A three-dimensional study of organelle interrelationships in regenerating rat liver. 3. Organelles related to bile.

A number of cell structures are described which show a morphological relationship to the bile canaliculi. Two types of peribiliary vesicles are identified: osmication positive ones occurring between the bile canaliculi and the osmicated immature Golgi cisternae and probably deriving from the latter, and osmication negative ones related to MVB, on which they appear as buds. Small coated vesicles are seen attached to this second type. Large lacunae may originate from MVB, as suggested by the MVB-like internal vesicles they may contain. Some stay in luminal continuity with the bile canaliculi. Canalicular coated vesicles are seen as parts of the canalicular plasma membrane and free in the cytoplasm.     

The plasticity of multivesicular bodies and the regulation of antigen presentation

Multivesicular bodies (MVBs) are ubiquitous endocytic organelles containing numerous 50-80 nm vesicles. MVBs are very dynamic in shape and function. In antigen presenting cells (APCs), MVBs play a central role in the loading of major histocompatibility complex class II (MHC II) with antigenic peptides. How MHC II is transported from MVBs to the cell surface is only partly understood. One way involves direct fusion of MVBs with the plasma membrane. As a consequence, their internal vesicles are secreted as so-called exosomes. An alternative has been illustrated in maturing dendritic cells (DCs). Here, MVBs are reshaped into long tubules by back fusion of the internal vesicles with the MVB limiting membrane. Vesicles derived from the tips of these tubules then carry MHC II to the cell surface.     

Human herpesvirus-6 induces MVB formation, and virus egress occurs by an exosomal release pathway

The final envelopment of most herpesviruses occurs at Golgi or post-Golgi compartments, such as the trans Golgi network (TGN); however, the final envelopment site of human herpesvirus 6 (HHV-6) is uncertain. In this study, we found novel pathways for HHV-6 assembly and release from T cells that differed, in part, from those of alphaherpesviruses. Electron microscopy showed that late in infection, HHV-6-infected cells were larger than uninfected cells and contained many newly formed multivesicular body (MVB)-like compartments that included small vesicles. These MVBs surrounded the Golgi apparatus. Mature virions were found in the MVBs and MVB fusion with plasma membrane, and the release of mature virions together with small vesicles was observed at the cell surface. Immunoelectron microscopy demonstrated that the MVBs contained CD63, an MVB/late endosome marker, and HHV-6 envelope glycoproteins. The viral glycoproteins also localized to internal vesicles in the MVBs and to secreted vesicles (exosomes). Furthermore, we found virus budding at TGN-associated membranes, which expressed CD63, adaptor protein (AP-1) and TGN46, and CD63 incorporation into virions. Our findings suggest that mature HHV-6 virions are released together with internal vesicles through MVBs by the cellular exosomal pathway. This scenario has significant implications for understanding HHV-6's maturation pathway.     

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.     

Tetraspan cargo adaptors usher GPI-anchored proteins into multivesicular bodies

Ubiquitinated membrane proteins are sorted into intralumenal endosomal vesicles on their way for degradation in lysosomes. Here we summarize the discovery of the Cos proteins, which work to organize and segregate ubiquitinated cargo prior to its incorporation into intralumenal vesicles of the multivesicular body (MVB). Importantly, cargoes such as GPI-anchored proteins (GPI-APs) that cannot undergo ubiquitination, rely entirely on Cos proteins for sorting into intralumenal vesicles using the same pathway that depends on ESCRTs and ubiquitin ligases that typical polytopic membrane proteins do. Here we show Cos proteins provide functions as not only adaptor proteins for ubiquitin ligases, but also as cargo carriers that can physically usher a variety of other proteins into the MVB pathway. We then discuss the significance of this new sorting model and the broader implications for this cargo adaptor mechanism, whereby yeast Cos proteins, and their likely animal analogs, provide a ubiquitin sorting signal in trans to enable sorting of a membrane protein network into intralumenal vesicles.     

Endosomes, exosomes and Trojan viruses

Retroviruses are enveloped viruses that are generally assumed to bud at the plasma membrane of infected cells. Recently it has become apparent that some of these viruses use the endocytic pathway to coordinate their assembly and release. In addition, these and some other enveloped viruses exploit the machinery that generates the internal membranes of multivesicular bodies (MVB). These observations and others have led to the suggestion that retroviruses be regarded as "viral exosomes". Here we discuss this concept and the emerging evidence that compartments of the endocytic pathway play important roles in the biogenesis of both the internal vesicles of MVB and viruses.     

Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes

Ubiquitination directs the sorting of cell surface receptors and other integral membrane proteins into the multivesicular body (MVB) pathway. Cargo proteins are subsequently deubiquitinated before their enclosure within MVB vesicles. In Saccharomyces cerevisiae, Bro1 functions at a late step of MVB sorting and is required for cargo protein deubiquitination. We show that the loss of Bro1 function is suppressed by the overexpression of DOA4, which encodes the ubiquitin thiolesterase required for the removal of ubiquitin from MVB cargoes. Overexpression of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endosomal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes. We further demonstrate that Doa4 interacts with Bro1 on endosomal membranes and that the recruitment of Doa4 to endosomes requires Bro1. Thus, our results point to a key role for Bro1 in coordinating the timing and location of deubiquitination by Doa4 in the MVB pathway.     

Ist1 regulates Vps4 localization and assembly

The ESCRT protein complexes are recruited from the cytoplasm and assemble on the endosomal membrane into a protein network that functions in sorting of ubiquitinated transmembrane proteins into the multivesicular body (MVB) pathway. This transport pathway packages cargo proteins into vesicles that bud from the MVB limiting membrane into the lumen of the compartment and delivers these vesicles to the lysosome/vacuole for degradation. The dissociation of ESCRT machinery by the AAA-type ATPase Vps4 is a necessary late step in the formation of MVB vesicles. This ATP-consuming step is regulated by several Vps4-interacting proteins, including the newly identified regulator Ist1. Our data suggest that Ist1 has a dual role in the regulation of Vps4 activity: it localizes to the ESCRT machinery via Did2 where it positively regulates recruitment of Vps4 and it negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer, in which Vps4 cannot bind to the ESCRT machinery. The activity of the MVB pathway might be in part determined by outcome of these two competing activities.     

A new role of diacylglycerol kinase alpha on the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes

Exosomes are small membrane vesicles that intracellularly accumulate into late or multivesicular endosomes (multivesicular bodies, MVB). Exosomes have a particular lipid and protein content, reflecting their origin as intraluminal vesicles of late endosomes. The stimulation of several hematopoietic cells induces the fusion of the limiting membrane of the MVB with the plasma membrane, leading to the release of exosomes towards the extracellular environment. In T lymphocytes, stimulation of the T cell receptor (TCR) induces the fusion of the MVBs with the plasma membrane and exosomes carrying several bio-active proteins are secreted. Among these proteins, the pro-apoptotic protein Fas ligand (FasL) is released as a non-proteolysed form (mFasL), associated to the exosomes. These mFasL-bearing exosomes may trigger the apoptosis of T lymphocytes. Here, we present evidences supporting a role of diacylglycerol kinase alpha (DGKalpha), a diacylglycerol (DAG)-consuming enzyme, on the secretion of exosomes carrying mFasL, and the subsequent activation-induced cell death (AICD) on a T cell line and primary T lymphoblasts.     

Vacuolar degradation of two integral plasma membrane proteins, AtLRR84A and OsSCAMP1, is cargo ubiquitination-independent and prevacuolar compartment-mediated in plant cells

In plant cells, how integral plasma membrane (PM) proteins are degraded in a cargo ubiquitination-independent manner remains elusive. Here, we studied the degradative pathway of two plant PM proteins: AtLRR84A, a type I integral membrane protein belonging to the leucine-rich repeat receptor-like kinase protein family, and OsSCAMP1 (rice secretory carrier membrane protein 1), a tetraspan transmembrane protein located on the PM and trans-Golgi network (TGN) or early endosome (EE). Using wortmannin and ARA7(Q69L) mutant that could enlarge the multivesicular body (MVB) or prevacuolar compartment (PVC) as tools, we demonstrated that, when expressed as green fluorescent protein (GFP) fusions in tobacco BY-2 or Arabidopsis protoplasts, both AtLRR84A and OsSCAMP1 were degraded in the lytic vacuole via the internal vesicles of MVB/PVC in a cargo ubiquitination-independent manner. Such MVB/PVC-mediated vacuolar degradation of PM proteins was further supported by immunocytochemical electron microscopy (immunoEM) study showing the labeling of the fusions on the internal vesicles of the PVC/MVB. Thus, cargo ubiquitination-independent and PVC-mediated degradation of PM proteins in the vacuole is functionally operated in plant cells.     

Direct binding to Rsp5 mediates ubiquitin-independent sorting of Sna3 via the multivesicular body pathway

The sorting of most integral membrane proteins into the lumenal vesicles of multivesicular bodies (MVBs) is dependent on the attachment of ubiquitin (Ub) to their cytosolic domains. However, Ub is not required for sorting of Sna3, an MVB vesicle cargo protein in yeast. We show that Sna3 circumvents Ub-mediated recognition by interacting directly with Rsp5, an E3 Ub ligase that catalyzes monoubiquitination of MVB vesicle cargoes. The PPAY motif in the C-terminal cytosolic domain of Sna3 binds the WW domains in Rsp5, and Sna3 is polyubiquitinated as a consequence of this association. However, Ub does not appear to be required for transport of Sna3 via the MVB pathway because its sorting occurs under conditions in which its ubiquitination is impaired. Consistent with Ub-independent function of the MVB pathway, we show by electron microscopy that the formation of MVB vesicles does not require Rsp5 E3 ligase activity. However, cells expressing a catalytically disabled form of Rsp5 have a greater frequency of smaller MVB vesicles compared with the relatively broad distribution of vesicles seen in MVBs of wild-type cells, suggesting that the formation of MVB vesicles is influenced by Rsp5-mediated ubiquitination.     

Cell‐Free Reconstitution of Multivesicular Body Formation and Receptor Sorting

The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell‐free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal‐transducing adaptor molecule (STAM), or addition of mutated ATPase‐deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process.     

Membrane-bound and fluid-phase macromolecules enter separate prelysosomal compartments in absorptive cells of suckling rat ileum

The absorptive cell of the suckling rat ileum is specialized for the uptake and digestion of milk macromolecules from the intestinal lumen. The apical cytoplasm contains an extensive tubulocisternal system, a variety of vesicles and multivesicular bodies (MVB), and a giant phagolysosomal vacuole where digestion is completed. To determine if sorting of membrane-bound and fluid-phase macromolecules occurs in this elaborate endocytic system, we infused adsorptive and soluble tracers into ligated intestinal loops in vivo and examined their fates. Lysosomal compartments were identified by acid phosphatase histochemistry. Native ferritin and two ferritin-lectin conjugates that do not bind to ileal membranes (Con A, UEAI) served as soluble tracers. Horseradish peroxidase binds to ileal membranes and thus was not useful as a fluid-phase tracer in this system. Cationized ferritin and a lectin that binds to terminal B-D-galactosyl sites on ileal membranes (Ricinus communis agglutinin [RCAI]-ferritin) were used as tracer ligands. All tracers entered the wide apical invaginations of the luminal cell surface and were transported intracellularly. Membrane-bound tracers were found in coated pits and vesicles, and throughout the tubulocisternal system (where cationized ferritin is released from the membrane) and later, in large clear vesicles and MVB. In contrast, fluid-phase tracers appeared within 5 min in vesicles of various sizes and were not transported through the tubulocisternae, rather, they were concentrated in a separate population of vesicles of increasing size that contained amorphous dense material. Large clear vesicles, large dense vesicles, and MVB eventually fused with the giant supranuclear vacuole. Acid phosphatase activity was present in MVB and in the giant vacuole but was not present in most large vesicles or in the tubulocisternae. These results demonstrate that membrane-bound and soluble protein are transported to a common lysosomal destination via separate intracellular routes involving several distinct prelysosomal compartments.     

Multivesicular bodies: a mechanism to package lytic and storage functions in one organelle?

Multivesicular bodies (MVBs) are endosomes or prevacuolar compartments. The lumens of their internal vesicles are thought to be topologically equivalent to cytoplasm and their membranes direct proteins and lipids for degradation. Here, we describe a new MVB function; in certain plant MVBs, the internal vesicles contain lytic enzymes and the surrounding 'soup' is a storage compartment. Separate vesicular pathways deliver proteins to the storage and lytic compartments. Recent data indicate that mammalian secretory lysosomes also have two compartments served by separate vesicular pathways. The formation of separate storage and lytic compartments within MVBs poses problems for membrane organization and topology that have not previously been considered in the literature. We offer a hypothetical model to address these problems.     

RAL-1 controls multivesicular body biogenesis and exosome secretion

Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1–deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release.     

Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells

Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.     

Ultrastructural study on the extrusion of multivesicular bodies from cardiac cells.

Cardiac conducting fibers taken from 12 dogs were studied in the electron microscope. The majority of samples contained a moderate number of multivesicular bodies (MVB's) and an extremely high number occurred in one dog. Single MVB's were found in the perinuclear region and in the cytoplasm separating myofibrils. A high accumulation of MVB's occurred at the cell periphery within cytoplasmic stalks extending into the extracellular space, in the intercalated disc, and also outside of the cell. The latter appeared to be extruded from the cell in the following way: 1) peripheral cytoplasm formed stalk-like extensions which contained MVB's; 2) the stalks detached from the cell; 3) vacuolar and sarcolemmal membranes surrounding the MVB disintegrated; 4) vesicles released from MVB adhered to the cellular surface and the coat of their membrane blended with the coat of the sarcolemma. The above pattern seems to be the same in those parts of sarcolemma which face the interstitial tissue and in the intercalated disc.     

Distinct roles for Tsg101 and Hrs in multivesicular body formation and inward vesiculation

In mammalian cells, epidermal growth factor (EGF) stimulation promotes multivesicular body (MVB) formation and inward vesiculation within MVB. Annexin 1 is required for EGF-stimulated inward vesiculation but not MVB formation, demonstrating that MVB formation (the number of MVBs/unit cytoplasm) and inward vesiculation (the number of internal vesicles/MVB) are regulated by different mechanisms. Here, we show that EGF-stimulated MVB formation requires the tumor susceptibility gene, Tsg101, a component of the ESCRT (endosomal sorting complex required for transport) machinery. Depletion of Tsg101 potently inhibits EGF degradation and MVB formation and causes the vacuolar domains of the early endosome to tubulate. Although Tsg101 depletion inhibits MVB formation and alters the morphology of the early endosome in unstimulated cells, these effects are much greater after EGF stimulation. In contrast, depletion of hepatocyte growth factor receptor substrate (Hrs) only modestly inhibits EGF degradation, does not induce tubulation of the early endosome, and causes the generation of enlarged MVBs that retain the ability to fuse with the lysosome. Together, these results indicate that Tsg101 is required for the formation of stable vacuolar domains within the early endosome that develop into MVBs and Hrs is required for the accumulation of internal vesicles within MVBs and that both these processes are up-regulated by EGF stimulation.     

MVB vesicle formation: ESCRT-dependent, ESCRT-independent and everything in between

Multivesicular bodies (MVBs) are endosomes that have internalized portions of the limiting membrane into the compartment, thereby forming intralumenal vesicles. This vesicle formation is unusual in that it is directed away from the cytoplasm, which requires a unique mechanism unlike any mechanism described for other vesicle formation events. The best contenders for the machinery that drives MVB vesicle formation are the ESCRT protein complexes. However, increasing evidence suggests that lipids may play a key role in this membrane-deformation process. This review attempts to combine the seemingly contradictory findings into a MVB vesicle formation model that is based on a lipid-driven and ESCRT-regulated mechanism.     

Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies

Doa4 is a ubiquitin-specific protease in Saccharomyces cerevisiae that deubiquitinates integral membrane proteins sorted into the lumenal vesicles of late-endosomal multivesicular bodies (MVBs). We show that the non-catalytic N terminus of Doa4 mediates its recruitment to endosomes through its association with Bro1, which is one of several highly conserved class E Vps proteins that comprise the core MVB sorting machinery. In turn, Bro1 directly stimulates deubiquitination by interacting with a YPxL motif in the catalytic domain of Doa4. Mutations in either Doa4 or Bro1 that disrupt catalytic activation of Doa4 impair deubiquitination and sorting of MVB cargo proteins and lead to the formation of lumenal MVB vesicles that are predominantly small compared with the vesicles seen in wild-type cells. Thus, by recruiting Doa4 to late endosomes and stimulating its catalytic activity, Bro1 fulfills a novel dual role in coordinating deubiquitination in the MVB pathway.