles were substantially enriched in cholesterol relative to the endosomal compartments of untransfected cells, indicating that expression of mutant hVPS4 gives rise to a kinetic block in postendosomal cholesterol sorting. The phenotype described here is largely consistent with the defects in vacuolar sorting associated with class E vps mutants in yeast, and a role for mammalian VPS4 is discussed in this context.     

Multivesicular body morphogenesis requires phosphatidyl-inositol 3-kinase activity

Multivesicular bodies are endocytic compartments containing multiple small vesicles that originate from the invagination and ‘pinching off’ of the limiting membrane into the luminal space [1], [2], [3]. The molecular mechanisms responsible for the formation of these compartments are unknown. In the human melanoma cell line Mel JuSo, newly synthesised major histocompatibility complex (MHC) class II molecules accumulate in multivesicular early lysosomes [4]. The phosphatidylinositol (PI) 3-kinase inhibitor wortmannin induced the transient vacuolation of early MHC class II compartments, but also of early and late endosomes. We demonstrate that endocytic membrane influx is required for the wortmannin-induced swelling of vesicles. The wortmannin-induced vacuoles contained a reduced number of intraluminal vesicles that were linked to the limiting membrane by membraneous connections. These data suggest that wortmannin inhibits the invagination and/or pinching off of intraluminal vesicles and provide evidence of a role for PI 3-kinase in multivesicular body morphogenesis. We propose that the wortmannin-induced vacuolation occurs as a result of the inability of multivesicular bodies to store endocytosed membranes as intraluminal vesicles thereby causing the formation of large ‘empty’ vacuoles.     

3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factor II receptor from the early endosome to the TGN

3-Methyladenine (3-MA), a well-known inhibitor of autophagic sequestration, can also prevent class III phosphatidylinositide (PI) 3-kinase activity, which is required for many processes in endosomal membrane trafficking. Although much is known about the effects of other PI 3-kinase inhibitors, such as wortmannin and LY294002, on endosomal membrane trafficking, little is known about those of 3-MA. Here we show that the treatment of cells with 3-MA results in a specific redistribution of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR300) from the trans-Golgi network (TGN) to early/recycling endosomal compartments containing internalized transferrin. Importantly, in contrast to wortmannin and LY294002, 3-MA did not cause the enlargement of late endosomal/lysosomal compartments. The results suggest that the effect of 3-MA is restricted to the retrieval of MPR300 from early/recycling endosomes.     

Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking.     

The concerted action of the Helicobacter pylori cytotoxin VacA and of the v-ATPase proton pump induces swelling of isolated endosomes

The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori, the bacterium associated to gastroduodenal ulcers and stomach cancers. VacA induces formation of cellular vacuoles that originate from late endosomal compartments. VacA forms an anion-selective channel and its activity has been suggested to increase the osmotic pressure in the lumen of these acidic compartments, driving their swelling to vacuoles. Here, we have tested this proposal on isolated endosomes that allow one to manipulate at will the medium. We have found that VacA enhances the v-ATPase proton pump activity and the acidification of isolated endosomes in a Cl- dependent manner. Other counter-anions such as pyruvate, Br-, I- and SCN- can be transported by VacA with stimulation of the v-ATPase. The VacA action on isolated endosomes is associated with their increase in size. Single amino acid substituted VacA with no channel-forming and vacuolating activity is unable to induce swelling of endosomes. These data provide a direct evidence that the transmembrane VacA channel mediates an influx of anions into endosomes that stimulates the electrogenic v-ATPase proton pump, leading to their osmotic swelling and transformation into vacuoles.     

The relationship between lumenal and limiting membranes in swollen late endocytic compartments formed after wortmannin treatment or sucrose accumulation

Immunofluorescence and electron microscopy were used to evaluate the formation of swollen endosomes in NRK cells after treatment with wortmannin or sucrose and to study the relationship between lumenal and limiting membrane. Both treatments resulted in the formation of two populations of swollen late endocytic vacuoles, positive for lysosomal glycoproteins or cation-independent mannose 6-phosphate receptors, but those induced by wortmannin were characterised by time-dependent accumulation of lumenal vesicles, whereas those induced by sucrose uptake did not accumulate lumenal vesicles. In both cases, the distribution of the late endosomal marker, lysobisphosphatidic acid, remained unchanged and was present within the lumen of the swollen vacuoles. Consumption of plasma membrane and peripheral early endosomes, and the appearance of transferrin receptors in swollen late endosomes, indicated that continued membrane influx from early endocytic compartments, together with inhibition of membrane traffic out of the swollen compartments, is sufficient to account for the observed phenotype of cells treated with wortmannin. The accumulation of organelles with the characteristic morphology of endocytic carrier vesicles in cells that have taken up sucrose offers an explanation for the paucity of lumenal vesicles in swollen sucrosomes. Our data suggest that in fibroblast cells the swollen endosome phenotype induced by wortmannin is a consequence of endocytic membrane influx, coupled with the failure to recycle membrane to other cellular destinations, and not the inhibition of multivesicular body biogenesis.     

The endosomal Na(+)/H(+) exchanger contributes to multivesicular body formation by regulating the recruitment of ESCRT-0 Vps27p to the endosomal membrane

Multivesicular bodies (MVBs) are late endosomal compartments containing luminal vesicles (MVB vesicles) that are formed by inward budding of the endosomal membrane. In budding yeast, MVBs are an important cellular mechanism for the transport of membrane proteins to the vacuolar lumen. This process requires a class E subset of vacuolar protein sorting (VPS) genes. VPS44 (allelic to NHX1) encodes an endosome-localized Na(+)/H(+) exchanger. The function of the VPS44 exchanger in the context of vacuolar protein transport is largely unknown. Using a cell-free MVB formation assay system, we demonstrated that Nhx1p is required for the efficient formation of MVB vesicles in the late endosome. The recruitment of Vps27p, a class E Vps protein, to the endosomal membrane was dependent on Nhx1p activity and was enhanced by an acidic pH at the endosomal surface. Taken together, we propose that Nhx1p contributes to MVB formation by the recruitment of Vps27p to the endosomal membrane, possibly through Nhx1p antiporter activity.     

Autophagy-related vacuoles in mouse gallbladder epithelium

The mouse gallbladder epithelial cells contain very heterogeneous vacuolar population. In an attempt to classify these vacuoles we identified NADPase and TPPase activity as well as the location of HRP which is used as the endocytotic marker. The results of the present study show that the vacuoles can be classified into three categories: (1) the vacuoles predominantly containing loose membrane coils related to the nascent autophagic vacuoles, (2) vacuoles containing densely packed membranes and exhibiting a positive HRP reaction, indicating the convergence of endocytotic and autophagic pathway, and (3) vacuoles composed of degraded membrane structures and containing the reaction product of NADPase activity, showing that the fusion of the lysosomes with the autophagosome-endosome took place. The highly developed cis, medial and trans Golgi compartments reflect the biosynthetic and endocytotic activity of the gallbladder epithelium.     

Lamellar bodies of rat alveolar type 2 cells have late endosomal marker proteins on their limiting membranes

Alveolar type 2 cells are known to take up surfactant phospholipids and proteins from the alveolar space and recycle them into secretory organelles via a receptor-mediated endocytic pathway. To clarify the intracellular route(s) through which materials ingested by the cells are processed, we examined the immunocytochemical localization of late endosomal and lysosomal membrane markers, rab 7 and lamp 1 proteins, within rat alveolar type 2 cells. The limiting membranes of lamellar bodies (LBs) showed positive immunoreactivity for both proteins, whereas multivesicular bodies (MVBs) exhibited positive immunoreactivity only for lamp 1 protein on free vesicles in the MVB lumen. From these findings, it is suggested that LBs are not only secretory granules, but also constitute one of the late endosomal compartments of the cells and that MVBs of this cell type may be targeted to cell organelle(s) other than lysosomes.     

Regulation of ROCKII by localization to membrane compartments and binding to DynaminI

ROCKII kinase activity is known to be regulated by Rho GTPase binding; however, the context-specific regulation of ROCKII is not clearly understood. We pursued the C-terminal PH domain as a candidate domain for regulating ROCKII function. A proteomics-based screen identified potential ROCKII signaling partners, a large number of which were associated with membrane dynamics. We used subcellular fractionation to demonstrate that ROCKII is localized to both the plasma membrane and internal endosomal membrane fractions, and then used microscopy to show that the C-terminal PH domain can localize to internal or peripheral membrane compartments, depending on the cellular context. Co-immunoprecipitation demonstrated that Dynamin1 is a novel ROCKII binding partner. Furthermore, blocking Dynamin function with a dominant negative mutant mimicked the effect of inhibiting ROCK activity on the actin cytoskeleton. Our data suggest that ROCKII is regulated by localization to specific membrane compartments and its novel binding partner, Dynamin1.     

Vacuolar membrane dynamics in the filamentous fungus Aspergillus oryzae

Vacuoles in filamentous fungi are highly pleomorphic and some of them, e.g., tubular vacuoles, are implicated in intra- and intercellular transport. In this report, we isolated Aovam3, the homologue of the Saccharomyces cerevisiae VAM3 gene that encodes the vacuolar syntaxin, from Aspergillus oryzae. In yeast complementation analyses, the expression of Aovam3 restored the phenotypes of both Deltavam3 and Deltapep12 mutants, suggesting that AoVam3p is likely the vacuolar and/or endosomal syntaxin in A. oryzae. FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)pyridinium dibromide] and CMAC (7-amino-4-chloromethylcoumarin) staining confirmed that the fusion protein of enhanced green fluorescent protein (EGFP) with AoVam3p (EGFP-AoVam3p) localized on the membrane of the pleomorphic vacuolar networks, including large spherical vacuoles, tubular vacuoles, and putative late endosomes/prevacuolar compartments. EGFP-AoVam3p-expressing strains allowed us to observe the dynamics of vacuoles with high resolutions, and moreover, led to the discovery of several new aspects of fungal vacuoles, which have not been discovered so far with conventional staining methods, during different developmental stages. In old hyphae, EGFP fluorescence was present in the entire lumen of large vacuoles, which occupied most of the cell, indicating that degradation of cytosolic materials had occurred in such hyphae via an autophagic process. In hyphae that were not in contact with nutrients, such as aerial hyphae and hyphae that grew on a glass surface, vacuoles were composed of small punctate structures and tubular elements that often formed reticulum-like networks. These observations imply the presence of so-far-unrecognized roles of vacuoles in the development of filamentous fungi.     

The role of phosphatidylinositol-3-kinases P85/P110 and hVPS34 in endocytosis of EGF-receptor complexes

The previous data (Zheleznova et al., 2001) did not enable the authors to conclude which particular wortmannin sensitive PI-3-kinase--p85/p110 (I class PI-3-K) or hVPS34 (III class PI-3-K)--may be involved in the regulation of EGF-receptor endocytosis. In the present work, we have shown that upon stimulation of EGF-receptor endocytosis additional structures stained with antibody against p85 appear in A431 cells, but the p85-positive compartment never co-localized with EGF-receptor-containing compartments either in control or in wortmannin-treated cells. At the same time, wortmannin treatment prevented association of hVPS34 with endosomal membranes. We have also found that early endosomal markers--Rab5 and EEA1 (membrane association of the latter depends on Rab5 and hVPS34)--co-localized with EGF-receptor in the juxtranuclear region during late stages of endocytosis, both in control and upon wortmannin treatment. These observations favor our suggestions that the transition of EGF-receptors from early to late endosomes may occur directly in this juxtranuclear region and be tightly associated with the formation of so called multivesicular bodies (MVB), which are late endosomes per se. We suggest that wortmannin may have no effect on early EEA1-dependent stage of the receptor endocytosis but blocks a transition of EGF-receptor complexes into the late endosomes by inhibiting activity of hVPS34 and removing it from membranes. The hVPS34 product PI-3-K, according to the known data, is involved in the formation of internal vesicles of MVB. Accumulation of EGF-receptors in these vesicles is believed to be necessary for the receptor degradation.     

Sonic hedgehog induces the segregation of patched and smoothened in endosomes

BACKGROUND: Sonic hedgehog (Shh) signal transduction involves the ligand binding Patched1 (Ptc1) protein and a signaling component, Smoothened (Smo). A select group of compounds inhibits both Shh signaling, regulated by Ptc1, and late endosomal lipid sorting, regulated by the Ptc-related Niemann-Pick C1 (NPC1) protein. This suggests that Ptc1 regulates Smo activity through a common late endosomal sorting pathway also utilized by NPC1. During signaling, Ptc accumulates in endosomal compartments, but it is unclear if Smo follows Ptc into the endocytic pathway.RESULTS: We characterized the dynamic subcellular distributions of Ptc1, Smo, and activated Smo mutants individually and in combination. Ptc1 and Smo colocalize extensively in the absence of ligand and are internalized together after ligand binding, but Smo becomes segregated from Ptc1/Shh complexes destined for lysosomal degradation. In contrast, activated Smo mutants do not colocalize with nor are cotransported with Ptc1. Agents that block late endosomal transport and protein sorting inhibit the ligand-induced segregation of Ptc1 and Smo. We show that, like NPC1-regulated lipid sorting, Shh signal transduction is blocked by antibodies that specifically disrupt the internal membranes of late endosomes, which provide a platform for protein and lipid sorting.CONCLUSIONS: These data support a model in which Ptc1 inhibits Smo only when in the same compartment. Ligand-induced segregation allows Smo to signal independently of Ptc1 after becoming sorted from Ptc1/Shh complexes in the late endocytic pathway.     

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.     

A histochemical study of variations in the localization of 5′-nucleotidase activity in the acinar cell of the rat exocrine pancreas over the twenty-four hour period

The circadian variation of 5'-nucleotidase (AMPase) activity was studied in rat pancreatic exocrine cells. The localization of this enzyme, often associated with the plasmalemma, was studied by ultracytochemical methods at six time points over the 24-h period. The localization of AMPase activity exhibited a clear-cut circadian variation. During the light span strong activity was observed on the luminal plasmalemma, negative or weak activity on the baso-lateral plasmalemma and clearly visible activity on intracellular structures such as cytoplasmic vacuoles (fragmentation-like vesicles), dilated rims of the Golgi cisternae (or cisternal ends of the Golgi stacks), condensing vacuoles and lysosomal bodies. During the dark span the activity was detectable only on the baso-lateral plasmalemma. The fact that AMPase activity could not be found on the luminal plasmalemma during the dark span suggests that the luminal membranes may be replaced by the membranes of secretory granules, which do not display AMPase activity. The intracellular localization of AMPase activity during the light span, especially at 08.00 h, includes all cytoplasmic compartments which have hitherto been associated with the intracellular pathway for membrane retrieval from the plasmalemma. Moreover, the appearance of the activity in the dilated rims of the Golgi stack and condensing vacuoles indicates that these compartments may constitute a functional unit.     

Association of annexin 2 with recycling endosomes requires either calcium- or cholesterol-stabilized membrane domains

Annexin 2 is a Ca2+- and phospholipid-binding protein previously identified on endosomal membranes and the plasma membrane. Inferred from this location and its stimulatory effect on membrane transport annexin 2 has been proposed to play a role in the structural organization and dynamics of endosomal membranes. Validation of this view requires a detailed analysis of the distribution of annexin 2 over the endosomal compartment and a characterization of the parameters governing this distribution. Towards this end we have devised an immunoisolation protocol to purify annexin 2-positive membrane vesicles from subcellular fractions of BHK cells containing early endosomes. We show that this approach leads to the isolation of intact endosomal vesicles containing internalized fluid-phase marker and that the immunoisolated membranes are positive for the transferrin receptor and Rab4 but not for the early endosomal antigen EEA1. A distinct and non-uniform distribution of annexin 2 over the early endosomal compartment is also observed in immunoelectron microscopy analyses of whole-mount specimens of BHK cells. Annexin 2 antibodies labeled transferrin receptor-containing tubular early endosomal structures, but not EEAl-positive endosomal vacuoles. We also observed that the Ca2+-independent association of annexin 2 with endosomal membranes was disrupted by the cholesterol-binding glycerid saponin, while Ca2+ could trigger annexin 2 binding to saponin-treated endosomal membranes. Thus, either Ca2+- or cholesterol-stabilized membrane domains are required for the binding of annexin 2 to endosomes suggesting that both factors may regulate this interaction.