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1.
Distinct roles for Tsg101 and Hrs in multivesicular body formation and inward vesiculation
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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. 相似文献
2.
Shrawan Kumar Mageswaran Megan Gorringe Dixon Matt Curtiss James P. Keener Markus Babst 《Traffic (Copenhagen, Denmark)》2014,15(2):212-229
The ESCRT (endosomal sorting complex required for transport) machinery is known to sort ubiquitinated transmembrane proteins into vesicles that bud into the lumen of multivesicular bodies (MVBs). Although the ESCRTs themselves are ubiquitinated they are excluded from the intraluminal vesicles and recycle back to the cytoplasm for further rounds of sorting. To obtain insights into the rules that distinguish ESCRT machinery from cargo we analyzed the trafficking of artificial ESCRT‐like protein fusions. These studies showed that lowering ESCRT‐binding affinity converts a protein from behaving like ESCRT machinery into cargo of the MVB pathway, highlighting the close relationship between machinery and the cargoes they sort. Furthermore, our findings give insights into the targeting of soluble proteins into the MVB pathway and show that binding to any of the ESCRTs can mediate ubiquitin‐independent MVB sorting. 相似文献
3.
Mitsui K Koshimura Y Yoshikawa Y Matsushita M Kanazawa H 《The Journal of biological chemistry》2011,286(43):37625-37638
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. 相似文献
4.
Progression of activated EGF receptor (EGFR) through the endocytic pathway regulates EGFR signaling. Here we show that a non-ubiquitinated EGFR mutant, unable to bind the endosomal-sorting complex required for transport (ESCRT) component, Hrs, is not efficiently targeted onto intraluminal vesicles (ILVs) of multivesicular endosomes/bodies (MVBs). Moreover, ubiquitination and ESCRT engagement of activated EGFR are required for EGF-stimulated ILV formation. Non-ubiquitinated EGFRs enter clathrin-coated tubules emanating from MVBs and show enhanced recycling to the plasma membrane, compared to wild-type EGFR. 相似文献
5.
ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting
Dores MR Chen B Lin H Soh UJ Paing MM Montagne WA Meerloo T Trejo J 《The Journal of cell biology》2012,197(3):407-419
The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs. 相似文献
6.
Multivesicular endosomes (MVBs) are major sorting platforms for membrane proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydrolase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and ILV formation are achieved by the concerted function of endosomal sorting complex required for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/Phk2 kinases, two yeast orthologues of the 3-phosphoinositide–dependent kinase, phosphorylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine 613 and demonstrate that this phosphorylation is required for proper Vps27 function. Indeed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27S613A, MVB sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the Vps28–green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT complex recruitment at the endosomal membrane. 相似文献
7.
Ist1 regulates Vps4 localization and assembly 总被引:1,自引:1,他引:0
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. 相似文献
8.
Multivesicular bodies: co-ordinated progression to maturity 总被引:2,自引:0,他引:2
Multivesicular endosomes/bodies (MVBs) sort endocytosed proteins to different destinations. Many lysosomally directed membrane proteins are sorted onto intralumenal vesicles, whilst recycling proteins remain on the perimeter membrane from where they are removed via tubular extensions. MVBs move to the cell centre during this maturation process and, when all recycling proteins have been removed, fuse with lysosomes. Recent advances have identified endosomal-sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways in intralumenal vesicle formation and mechanisms for sorting recycling cargo into tubules. Cytoskeletal motors, through interactions with these machineries and by regulating MVB movement, help to co-ordinate events leading to a mature, fusion-competent MVB. 相似文献
9.
内吞体分选转运复合体(Endosomal sorting complex required for transport,ESCRT)主要识别泛素化修饰的膜蛋白,介导内吞小泡出芽和多泡体(Multivesicular bodies,MVBs)的形成。此外,以类似的拓扑方式,ESCRT也参与胞质分裂、自体吞噬、以及包膜病毒的出芽等过程。已有的研究表明,大量的反转录病毒和RNA病毒含有晚期结构域(Late-domains),该结构域与ESCRT组分相互作用,将ESCRT-Ⅲ和VPS4等募集在病毒组装与出芽区域,并利用ESCRT-Ⅲ使病毒粒子得以释放。最近,有研究发现,一些DNA包膜病毒、如乙肝病毒、疱疹病毒和杆状病毒等的出芽释放也依赖于宿主细胞ESCRT系统,但其机理尚需深入研究。 相似文献
10.
Endosomal sorting complexes required for transport (ESCRT) have been implicated in topologically similar but diverse cellular and pathological processes including multivesicular body (MVB) biogenesis, cytokinesis and enveloped virus budding. Although receptor sorting at the endosomal membrane producing MVBs employs the regulated assembly of ESCRT-0 followed by ESCRT-I, -II, -III and the vacuolar protein sorting (VPS)4 complex, other ESCRT-catalyzed processes require only a subset of complexes which commonly includes ESCRT-III and VPS4. Recent progress has shed light on the pathway of ESCRT assembly and highlights the separation of tasks of different ESCRT complexes and associated partners. The emerging picture suggests that among all ESCRT-catalyzed processes, divergent pathways lead to ESCRT-III assembly within the neck of a budding structure catalyzing membrane fission. 相似文献
11.
A concentric circle model of multivesicular body cargo sorting 总被引:4,自引:0,他引:4
Targeting of ubiquitylated transmembrane proteins into luminal vesicles of endosomal multivesicular bodies (MVBs) depends on their recognition by endosomal sorting complexes required for transport (ESCRTs), which are also required for MVB vesicle formation. The model originally proposed for how ESCRTs function succinctly summarizes much of the protein-protein interaction and genetic data but oversimplifies the coordination of cargo recognition and cannot explain why ESCRTs are required for the budding of MVB vesicles. Recent structural and functional studies of ESCRT complexes suggest an alternative model that might direct the next series of breakthroughs in understanding protein sorting through the MVB pathway. 相似文献
12.
Membrane budding is essential for the egress of many enveloped viruses, and this process shares similarities with the biogenesis of multivesicular bodies (MVBs). In eukaryotic cells, the budding of intraluminal vesicles (IVLs) is mediated by the endosomal sorting complex required for transport (ESCRT) machinery and some viruses require ESCRT machinery components or functions to bud from host cells. Baculoviruses, such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), enter host cells by clathrin-mediated endocytosis. Viral DNA replication and nucleocapsid assembly occur within the nucleus. Some progeny nucleocapsids are subsequently trafficked to, and bud from, the plasma membrane, forming budded virions (BV). To determine whether the host ESCRT machinery is important or necessary for AcMNPV replication, we cloned a cDNA of Spodoptera frugiperda VPS4, a key regulator for disassembly and recycling of ESCRT III. We then examined viral infection and budding in the presence of wild-type (WT) or dominant negative (DN) forms of VPS4. First, we used a viral complementation system, in combination with fluorescent tags, to examine the effects of transiently expressed WT or DN VPS4 on viral entry. We found that dominant negative VPS4 substantially inhibited virus entry. Entering virus was observed within aberrant compartments containing the DN VPS4 protein. We next used recombinant bacmids expressing WT or DN VPS4 proteins to examine virus egress. We found that production of infectious AcMNPV BV was substantially reduced by expression of DN VPS4 but not by WT VPS4. Together, these results indicate that a functional VPS4 is necessary for efficient AcMNPV BV entry into, and egress from, insect cells. 相似文献
13.
Sheliang Wang Toshiaki Ito Masataka Uehara Satoshi Naito Junpei Takano 《Journal of plant research》2015,128(5):863-873
Endomembrane organization is essential for cell physiology. We previously identified an Arabidopsis thaliana mutant in which a plasma membrane (PM) marker GFP-NIP5;1 and trans-Golgi network/early endosome (TGN/EE) markers were accumulated in intracellular aggregates in epidermal cells of the root elongation zone. The mutant was identified as an allele of UDP-glucose epimerase 4 (UGE4)/root hair defective 1/root epidermal bulgar 1, which was previously described as a mutant with swollen root epidermal cells and has an altered sugar composition in cell wall polysaccharides. Importantly, these defects including aggregate formation were restored by supplementation of d-galactose in the medium. These results suggested that UDP-d-galactose synthesis by UGE4 is important for endomembrane organization in addition to cell wall structure. Here, we further investigated the nature of the aggregates using various markers of endomembrane compartments and BOR1-GFP, which traffics from PM to vacuole in response to high-B supply. The markers of multi-vesicular bodies/late endosomes (MVB/LEs) and BOR1-GFP were strongly accumulated in the intracellular aggregates, while those of the endoplasmic reticulum (ER), the vacuolar membrane, and the Golgi were only slightly affected in the uge4 mutant. The abnormal localizations of these markers in the uge4 mutant differed from the effects of inhibitors of actin and microtubule polymerization, although they also affected endomembrane organization. Furthermore, electron microscopy analysis revealed accumulation of abnormal high-electron-density vesicles in elongating epidermal cells. The abnormal vesicles were often associated or interconnected with TGN/EEs and contained ADP-ribosylation factor 1, which is usually localized to the Golgi and the TGN/EEs. On the other hand, structures of the ER, Golgi apparatus, and MVB/LEs were apparently normal in uge4 cells. Together, our data indicate the importance of UDP-d-galactose synthesis by UGE4 for the organization and function of endomembranes, especially TGN/EEs, which are a sorting station of the secretory and vacuolar pathways. 相似文献
14.
Schluter C Lam KK Brumm J Wu BW Saunders M Stevens TH Bryan J Conibear E 《Molecular biology of the cell》2008,19(4):1282-1294
Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. 相似文献
15.
The sorting of transmembrane cargo proteins into the lumenal vesicles of multivesicular bodies (MVBs) depends on the recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomal membranes. The subsequent dissociation of ESCRT complexes from endosomes requires Vps4, a member of the AAA family of adenosine triphosphatases. We show that Did2 directs Vps4 activity to the dissociation of ESCRT-III but has no role in the dissociation of ESCRT-I or -II. Surprisingly, vesicle budding into the endosome lumen occurs in the absence of Did2 function even though Did2 is required for the efficient sorting of MVB cargo proteins into lumenal vesicles. This uncoupling of MVB cargo sorting and lumenal vesicle formation suggests that the Vps4-mediated dissociation of ESCRT-III is an essential step in the sorting of cargo proteins into MVB vesicles but is not a prerequisite for the budding of vesicles into the endosome lumen. 相似文献
16.
MR Dores MM Paing H Lin WA Montagne A Marchese J Trejo 《Molecular biology of the cell》2012,23(18):3612-3623
The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail-localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting. 相似文献
17.
Efficient cargo sorting by ESCRT-I and the subsequent release of ESCRT-I from multivesicular bodies requires the subunit Mvb12
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The endosomal sorting complex required for transport (ESCRT)-I protein complex functions in recognition and sorting of ubiquitinated transmembrane proteins into multivesicular body (MVB) vesicles. It has been shown that ESCRT-I contains the vacuolar protein sorting (Vps) proteins Vps23, Vps28, and Vps37. We identified an additional subunit of yeast ESCRT-I called Mvb12, which seems to associate with ESCRT-I by binding to Vps37. Transient recruitment of ESCRT-I to MVBs results in the rapid degradation of Mvb12. In contrast to mutations in other ESCRT-I subunits, which result in strong defects in MVB cargo sorting, deletion of MVB12 resulted in only a partial sorting phenotype. This trafficking defect was fully suppressed by overexpression of the ESCRT-II complex. Mutations in MVB12 did not affect recruitment of ESCRT-I to MVBs, but they did result in delivery of ESCRT-I to the vacuolar lumen via the MVB pathway. Together, these observations suggest that Mvb12 may function in regulating the interactions of ESCRT-I with cargo and other proteins of the ESCRT machinery to efficiently coordinate cargo sorting and release of ESCRT-I from the MVB. 相似文献
18.
Mori Y Koike M Moriishi E Kawabata A Tang H Oyaizu H Uchiyama Y Yamanishi K 《Traffic (Copenhagen, Denmark)》2008,9(10):1728-1742
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. 相似文献
19.
Babst M 《Current opinion in cell biology》2011,23(4):452-457
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. 相似文献
20.
Multivesicular endosomes/bodies (MVBs) contain intraluminal vesicles (ILVs) that bud away from the cytoplasm. Multiple mechanisms of ILV formation have been identified, but the relationship between different populations of ILVs and MVBs remains unclear. Here, we show in HeLa cells that different ILV subpopulations can be distinguished by size. EGF stimulation promotes the formation of large ESCRT‐dependent ILVs, whereas depletion of the ESCRT‐0 component, Hrs, promotes the formation of a uniformly sized population of small ILVs, the formation of which requires CD63. CD63 has previously been implicated in ESCRT‐independent sorting of PMEL in MVBs and transfected PMEL is present on the small ILVs that form on Hrs depletion. Upregulation of CD63‐dependent ILV formation by Hrs depletion indicates that Hrs and CD63 regulate competing machineries required for the generation of distinct ILV subpopulations. Taken together our results indicate that ILV size is influenced by their cargo and mechanism of formation and suggest a competitive relationship between ESCRT‐dependent and ‐independent mechanisms of ILV formation within single MVBs. 相似文献