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Stanley N Cohen - One of the best experts on this subject based on the ideXlab platform.
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Identification of TSG101 functional domains and p21 loci required for TSG101-mediated p21 gene regulation
PloS one, 2013Co-Authors: Yu Shiuan Lin, Stanley N Cohen, Yin Ju Chen, Tzu-hao ChengAbstract:TSG101 (tumor susceptibility gene 101) is a multi-domain protein known to act in the cell nucleus, cytoplasm, and periplasmic membrane. Remarkably, TSG101, whose location within cells varies with the stage of the cell cycle, affects biological events as diverse as cell growth and proliferation, gene expression, cytokinesis, and endosomal trafficking. The functions of TSG101 additionally are recruited for viral and microvesicle budding and for intracellular survival of invading bacteria. Here we report that the TSG101 protein also interacts with and down-regulates the promoter of the p21CIP1/WAF1tumor suppressor gene, and identify a p21 locus and TSG101 domains that mediate this interaction. TSG101 deficiency in Saos-2 human osteosarcoma cells was accompanied by an increased abundance of p21 mRNA and protein and the retardation of cell proliferation. A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP) analysis and p21-driven luciferase reporter gene expression, respectively. Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB) in the repression of p21 promoter activity. Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking.
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TSG101 Control of Human Immunodeficiency Virus Type 1 Gag Trafficking and Release
Journal of virology, 2003Co-Authors: A. Goff, Stanley N Cohen, Lorna S. Ehrlich, Carol A. CarterAbstract:All retroviruses have in common three genes, gag, pol, and env, which specify the structural and enzymatic functions of the virus (reviewed in reference 50). The gag gene alone is sufficient for assembly and release of immature virus-like particles (VLPs) from infected cells. Maturation to form the infectious particle requires a protease (PR) encoded in pol. The gag-encoded protein (Gag) contains distinct domains involved in assembly and release. The region required for release by budding from the plasma membrane is called the late (L) domain (20, 25). The L domain of Gag is a Pro-rich motif that is highly conserved in retroviruses (17, 20, 25, 53, 54, 56, 58; reviewed in reference 16); other enveloped viruses, including rhabdoviruses, filoviruses, and Epstein-Barr virus, and cellular proteins also have Pro-rich motifs (17, 21, 22, 26, 27, 39). The L domains in retroviruses differ in amino acid sequence and location within the respective viral structural proteins but are functionally exchangeable (8, 18, 41, 54, 57), suggesting commonality of function. Recently, the PTAPP motif, or L domain, of human immunodeficiency virus type 1 (HIV-1), which is located in the C-terminal p6 region of the Gag precursor polyprotein, was found to bind to the protein product of the TSG101 gene (19, 37, 51). TSG101 was originally identified by the reversible neoplasia associated with its functional inactivation in murine fibroblasts (34). A cell line deficient in TSG101 (SL6) (34) shows a variety of nuclear, microtubule, and mitotic spindle abnormalities (55, 59), and TSG101 null mutant mice show defective cell proliferation and early embryonic death (45). Sequence analysis has suggested (31, 34, 42), and experimental evidence has shown, that TSG101 can function in both the modulation of transcription (24, 49, 52) and the inhibition of ubiquitination and protein decay (33). The latter effects are mediated by an N-terminal region that contains a ubiquitin (Ub) conjugase (E2)-like domain. The domain lacks the active-site Cys residue crucial to Ub conjugation but binds Ub elsewhere in this region (43). The N-terminal domain of TSG101 also is the minimal binding region required for its interaction with HIV-1 Gag (19, 37, 51). TSG101 appears to be ubiquitous in the cell (6, 55) and exhibits cell cycle-dependent localization in the Golgi complex (55), suggesting that it is highly dynamic. TSG101-deficient SL6 cells show defective endosomal trafficking (4), and an orthologue of TSG101, Stp22p/Vps23, is a class E vacuolar protein sorting (Vps) protein in the endosomal sorting machinery of Saccharomyces cerevisiae (4, 6, 35). Both the mammalian and the yeast proteins have been shown to recognize Ub and act in the removal of endosomal protein-Ub conjugates through a multivesicular body (MVB) (5, 29). The C-terminal region of TSG101 contains a binding site for Vps28, and an upstream coiled-coil domain may facilitate Vps37 binding (6, 29, 38). Together, the three proteins form a complex, ESCRT-1 (endocytic sorting complex required for trafficking). The ability of TSG101 to bind Ub is critical to the function of the complex. ESCRT complexes 2 and 3 have also been described recently (2, 3). Recent studies have demonstrated that TSG101 is required for HIV-1 production (7, 9, 19, 37), implicating the endocytic pathway in this process. During investigation of the effects of perturbation of the TSG101 protein level on Gag assembly, we found that TSG101 overexpression diminished Gag release, an effect similar to that of p6 deletion. This observation suggested a means of identifying the basis for the TSG101 binding requirement in Gag release and prompted us to examine cells for alterations associated with p6 deletion and TSG101 overexpression. We found that wild-type (wt) Gag was stably associated with a particulate fraction enriched in MVBs and plasma membrane components where Gag precursors lacking the p6 region (Δp6) did not stably accumulate. Expression of full-length or truncated TSG101 proteins inhibited association of wt Gag with this fraction, promoted TSG101-Gag colocalization with perinuclear endosomes, and diminished VLP release. Disruption of the PTAP-binding function of TSG101 resulted in perinuclear sequestration of TSG101-Gag complexes. Disruption of the Ub-binding function of TSG101, which is required for correct sorting of cargo in endocytic trafficking, prevented TSG101-Gag colocalization in both the perinuclear and plasma membrane regions and was very inhibitory to VLP release. These results identify an endosome-enriched subcellular fraction in which Gag accumulation is dependent on both p6 and TSG101, and they also indicate that Gag trafficking and VLP release require the PTAP- and Ub-binding functions of TSG101.
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TSG101 interaction with hrs mediates endosomal trafficking and receptor down regulation
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Quan Lu, Lila Weiqiao Hope, M A Brasch, Christoph Reinhard, Stanley N CohenAbstract:Down-regulation of mitogenic signaling in mammalian cells relies in part on endosomal trafficking of activated receptors into lysosomes, where the receptors are degraded. These events are mediated by ubiquitination of the endosomal cargo and its consequent sorting into multivesicular bodies that form at the surfaces of late endosomes. Tumor susceptibility gene 101 (TSG101) recently was found to be centrally involved in this process. Here we report that TSG101 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), an early endosomal protein, and that disruption of this interaction impedes endosomal trafficking and endocytosis-mediated degradation of mitogenic receptors. TSG101/HRS interaction occurs between a ubiquitin-binding domain of TSG101 and two distinct proline-rich regions of HRS, and is modulated by a C-terminal TSG101 sequence that resembles a motif targeted in HRS. Mutational perturbation of TSG101/HRS interaction prevented delivery of epidermal growth factor receptor (EGFR) to late endosomes, resulted in the cellular accumulation of ubiquitinated EGFR in early endosomes, and inhibited ligand-induced down-regulation of EGFR. Our results reveal the TSG101 interaction with HRS as a crucial step in endocytic down-regulation of mitogenic signaling and suggest a role for this interaction in linking the functions of early and late endosomes.
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TSG101 interaction with hrs mediates endosomal trafficking and receptor down regulation
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Lila Weiqiao Hope, M A Brasch, Christoph Reinhard, Stanley N CohenAbstract:Down-regulation of mitogenic signaling in mammalian cells relies in part on endosomal trafficking of activated receptors into lysosomes, where the receptors are degraded. These events are mediated by ubiquitination of the endosomal cargo and its consequent sorting into multivesicular bodies that form at the surfaces of late endosomes. Tumor susceptibility gene 101 (TSG101) recently was found to be centrally involved in this process. Here we report that TSG101 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), an early endosomal protein, and that disruption of this interaction impedes endosomal trafficking and endocytosis-mediated degradation of mitogenic receptors. TSG101/HRS interaction occurs between a ubiquitin-binding domain of TSG101 and two distinct proline-rich regions of HRS, and is modulated by a C-terminal TSG101 sequence that resembles a motif targeted in HRS. Mutational perturbation of TSG101/HRS interaction prevented delivery of epidermal growth factor receptor (EGFR) to late endosomes, resulted in the cellular accumulation of ubiquitinated EGFR in early endosomes, and inhibited ligand-induced down-regulation of EGFR. Our results reveal the TSG101 interaction with HRS as a crucial step in endocytic down-regulation of mitogenic signaling and suggest a role for this interaction in linking the functions of early and late endosomes.
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negative regulation of cell growth and differentiation by TSG101 through association with p21 cip1 waf1
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Cristina Mammucari, Stanley N Cohen, Arianna Nenci, Sara Cabodi, Paolo G DottoAbstract:TSG101 was discovered in a screen for tumor susceptibility genes and has since been shown to have a multiplicity of biological effects. However, the basis for TSG101's ability to regulate cell growth has not been elucidated. We report here that the TSG101 protein binds to the cyclin/cyclin-dependent kinase (CDK) inhibitor (CKI) p21Cip1/WAF1 and increases stability of the p21 protein in HEK293F cells and differentiating primary keratinocytes, suppressing differentiation in a p21-dependent manner. In proliferating keratinocytes where the p21 protein is relatively stable, TSG101 does not affect the stability or expression of p21 but shows p21-dependent recruitment to cyclin/CDK complexes, inhibits cyclin/CDK activity, and causes strong growth suppression to a much greater extent in p21+/+ than in p21−/− cells. Conversely, suppression of endogenous TSG101 expression by an antisense TSG101 cDNA causes doubling of the fraction of keratinocytes in the S phase of the cell cycle as occurs during p21 deficiency. Our results indicate that TSG101 has a direct role in the control of growth and differentiation in primary epithelial cells, and that p21 is an important mediator of these TSG101 functions.
Kay Uwe Wagner - One of the best experts on this subject based on the ideXlab platform.
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The Multifaceted Roles of the Tumor Susceptibility Gene 101 (TSG101) in Normal Development and Disease.
Cancers, 2020Co-Authors: Rosa-maria Ferraiuolo, Aleata A. Triplett, Karoline C. Manthey, Marissa J. Stanton, Kay Uwe WagnerAbstract:The multidomain protein encoded by the Tumor Susceptibility Gene 101 (TSG101) is ubiquitously expressed and is suggested to function in diverse intracellular processes. In this review, we provide a succinct overview of the main structural features of the protein and their suggested roles in molecular and cellular functions. We then summarize, in more detail, key findings from studies using genetically engineered animal models that demonstrate essential functions of TSG101 in cell proliferation and survival, normal tissue homeostasis, and tumorigenesis. Despite studies on cell lines that provide insight into the molecular underpinnings by which TSG101 might function as a negative growth regulator, a biologically significant role of TSG101 as a tumor suppressor has yet to be confirmed using genuine in vivo cancer models. More recent observations from several cancer research teams suggest that TSG101 might function as an oncoprotein. A potential role of post-translational mechanisms that control the expression of the TSG101 protein in cancer is being discussed. In the final section of the review, we summarize critical issues that need to be addressed to gain a better understanding of biologically significant roles of TSG101 in cancer.
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a knockout of the TSG101 gene leads to decreased expression of erbb receptor tyrosine kinases and induction of autophagy prior to cell death
PLOS ONE, 2012Co-Authors: Chantey R Morris, Kay Uwe Wagner, Karoline C. Manthey, Marissa J. StantonAbstract:The Tumor Susceptibility Gene 101 (TSG101) encodes a multi-domain protein that mediates a variety of molecular and biological processes including the trafficking and lysosomal degradation of cell surface receptors. Conventional and conditional knockout models have demonstrated an essential requirement of this gene for cell cycle progression and cell viability, but the consequences of a complete ablation of TSG101 on intracellular processes have not been examined to date. In this study, we employed mouse embryonic fibroblasts that carry two TSG101 conditional knockout alleles to investigate the expression of ErbB receptor tyrosine kinases as well as stress-induced intracellular processes that are known to be associated with a defect in growth and cell survival. The conditional deletion of the TSG101 gene in this well-controlled experimental model resulted in a significant reduction in the steady-state levels of the EGFR and ErbB2 but a stress-induced elevation in the phosphorylation of mitogen activated protein (MAP) kinases independent of growth factor stimulation. As part of an integrated stress response, TSG101-deficient cells exhibited extensive remodeling of actin filaments and greatly enlarged lysosomes that were enriched with the autophagy-related protein LC3. The increase in the transcriptional activation and expression of LC3 and its association with Lamp1-positive lysosomes in a PI3K-dependent manner suggest that TSG101 knockout cells utilize autophagy as a survival mechanism prior to their ultimate death. Collectively, this study shows that a knockout of the TSG101 gene causes complex intracellular changes associated with stress response and cell death. These multifaceted alterations need to be recognized as they have an impact on defining particular functions for TSG101 in processes such as signal transduction and lysosomal/endosomal trafficking.
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TSG101 is upregulated in a subset of invasive human breast cancers and its targeted overexpression in transgenic mice reveals weak oncogenic properties for mammary cancer initiation
Oncogene, 2007Co-Authors: M J Stanton, W W West, G L Todd, Kay Uwe WagnerAbstract:Previous studies reported that the Tumor Susceptibility Gene 101 (TSG101) is upregulated in selected human malignancies, and the expression of exogenous TSG101 was suggested to transform immortalized fibroblasts in culture. To date, the potential oncogenic properties of TSG101 have not been examined in vivo owing to the lack of appropriate model systems. In this study, we show that TSG101 is highly expressed in a subset of invasive human breast cancers. Based on this observation, we generated the first transgenic mouse model with a targeted overexpression of TSG101 in the developing mammary gland to test whether exogenous TSG101 is capable of initiating tumorigenesis. Normal functionality of exogenous TSG101 was tested by rescuing the survival of TSG101-deficient mammary epithelial cells in conditional knockout mice. The overexpression of TSG101 resulted in increased phosphorylation of the epidermal growth factor receptor and downstream activation of MAP kinases. Despite an increase in the activation of these signal transducers, the mammary gland of females expressing exogenous TSG101 developed normally throughout the reproductive cycle. In aging females, the overexpression of TSG101 seemed to increase the susceptibility of mammary epithelia toward malignant transformation. However, owing to the long latency of tumor formation and the sporadic occurrence of bona fide mammary cancers, we conclude that the TSG101 protein has only weak oncogenic properties. Instead of cancer initiation, it is therefore likely that TSG101 plays a more predominant role in the progression of a subset of spontaneously arising breast cancers.
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Cell Cycle Arrest and Cell Death Are Controlled by p53-dependent and p53-independent Mechanisms in TSG101-deficient Cells
The Journal of biological chemistry, 2004Co-Authors: Marissa J. Carstens, Aleata A. Triplett, Andrea Krempler, Maarten Van Lohuizen, Kay Uwe WagnerAbstract:Our previous studies have shown that cells conditionally deficient in TSG101 arrested at the G1/S cell cycle checkpoint and died. We created a series of TSG101 conditional knock-out cell lines that lack p53, p21Cip1, or p19Arf to determine the involvement of the Mdm2-p53 circuit as a regulator for G1/S progression and cell death. In this new report we show that the cell cycle arrest in TSG101-deficient cells is p53-dependent, but a null mutation of the p53 gene is unable to maintain cell survival. The deletion of the Cdkn1a gene in TSG101 conditional knock-out cells resulted in G1/S progression, suggesting that the p53-dependent G1 arrest in the TSG101 knock-out is mediated by p21Cip1. The Cre-mediated excision of TSG101 in immortalized fibroblasts that lack p19Arf seemed not to alter the ability of Mdm2 to sequester p53, and the p21-mediated G1 arrest was not restored. Based on these findings, we propose that the p21-dependent cell cycle arrest in TSG101-deficient cells is an indirect consequence of cellular stress and not caused by a direct effect of TSG101 on Mdm2 function as previously suggested. Finally, the deletion of TSG101 from primary tumor cells that express mutant p53 and that lack p21Cip1 expression results in cell death, suggesting that additional transforming mutations during tumorigenesis do not affect the important role of TSG101 for cell survival.
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targeted deletion of the TSG101 gene results in cell cycle arrest at g1 s and p53 independent cell death
Journal of Biological Chemistry, 2002Co-Authors: Andrea Krempler, Malinda D. Henry, Aleata A. Triplett, Kay Uwe WagnerAbstract:The tumor susceptibility gene 101 (TSG101) was originally discovered in a screen for potential tumor suppressors using insertional mutagenesis in immortalized fibroblasts. To investigate essential functions of this gene in cell growth and neoplastic transformation, we derived primary mouse embryonic fibroblasts from TSG101 conditional knockout mice. Expression of Cre recombinase from a retroviral vector efficiently down-regulated TSG101. The deletion of TSG101 caused growth arrest and cell death but did not result in increased proliferation and cellular transformation. Inactivation of p53 had no influence on the deleterious phenotype, but TSG101(-/-) cells were rescued through expression of exogenous TSG101. Fluorescence-activated cell sorting, proliferation assays, and Western blot analysis of crucial regulators of the cell cycle revealed that TSG101 deficiency resulted in growth arrest at the G(1)/S transition through inactivation of cyclin-dependent kinase 2. As a consequence, DNA replication was not initiated in TSG101-deficient cells. Our results clearly demonstrate that TSG101 is not a primary tumor suppressor in mouse embryonic fibroblasts. However, the protein is crucial for cell proliferation and cell survival.
Philip G. Woodman - One of the best experts on this subject based on the ideXlab platform.
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TSG101 mammalian vps23 and mammalian vps28 interact directly and are recruited to vps4 induced endosomes
Journal of Biological Chemistry, 2001Co-Authors: Naomi Bishop, Philip G. WoodmanAbstract:Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.
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TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes.
The Journal of biological chemistry, 2000Co-Authors: Naomi Bishop, Philip G. WoodmanAbstract:Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.
Carol A. Carter - One of the best experts on this subject based on the ideXlab platform.
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RNA Binding Suppresses TSG101 Recognition of Ub-Modified Gag and Facilitates Recruitment to the Plasma Membrane.
Viruses, 2020Co-Authors: Susan M. Watanabe, Madeleine Strickland, Nico Tjandra, Carol A. CarterAbstract:The ESCRT-I factor TSG101 is essential for sorting endocytic cargo and is exploited by viral pathogens to facilitate egress from cells. Both the nucleocapsid (NC) domain and p6 domain in HIV-1 Gag contribute to recruitment of the protein. However, the role of NC is unclear when the P(S/T)AP motif in p6 is intact, as the motif recruits TSG101 directly. The zinc fingers in NC bind RNA and membrane and are critical for budding. TSG101 can substitute for the distal ZnF (ZnF2) and rescue budding of a mutant made defective by deletion of this element. Here, we report that the ubiquitin (Ub) E2 variant (UEV) domain in TSG101 binds tRNA in vitro. We confirmed that TSG101 can substitute for ZnF2 when provided at the viral assembly site as a chimeric Gag-TSG101 protein (Gag-ΔZnF2-TSG101) and rescue budding. The UEV was not required in this context; however, mutation of the RNA binding determinants in UEV prevented TSG101 recruitment from the cell interior when Gag and TSG101 were co-expressed. The same TSG101 mutations increased recognition of Gag-Ub, suggesting that tRNA and Ub compete for binding sites. This study identifies a novel TSG101 binding partner that may contribute to its function in recognition of Ub-modified cargo.
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TSG101 escrt i recruitment regulated by the dual binding modes of k63 linked diubiquitin
Social Science Research Network, 2020Co-Authors: Madeleine Strickland, Carol A. Carter, Susan Watanabe, Steven M. Bonn, Christina M. Camara, David Fushman, Nico TjandraAbstract:The ESCRT-I protein TSG101 plays a critical role in viral budding and endocytic sorting. Although TSG101 is known to recognize monoubiquitin (Ub1), here we show for the first time that it can also bind several diubiquitins (Ub2), with a preference for K63-linked Ub2. The NMR structure of the TSG101:K63-Ub2 complex showed that while the Ub1-binding site accommodates the distal domain of Ub2, the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site resulted in a distinct phenotype with respect to ubiquitin-dependent recruitment. Mutation in the vestigial active site abrogated the interaction between TSG101 and the HIV-1 protein Gag, while mutation at the N-terminal helix increased the population of Gag-TSG101 in the cell interior. Given the broad involvement of TSG101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo.
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TSG101/ESCRT-I Recruitment Regulated by the Dual Binding Modes of K63-Linked Diubiquitin
SSRN Electronic Journal, 2020Co-Authors: Madeleine Strickland, Carol A. Carter, Susan Watanabe, Steven M. Bonn, Christina M. Camara, David Fushman, Nico TjandraAbstract:The ESCRT-I protein TSG101 plays a critical role in viral budding and endocytic sorting. Although TSG101 is known to recognize monoubiquitin (Ub1), here we show for the first time that it can also bind several diubiquitins (Ub2), with a preference for K63-linked Ub2. The NMR structure of the TSG101:K63-Ub2 complex showed that while the Ub1-binding site accommodates the distal domain of Ub2, the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site resulted in a distinct phenotype with respect to ubiquitin-dependent recruitment. Mutation in the vestigial active site abrogated the interaction between TSG101 and the HIV-1 protein Gag, while mutation at the N-terminal helix increased the population of Gag-TSG101 in the cell interior. Given the broad involvement of TSG101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo.
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TSG101 can replace Nedd4 function in ASV Gag release but not membrane targeting
Virology, 2008Co-Authors: Gisselle N. Medina, Yongjun Zhang, Yi Tang, Jonathan Leis, Lorna S. Ehrlich, Andrew Pincetic, Carol A. CarterAbstract:The Late (L) domain of the avian sarcoma virus (ASV) Gag protein binds Nedd4 ubiquitin ligase E3 family members and is the determinant of efficient virus release in avian and mammalian cells. We previously demonstrated that Nedd4 and TSG101 constitutively interact raising the possibility that Nedd4 links ASV Gag to the ESCRT machinery. We now demonstrate that covalently linking TSG101 to ASV Gag lacking the Nedd4 binding site (Δp2b-TSG101) ablates the requirement for Nedd4, but the rescue of budding occurs by use of a different budding mechanism than that used by wild type ASV Gag. The evidence that TSG101 and Nedd4 direct release by different pathways is: (i) Release of the virus-like particles (VLPs) assembled from Gag in DF-1, an avian cell line, was resistant to dominant-negative interference by a TSG101 mutant previously shown to inhibit release of both HIV and Mo-MLV. (ii) Release of VLPs from DF-1 cells was resistant to siRNA-mediated depletion of the endogenous pool of TSG101 in these cells. (iii) VLPs assembled from wild-type ASV Gag exhibited highly efficient release from endosome-like membrane domains enriched in the tetraspanin protein CD63 or a fluorescent analogue of the phospholipid phosphatidylethanolamine. However, the VLPs assembled from the L domain mutant Δp2b or a chimeric Δp2b-TSG101 Gag lacked these domain markers even though the chimeric Gag was released efficiently compared to the Δp2b mutant. These results suggest that TSG101 and Nedd4 facilitate Gag release through functionally exchangeable but independent routes and that TSG101 can replace Nedd4 function in facilitating budding but not directing through the same membranes.
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the functionally exchangeable l domains in rsv and hiv 1 gag direct particle release through pathways linked by TSG101
Traffic, 2005Co-Authors: Gisselle N. Medina, Yongjun Zhang, Yi Tang, Eva Gottwein, Marcy L. Vana, Fadila Bouamr, Jonathan Leis, Carol A. CarterAbstract:The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag bind TSG101 and Nedd4, respectively. TSG101 and Nedd4 function in endocytic trafficking, and studies show that expression of TSG101 or Nedd4 fragments interfere with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co-expression with mutated forms of Vps4, Nedd4 and TSG101. A dominant-negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV-1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and TSG101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with TSG101. Deletion of the N-terminal region of TSG101 or the HECT domain of Nedd4 did not prevent interaction; however, three-dimensional spatial imaging suggested that the interaction of RSV Gag with full-length TSG101 and N-terminally truncated TSG101 was not the same. Co-expression of RSV Gag with the TSG101 C-terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while TSG101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through TSG101–Nedd4 interaction.
Naomi Bishop - One of the best experts on this subject based on the ideXlab platform.
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TSG101 mammalian vps23 and mammalian vps28 interact directly and are recruited to vps4 induced endosomes
Journal of Biological Chemistry, 2001Co-Authors: Naomi Bishop, Philip G. WoodmanAbstract:Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.
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TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes.
The Journal of biological chemistry, 2000Co-Authors: Naomi Bishop, Philip G. WoodmanAbstract:Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221- amino acid cytosolic protein that interacts with TSG101/mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.
