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Reinhild Prange - One of the best experts on this subject based on the ideXlab platform.
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γ2-Adaptin is functioning in the late endosomal sorting pathway and interacts with ESCRT-I and -III subunits.
Biochimica et biophysica acta, 2010Co-Authors: Tatjana Döring, Katherina Gotthardt, Jens Stieler, Reinhild PrangeAbstract:γ2-Adaptin is a clathrin adaptor-related protein with unclear physiological function. Previous studies indicated that γ2-Adaptin might act within the multivesicular body (MVB) protein-sorting pathway that is central to receptor down-regulation, lysosome biogenesis, and budding of enveloped viruses. Here, we have analyzed the effects of excess and deficit γ2-Adaptin on exogenous and endogenous MVB cargoes and on the MVB machinery itself. Foreign cargoes, like retroviral Gags, are entrapped by overexpressed γ2-Adaptin in detergent-insoluble polymers and blocked in budding. When viral budding involves MVB/endosomal structures, excess γ2-Adaptin acts by accelerating lysosomal Gag destruction. Consistently, depletion of γ2-Adaptin avoids Gag routing to the lysosome and increases viral production. Functional studies with natural MVB cargoes support a role of γ2-Adaptin in MVB-to-lysosome transition. Furthermore, we show that different members of the endosomal sorting complex required for transport (ESCRT) that drive sorting from endosomes to lysosomes are sequestered upon γ2-Adaptin overexpression. If sequestered irreversibly, they are targeted to enhanced lysosomal degradation. The participation of γ2-Adaptin in MVB sorting is further suggested by our finding that it specifically interacts with the ESCRT subunits Vps28 and CHMP2A. These observations identify γ2-Adaptin as a critical factor in MVB trafficking, which likely is involved in endosome-to-lysosome maturation.
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γ2-Adaptin, a Ubiquitin-interacting Adaptor, Is a Substrate to Coupled Ubiquitination by the Ubiquitin Ligase Nedd4 and Functions in the Endosomal Pathway *
The Journal of biological chemistry, 2008Co-Authors: Martina Rost, Tatjana Döring, Reinhild PrangeAbstract:gamma2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that gamma2-Adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of gamma2-Adaptin. By mapping regions of Nedd4 involved in binding to gamma2-Adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of gamma2-Adaptin for subsequent ubiquitin conjugation. Unlike known coupled ubiquitination reactions, this novel type of interaction leads to mono- and multi/polyubiquitinated gamma2-Adaptin. In addition, we show that gamma2-Adaptin functions in the endosomal/multivesicular body (MVB) pathway. Depletion of gamma2-Adaptin impairs the degradation of internalized epidermal growth factor and results in defective MVB morphology characterized by significantly enlarged vesicles. These defects cannot be rescued by gamma1-Adaptin, a closely related homolog of gamma2-Adaptin, which is unable to bind ubiquitin. Together, these results indicate that gamma2-Adaptin may operate within the MVB sorting system in a manner different from that of classic Adaptins.
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Hepatitis B Virus Maturation Is Sensitive to Functional Inhibition of ESCRT-III, Vps4, and γ2-Adaptin
Journal of virology, 2007Co-Authors: Carsten Lambert, Tatjana Döring, Reinhild PrangeAbstract:Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor γ2-Adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPase-defective Vps4A or Vps4B mutants, HBV assembly and egress were potently blocked. Each of the MVB inhibitors arrested virus particle maturation by entrapping the viral core and large and small envelope proteins in detergent-insoluble membrane structures that closely resembled aberrant endosomal class E compartments. In contrast, HBV subvirus particle release was not affected by MVB inhibitors, hinting at different export routes used by viral and subviral particles. To further define the role γ2-Adaptin plays in HBV formation, we examined the effects of its overexpression in virus-replicating cells. Intriguingly, excess γ2-Adaptin blocked HBV production in a manner similar to the actions of CHMP and Vps4 mutants. Moreover, overexpressed γ2-Adaptin perturbed the endosomal morphology and diminished the budding of a retroviral Gag protein, implying that it may act as a principal inhibitor of the MVB sorting pathway. Together, these results demonstrate that HBV exploits the MVB machinery with the aid of γ2-Adaptin.
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γ2 Adaptin a novel ubiquitin interacting adaptor and nedd4 ubiquitin ligase control hepatitis b virus maturation
Journal of Biological Chemistry, 2006Co-Authors: Martina Rost, Tatjana Döring, Sylvia Mann, Carsten Lambert, Nicole Thomé, Reinhild PrangeAbstract:Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular γ2-Adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered γ2-Adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same γ2-adaptor and that disruption of the HBV/γ2-Adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of γ2-Adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both γ2-Adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with γ2-Adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.
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γ2-Adaptin, a Novel Ubiquitin-interacting Adaptor, and Nedd4 Ubiquitin Ligase Control Hepatitis B Virus Maturation *
The Journal of biological chemistry, 2006Co-Authors: Martina Rost, Tatjana Döring, Sylvia Mann, Carsten Lambert, Nicole Thomé, Reinhild PrangeAbstract:Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular γ2-Adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered γ2-Adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same γ2-adaptor and that disruption of the HBV/γ2-Adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of γ2-Adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both γ2-Adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with γ2-Adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.
Gregory S. Payne - One of the best experts on this subject based on the ideXlab platform.
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yeast epsin related proteins required for golgi endosome traffic define a γ Adaptin ear binding motif
Nature Cell Biology, 2003Co-Authors: Mara C. Duncan, Giancarlo Costaguta, Gregory S. PayneAbstract:Yeast epsin-related proteins required for Golgi–endosome traffic define a γ-Adaptin ear-binding motif
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Yeast epsin-related proteins required for Golgi–endosome traffic define a γ-Adaptin ear-binding motif
Nature cell biology, 2002Co-Authors: Mara C. Duncan, Giancarlo Costaguta, Gregory S. PayneAbstract:Yeast epsin-related proteins required for Golgi–endosome traffic define a γ-Adaptin ear-binding motif
Peter Schu - One of the best experts on this subject based on the ideXlab platform.
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Rabaptin‐5α/rabaptin‐4 serves as a linker between rab4 and γ1‐Adaptin in membrane recycling from endosomes
The EMBO journal, 2003Co-Authors: Magdalena Deneka, Peter Schu, Maaike Neeft, Ioana Popa, Masja Van Oort, Hein Sprong, Viola Oorschot, Judith Klumperman, Peter Van Der SluijsAbstract:Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-Adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-Adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- Adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-Adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-Adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-Adaptin regulate membrane recycling.
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rabaptin 5α rabaptin 4 serves as a linker between rab4 and γ1 Adaptin in membrane recycling from endosomes
The EMBO Journal, 2003Co-Authors: Magdalena Deneka, Peter Schu, Maaike Neeft, Ioana Popa, Masja Van Oort, Hein Sprong, Viola Oorschot, Judith Klumperman, Peter Van Der SluijsAbstract:Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-Adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-Adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- Adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-Adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-Adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-Adaptin regulate membrane recycling.
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μ1A‐Adaptin‐deficient mice: lethality, loss of AP‐1 binding and rerouting of mannose 6‐phosphate receptors
The EMBO journal, 2000Co-Authors: Christoph Meyer, Daniela Zizioli, Susanne Lausmann, Eeva-liisa Eskelinen, Jens Hamann, Paul Saftig, Kurt Von Figura, Peter SchuAbstract:The heterotetrameric AP-1 complex is involved in the formation of clathrin-coated vesicles at the trans-Golgi network (TGN) and interacts with sorting signals in the cytoplasmic tails of cargo molecules. Targeted disruption of the mouse mu1A-Adaptin gene causes embryonic lethality at day 13.5. In cells deficient in micro1A-Adaptin the remaining AP-1 Adaptins do not bind to the TGN. Polarized epithelial cells are the only cells of micro1A-Adaptin-deficient embryos that show gamma-Adaptin binding to membranes, indicating the formation of an epithelial specific AP-1B complex and demonstrating the absence of additional mu1A homologs. Mannose 6-phosphate receptors are cargo molecules that exit the TGN via AP-1-clathrin-coated vesicles. The steady-state distribution of the mannose 6-phosphate receptors MPR46 and MPR300 in mu1A-deficient cells is shifted to endosomes at the expense of the TGN. MPR46 fails to recycle back from the endosome to the TGN, indicating that AP-1 is required for retrograde endosome to TGN transport of the receptor.
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μ1a Adaptin deficient mice lethality loss of ap 1 binding and rerouting of mannose 6 phosphate receptors
The EMBO Journal, 2000Co-Authors: Christoph Meyer, Daniela Zizioli, Susanne Lausmann, Eeva-liisa Eskelinen, Jens Hamann, Paul Saftig, Kurt Von Figura, Peter SchuAbstract:The heterotetrameric AP-1 complex is involved in the formation of clathrin-coated vesicles at the trans-Golgi network (TGN) and interacts with sorting signals in the cytoplasmic tails of cargo molecules. Targeted disruption of the mouse µ1A-Adaptin gene causes embryonic lethality at day 13.5. In cells deficient in µ1A-Adaptin the remaining AP-1 Adaptins do not bind to the TGN. Polarized epithelial cells are the only cells of µ1A-Adaptin-deficient embryos that show γ-Adaptin binding to membranes, indicating the formation of an epithelial specific AP-1B complex and demonstrating the absence of additional µ1A homologs. Mannose 6-phosphate receptors are cargo molecules that exit the TGN via AP-1–clathrin-coated vesicles. The steady-state distribution of the mannose 6-phosphate receptors MPR46 and MPR300 in µ1A-deficient cells is shifted to endosomes at the expense of the TGN. MPR46 fails to recycle back from the endosome to the TGN, indicating that AP-1 is required for retrograde endosome to TGN transport of the receptor.
Tatjana Döring - One of the best experts on this subject based on the ideXlab platform.
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γ2-Adaptin is functioning in the late endosomal sorting pathway and interacts with ESCRT-I and -III subunits.
Biochimica et biophysica acta, 2010Co-Authors: Tatjana Döring, Katherina Gotthardt, Jens Stieler, Reinhild PrangeAbstract:γ2-Adaptin is a clathrin adaptor-related protein with unclear physiological function. Previous studies indicated that γ2-Adaptin might act within the multivesicular body (MVB) protein-sorting pathway that is central to receptor down-regulation, lysosome biogenesis, and budding of enveloped viruses. Here, we have analyzed the effects of excess and deficit γ2-Adaptin on exogenous and endogenous MVB cargoes and on the MVB machinery itself. Foreign cargoes, like retroviral Gags, are entrapped by overexpressed γ2-Adaptin in detergent-insoluble polymers and blocked in budding. When viral budding involves MVB/endosomal structures, excess γ2-Adaptin acts by accelerating lysosomal Gag destruction. Consistently, depletion of γ2-Adaptin avoids Gag routing to the lysosome and increases viral production. Functional studies with natural MVB cargoes support a role of γ2-Adaptin in MVB-to-lysosome transition. Furthermore, we show that different members of the endosomal sorting complex required for transport (ESCRT) that drive sorting from endosomes to lysosomes are sequestered upon γ2-Adaptin overexpression. If sequestered irreversibly, they are targeted to enhanced lysosomal degradation. The participation of γ2-Adaptin in MVB sorting is further suggested by our finding that it specifically interacts with the ESCRT subunits Vps28 and CHMP2A. These observations identify γ2-Adaptin as a critical factor in MVB trafficking, which likely is involved in endosome-to-lysosome maturation.
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γ2-Adaptin, a Ubiquitin-interacting Adaptor, Is a Substrate to Coupled Ubiquitination by the Ubiquitin Ligase Nedd4 and Functions in the Endosomal Pathway *
The Journal of biological chemistry, 2008Co-Authors: Martina Rost, Tatjana Döring, Reinhild PrangeAbstract:gamma2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that gamma2-Adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of gamma2-Adaptin. By mapping regions of Nedd4 involved in binding to gamma2-Adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of gamma2-Adaptin for subsequent ubiquitin conjugation. Unlike known coupled ubiquitination reactions, this novel type of interaction leads to mono- and multi/polyubiquitinated gamma2-Adaptin. In addition, we show that gamma2-Adaptin functions in the endosomal/multivesicular body (MVB) pathway. Depletion of gamma2-Adaptin impairs the degradation of internalized epidermal growth factor and results in defective MVB morphology characterized by significantly enlarged vesicles. These defects cannot be rescued by gamma1-Adaptin, a closely related homolog of gamma2-Adaptin, which is unable to bind ubiquitin. Together, these results indicate that gamma2-Adaptin may operate within the MVB sorting system in a manner different from that of classic Adaptins.
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Hepatitis B Virus Maturation Is Sensitive to Functional Inhibition of ESCRT-III, Vps4, and γ2-Adaptin
Journal of virology, 2007Co-Authors: Carsten Lambert, Tatjana Döring, Reinhild PrangeAbstract:Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor γ2-Adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPase-defective Vps4A or Vps4B mutants, HBV assembly and egress were potently blocked. Each of the MVB inhibitors arrested virus particle maturation by entrapping the viral core and large and small envelope proteins in detergent-insoluble membrane structures that closely resembled aberrant endosomal class E compartments. In contrast, HBV subvirus particle release was not affected by MVB inhibitors, hinting at different export routes used by viral and subviral particles. To further define the role γ2-Adaptin plays in HBV formation, we examined the effects of its overexpression in virus-replicating cells. Intriguingly, excess γ2-Adaptin blocked HBV production in a manner similar to the actions of CHMP and Vps4 mutants. Moreover, overexpressed γ2-Adaptin perturbed the endosomal morphology and diminished the budding of a retroviral Gag protein, implying that it may act as a principal inhibitor of the MVB sorting pathway. Together, these results demonstrate that HBV exploits the MVB machinery with the aid of γ2-Adaptin.
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γ2 Adaptin a novel ubiquitin interacting adaptor and nedd4 ubiquitin ligase control hepatitis b virus maturation
Journal of Biological Chemistry, 2006Co-Authors: Martina Rost, Tatjana Döring, Sylvia Mann, Carsten Lambert, Nicole Thomé, Reinhild PrangeAbstract:Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular γ2-Adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered γ2-Adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same γ2-adaptor and that disruption of the HBV/γ2-Adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of γ2-Adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both γ2-Adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with γ2-Adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.
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γ2-Adaptin, a Novel Ubiquitin-interacting Adaptor, and Nedd4 Ubiquitin Ligase Control Hepatitis B Virus Maturation *
The Journal of biological chemistry, 2006Co-Authors: Martina Rost, Tatjana Döring, Sylvia Mann, Carsten Lambert, Nicole Thomé, Reinhild PrangeAbstract:Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular γ2-Adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered γ2-Adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same γ2-adaptor and that disruption of the HBV/γ2-Adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of γ2-Adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both γ2-Adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with γ2-Adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.
Martina Rost - One of the best experts on this subject based on the ideXlab platform.
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γ2-Adaptin, a Ubiquitin-interacting Adaptor, Is a Substrate to Coupled Ubiquitination by the Ubiquitin Ligase Nedd4 and Functions in the Endosomal Pathway *
The Journal of biological chemistry, 2008Co-Authors: Martina Rost, Tatjana Döring, Reinhild PrangeAbstract:gamma2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that gamma2-Adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of gamma2-Adaptin. By mapping regions of Nedd4 involved in binding to gamma2-Adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of gamma2-Adaptin for subsequent ubiquitin conjugation. Unlike known coupled ubiquitination reactions, this novel type of interaction leads to mono- and multi/polyubiquitinated gamma2-Adaptin. In addition, we show that gamma2-Adaptin functions in the endosomal/multivesicular body (MVB) pathway. Depletion of gamma2-Adaptin impairs the degradation of internalized epidermal growth factor and results in defective MVB morphology characterized by significantly enlarged vesicles. These defects cannot be rescued by gamma1-Adaptin, a closely related homolog of gamma2-Adaptin, which is unable to bind ubiquitin. Together, these results indicate that gamma2-Adaptin may operate within the MVB sorting system in a manner different from that of classic Adaptins.
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γ2 Adaptin a novel ubiquitin interacting adaptor and nedd4 ubiquitin ligase control hepatitis b virus maturation
Journal of Biological Chemistry, 2006Co-Authors: Martina Rost, Tatjana Döring, Sylvia Mann, Carsten Lambert, Nicole Thomé, Reinhild PrangeAbstract:Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular γ2-Adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered γ2-Adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same γ2-adaptor and that disruption of the HBV/γ2-Adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of γ2-Adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both γ2-Adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with γ2-Adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.
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γ2-Adaptin, a Novel Ubiquitin-interacting Adaptor, and Nedd4 Ubiquitin Ligase Control Hepatitis B Virus Maturation *
The Journal of biological chemistry, 2006Co-Authors: Martina Rost, Tatjana Döring, Sylvia Mann, Carsten Lambert, Nicole Thomé, Reinhild PrangeAbstract:Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular γ2-Adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered γ2-Adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same γ2-adaptor and that disruption of the HBV/γ2-Adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of γ2-Adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both γ2-Adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with γ2-Adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.
