InteractiveFly: GeneBrief

tumor suppressor protein 101: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - tumor suppressor protein 101

Synonyms - erupted (ept)

Cytological map position - 73D1

Function - vesicular transport protein, signaling

Keywords - vacuolar protein sorting (VPS) pathway, regulation of JAK-STAT cascade, oncogene, Endosomal Sorting Complex Required for Transport-1 (ESCRT-1)

Symbol - TSG101

FlyBase ID: FBgn0036666

Genetic map position - 2L

Classification - amino-terminal ubiquitin-conjugating domain

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

The reproducible pattern of organismal growth during metazoan development is the product of genetically controlled signaling pathways. Patterned activation of these pathways shapes developing organs and dictates overall organismal shape and size. Patches of tissue that are mutant for the Drosophila Tsg101 ortholog, erupted, cause dramatic overexpression of adjacent wild-type tissue. Tsg101 proteins function in endosomal sorting and are required to incorporate late endosomes into multivesicular bodies. Drosophila cells with impaired Tsg101 function show accumulation of the Notch receptor in intracellular compartments marked by the endosomal protein Hrs. This causes increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired (upd), which encodes the secreted ligand of the JAK-STAT pathway. Activation of JAK-STAT signaling in surrounding wild-type cells correlates with their overgrowth. These findings define a pathway by which changes in endocytic trafficking can regulate tissue growth in a non-cell-autonomous manner (Moberg, 2005). Tsg101 possesses the ability to bind monoubiquitinated substrates (Garrus, 2001; Sundquist, 2004). These substrates are predicted to be the ubiquitinated cytoplasmic tails of membrane bound proteins, and this interaction is predicted to deliver cargos to the lysosome via multivesicular bodies (reviewed in Katzmann, 2002).

Organismal patterning requires that the fates of individual cells within a multicellular organ be coordinated with the fates of surrounding cells. In most cases, this is achieved by secreted morphogens produced by a small group of “organizing” cells that then trigger a coordinated response among receiving cells. This type of signaling mediates the specialization of a subset of cells within a larger pool of precursors and shapes developing organs via local effects on cell proliferation (Moberg, 2005).

A number of secreted factors that control proliferation have been identified, including epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Other signaling pathways that can influence cell proliferation during development include those activated by secreted proteins such as Hedgehog, members of the Wnt and bone morphogenic protein (BMP) families, as well as the pathway downstream of the Notch receptor. For each of these pathways to regulate tissue growth and cell proliferation, they must eventually be able to influence mechanisms that regulate cell growth and division (Moberg, 2005).

Extracellular mitogens are also implicated in the aberrant growth observed in many cancers. In addition to the well-characterized role of hormones in promoting or sustaining the growth of certain carcinomas (e.g., breast and prostate), there is increasing recent awareness of the importance of growth-stimulating properties of stromal cells that are associated with most cancers of epithelial origin. Several studies have documented the increased propensity of tumor-associated fibroblasts to accelerate tumor progression in animal models and to promote tumorigenic changes in normal epithelial cells. While it is likely that many of these effects are the result of factors secreted by the stromal cells, the precise nature of these signals have, in most cases, not been defined (Moberg, 2005).

Studies of Drosophila imaginal discs have contributed significantly to understanding of the mechanisms that regulate tissue growth during organismal development. Studies in Drosophila have also begun to link signaling molecules that function in patterning the imaginal disc (e.g., Hh, Dpp, and Wg) with regulation of tissue growth and cell cycle progression. Genetic lesions in pathways that regulate imaginal disc growth can alter the size of adult structures such as the eye and the wing, and a number of genetic screens have used these phenotypes to identify mutations that result in tissue overgrowth. As is the case with the study of mammalian tumors, most of these mutations enhance growth cell-autonomously and provide little insight into mechanisms that might underlie interactions between tumors and stromal cells in mammals (Moberg, 2005).

Mutations in erupted (ept) are described that enable clones of mutant cells to stimulate the overgrowth of surrounding wild-type tissue. ept mutations disrupt the function of the Drosophila ortholog of the mammalian Tumor susceptibility gene 101 (Tsg101), whose role in tumorigenesis is controversial, but which has a defined role in the endocytic pathway. Mutations in Tsg101 activate Notch signaling and cause overproduction of the secreted mitogen Unpaired (Upd). These studies thus define a mechanism by which alterations in trafficking through the endocytic pathway can trigger the secretion of a growth factor and cause overproliferation of neighboring cells (Moberg, 2005).

Mammalian Tsg101 was initially discovered because antisense Tsg101 expression allowed fibroblasts to form colonies in soft agar and produce tumors in nude mice (Li, 1996). However, the role, if any, of Tsg101 in mammalian tumorigenesis has remained controversial. The similarity of Tsg101 to yeast Vps23p has led to a better understanding of Tsg101 as a component of an endosomal pathway that routes monoubiquitinated proteins into multivesicular bodies and the lysosome (reviewed in Katzmann, 2002). Mutations in the Drosophila ortholog of Tsg101, erupted, do indeed result in tissue overgrowth. Surprisingly, overgrowth occurs in the wild-type tissue that surrounds mutant cells. Notch trapped in ept mutant endosomes is activated, and it stimulates production of the secreted growth factor Upd, which causes stat92E-dependent proliferation and overgrowth of surrounding wild-type cells (Moberg, 2005).

Models of endosome-mediated receptor internalization emphasize the role of monoubiquitination as a signal for routing proteins through the MVB pathway (Katzmann, 2002). The data indicate that this pathway plays an important role in limiting Notch levels in cells of the developing eye, and that the excess Notch that accumulates upstream of the block in Tsg101 mutants is active and localizes to an Hrs-positive compartment. This might imply that ligand bound Notch is normally trafficked through endocytic compartments, but that not all ligand bound receptors succeed in transmitting a signal to the nucleus. Blocking this pathway at a particular step might then arrest Notch in a compartment from which it is able to signal. This is consistent with a proposed model in which endocytosed Notch is the preferred substrate of the Presenilin (Psn)-dependent γ-secretase (Gupta-Rossi, 2004), although no enrichment of Psn was observed in ept mutant cells. Delta accumulates ectopically in ept mutant eye disc cells, suggesting that the mechanism of Notch activation in endosomes may be ligand dependent. Further experiments are needed to test the role of Ub, Delta, and the γ-secretase in activating Notch in ept mutant cells (Moberg, 2005).

Certain asymmetric cell fates in the nervous system are controlled by selective endosomal routing of Delta (Emery, 2005). As several manipulations that block Notch entry into early endosomes also compromise signaling (Gupta-Rossi, 2004; Hori, 2004), Notch activation during normal development may also be controlled by trafficking to a particular compartment. These data suggest that this compartment lies between the cell membrane and the point in the MVB pathway at which Tsg101 acts, and includes the Hrs-positive endosome. eyFLP-mediated mitotic recombination of hrs alleles do not provoke overgrowth in the developing eye. If further study confirms that hrs mutant cells accumulate Notch but do not activate it, it would suggest that Notch activation occurs in an endosomal compartment downstream of the block in hrs mutants, but upstream of the block in ept mutants (Moberg, 2005).

hrs inactivation has been shown to affect localization of a number of receptors, including Notch, Egfr, Patched, Smoothened, and Thickveins. This study focused on the effects of ept on Notch, but it is likely that endosomal sorting of other receptors is also disrupted in ept cells. Indeed, the ability of ept alleles to enhance phenotypes of two EGFR pathway components, rolled/MAPK and Gap1, is consistent with findings that mammalian Tsg101 regulates Egfr endosomal sorting (Babst, 2000: Bishop, 2002; Lu, 2003). The extent to which other pathways contribute to ept mutant phenotypes, and the degree to which ept affects receptor trafficking in tissues other than the developing eye, clearly merits further investigation (Moberg, 2005).

Notch activation is an important determinant of the size of the adult eye. Manipulations that increase Notch activity or activate Notch at ectopic sites (e.g., by generating fringe mutant clones) result in increased growth of the eye imaginal disc and larger adult eyes. Activated Notch proteins also appear to promote cell proliferation in the context of certain cancers (reviewed in Maillard, 2003). While there are likely to be a number of cell-autonomous Notch targets that effect growth, data presented here identify Upd and the JAK-STAT module as likely mediators of non-cell-autonomous growth phenotypes associated with Notch activation, and they show that loss of Tsg101 is sufficient to activate this pathway in cells of the eye-antennal disc. In light of these findings, it will be of interest to test whether Notch is able to regulate the JAK-STAT pathway in epithelial tissues other than the fly eye (Moberg, 2005).

From the first description of Tsg101 as a gene required to restrict oncogenic transformation and anchorage-independent growth of cultured mammalian cells (Li, 1996), a satisfying mechanistic explanation of how Tsg101 regulates cell proliferation has proved elusive. Another ESCRT-1 subunit, HCRP-1/hVps37A, is also implicated as a growth inhibitory gene (Bache, 2004), but its role in proliferation control is similarly unclear. Mutation of the Drosophila Tsg101 homolog, erupted, affects tissue growth in two different ways: (1) clones of ept cells promote the overgrowth of surrounding wild-type tissue; (2) eye discs composed almost entirely of ept mutant tissue continue to grow during an extended larval stage, and they become tumorous masses. Despite an apparent slow-growth phenotype, these ept mutant cells continue to proliferate beyond the developmental stage at which they would otherwise exit the cell cycle. This contrasts with the behavior of normal cells that stop growing when the tissue reaches the appropriate size, and suggests that ept cells are unable to respond to signals that normally sense and restrict organ size. This phenotype differs from that induced by eye-specific overexpression of upd (Bach, 2003), suggesting that it is not due only to mitogenic effects of Upd. In addition to this growth defect, ept mutant cells also display phenotypes consistent with defects in apicobasal polarity. Considered together, these cell-autonomous defects are quite similar to those associated with mutations in the 'neoplastic tumor suppressor genes' (nTSGs) scribble, discs-large, and lethal(2) giant larvae (reviewed in Bilder, 2004). While this similarity need not reflect a common mechanism of growth control between Tsg101 and the nTSGs, it is perhaps significant that all of these genes can now be linked to the control of epithelial cell polarity via the Crb pathway (Moberg, 2005).

Mice lacking Tsg101 function die as early embryos, and inactivation of Tsg101 in cultured cells impairs proliferation (Wagner, 2003). However, the data suggest the possibility that mammalian cells with reduced Tsg101 function could promote the growth of neighboring cells in vivo. While there is no link yet established in mammalian cells between ESCRT-1 function and a putative Notch-upd-stat pathway, mammalian Notch signaling has been shown to induce transcription of a number of cytokine genes (reviewed in Maillard, 2005) whose encoded factors signal through downstream JAK-STAT pathways. Intriguingly, a recent report has also found evidence that the genomic interval containing human Tsg101 shows a high rate of allelic imbalance in nontumor-derived stroma associated with breast carcinomas (Ellsworth, 2004). Thus, an evaluation of the growth-regulating properties of Tsg101 mutant mammalian cells may require the use of more complex culture systems that incorporate both wild-type and mutant cells, possibly derived from different cell types (Moberg, 2005).


REGULATION

Protein Interactions

Proteins that constitute the endosomal sorting complex required for transport (ESCRT) are necessary for the sorting of proteins into multivesicular bodies (MVBs) and the budding of several enveloped viruses, including HIV-1. The first of these complexes, ESCRT-I, consists of three proteins: Vps28p, Vps37p, and Vps23p or Tsg101 in mammals. A mutation was characterized in the Drosophila homolog of vps28. The dVps28 gene is essential: homozygous mutants die at the transition from the first to second instar. Removal of maternally contributed dVps28 causes early embryonic lethality. In such embryos lacking dVps28, several processes that require the actin cytoskeleton are perturbed, including axial migration of nuclei, formation of transient furrows during cortical divisions in syncytial embryos, and the subsequent cellularization. Defects in actin cytoskeleton organization also become apparent during sperm individualization in dVps28 mutant testis. Because dVps28 mutant cells contained MVBs, these defects are unlikely to be a secondary consequence of disrupted MVB formation and suggest an interaction between the actin cytoskeleton and endosomal membranes in Drosophila embryos earlier than previously appreciated (Sevrioukov, 2005).

In the Drosophila genome, a single gene, CG12770, exhibits significant homology to the yeast and mammalian Vps28 proteins. The cDNA GH04443 is derived from this locus and encodes a predicted protein of 210 amino acids that is 62% and 35% identical to its human (hVPS28) and yeast (ScVPS28) counterparts, respectively. There are no similarities to other protein sequence motifs in the database. An antibody raised against dVps28 recognizes a protein of the expected size that is widely expressed during Drosophila development and also in cultured Drosophila S2 cells. In S2 cells as well as in cells of the eye disc and in isolated spermatocysts, dVps28 protein was uniformly distributed throughout the cytosol with no obvious enrichment in any organelle (Sevrioukov, 2005).

To test whether the homology of Vps28 proteins extends to their biochemical activity, its binding to Vps23p/Tsg101 (Babst, 2000; Bishop, 2001) was examined. The Drosophila homolog dTsg101 is encoded by cDNA GH09529. Because antibodies are not yet available against endogenous dTsg101 protein, epitope-tagged versions of dTsg101 and dVps28 were coexpressed to test their interaction in S2 cells. Immunoprecipitation of dVps28 from S2 cells resulted in the coprecipitation of expressed HA-dTsg101, which was increased after coexpression of Myc-dVps28. These results indicated that, like its yeast and mammalian orthologs, dVps28 binds specifically to dTsg101 (Sevrioukov, 2005).

Consistent with ESCRT's conserved function from yeast to mammalian cells, loss of dVps28 function causes morphological changes in MVBs and developmental defects in the compound eye that indicate a subtle misregulation of cell signaling molecules. However, for the ligands and receptors, no significant changes were detected in their cell surface levels or their delivery to lysosomes in dVps28 cells, indicating that any changes were too subtle to be detected by the immunofluorescence methods used (Sevrioukov, 2005).

One possible explanation for this observation is that some Vps28 functions are partially fulfilled by another protein. It is not likely that this hypothetical protein is similar to Vps28p in sequence because no another Vps28-like molecule in the completed genome sequences of Drosophila melanogaster. Another possibility is that the dVps28l(2)k16503 allele does not completely inactivate dVps28 function. Although this possibility cannot be ruled out, it is unlikely for two reasons: (1) the dVps28l(2)k16503 allele is a strong mutation that causes lethality early in development at the transition from first to second instar, much earlier than a null mutation in the hrs gene, which regulates the sorting of receptors into MVBs and (2) the lethal phase and the loss of dVps28 protein were indistinguishable between larvae homozygous for dVps28l(2)k16503 and those that were hemizygous over a deficiency of the region. This indicates that the dVps28l(2)k16503 allele removes most if not all of dVps28 function (Sevrioukov, 2005).

Another possibility for the relatively mild phenotypes is the perdurance of dVps28 protein. This is suggested by the dramatic phenotypes after removal of the maternal contribution in mKO dVps28 embryos: many remained unfertilized and the remaining embryos were arrested in their development before reaching the cellular blastoderm. It is interesting to compare this phenotype to that of embryos lacking any maternal Hrs contribution. Hrs binds to mono-ubiquitinated membrane proteins, whose sorting into MVBs is thought to be mediated by Hrs binding to ESCRT-1. Consistent with this notion, mKO hrs embryos exhibit a reduced down-regulation of cell surface receptors and a resulting enhanced activation of the MAP kinase pathway. Importantly, these defects were observed after cellularization had been completed and during gastrulation. The finding that mKO dVps28 embryos exhibit major defects before completion of cellularization indicates functions of dVps28 in addition to the down-regulation of membrane proteins mediated by the interaction of ESCRT-I with Hrs (Sevrioukov, 2005).

Surprisingly, the earliest defect found in fertilized eggs was an uneven distribution of nuclei. In Drosophila, the first 13 nuclear divisions occur without accompanying cell divisions. The first five of these syncytial divisions occur close to the center of the embryo, with nuclei subsequently spreading out along the anterior-posterior axis. This process, referred to as axial expansion, depends on the function of the actin cytoskeleton. In mKO dVps28 embryos, this process often is disorganized resulting in an uneven distribution of nuclei (Sevrioukov, 2005).

Other dVps28 phenotypes emerged during cellularization. After axial expansion, the nuclei move to the embryo's cortex where the last four of the syncytial nuclear divisions occur. The resulting nuclei are surrounded by invaginating membranes in a process that requires the actin-myosin network. At this stage, two defects were evident in embryos lacking dVps28. Many of the cells that formed were droplet shaped instead of the usual cuboidal shape. The actin cytoskeleton is required for normal cellularization and interfering with is function, by cytochalasin, or altering the activity of the Rho or Cdc42 GTPases, interferes with normal cellularization. Furthermore pole cells, usually the first cells to form, were absent in most embryos. The lack of pole cells has previously been observed in embryos in which axial expansion is perturbed upon interference with the actin cytoskeleton (Sevrioukov, 2005).

All of these early phenotypes are consistent with a role of dVps28 in directly or indirectly organizing the actin cytoskeleton. A role for endosomes in actin remodeling during cellularization has previously been established in embryos mutant for nuf or rab11. The small GTPase Rab11 localizes to recycling endosomes, and Nuf is a homolog of Arfo2 that directly binds Rab11 and acts in recycling endosomes. Mutants eliminating either of these proteins cause gaps in which actin fails to be recruited to the furrows during cortical nuclear divisions, similar to the observations in mKO dVps28 embryos. In all three of these mutants, the actin defects may be a consequence of the failure to recruit Discontinuous actin hexagon (Dah: a membrane-associated protein that localizes to invaginating furrows in syncytial blastoderm embryos and during cellularization) to invaginating furrows. Dah has significant similarity to dystrobrevin and dystrophin. Dystrophin plays a critical role in anchoring the actin cytoskeleton to membranes, and consistent with a similar function for Dah, embryos lacking maternally contributed Dah fail to properly assemble the actin cytoskeleton at furrows (Sevrioukov, 2005 and references therein).

A subset of the defects in rab11 mutant embryos has been linked to a requirement for trafficking through the recycling endosome during cellularization. Consistent with this notion, defects in rab11 and nuf embryos only become apparent during cortical divisions. In mKO dVps28 embryos, by contrast, defects were detected in the distribution of nuclei, long before cellularization initiates. This indicates a function of dVps28 in actin remodeling independent of recycling to the cell surface and independent of Rab11 and Nuf. This is consistent with the finding that in mKO dVps28 embryos, recycling endosomes are not affected, as judged by Rab11 and Nuf localization, suggesting that dVps28 is required for furrow localization of Dah down-stream or in parallel to Rab11 and Nuf's function in recycling endosomes. Such a model is difficult to reconcile with the canonical function of Vps28 as an ESCRT-1 subunit involved in targeting proteins into the interior vesicles of late endosomes (Sevrioukov, 2005 and references therein).

It is unlikely that Dah mediates all effects of dVps28 on the actin cytoskeleton. No defects are observed in embryos lacking Dah before cycle 10, long after defects have become apparent in mKO dVps28 embryos. Furthermore, Dah is not required during spermatogenesis, another developmental context in which effects of dVps28 on the organization of the actin cytoskeleton were observed. Spermatogenesis in dVps28 mutant testis progresses until bundles of 64 syncytial spermatids are formed. These spermatids are separated by a process called individualization that requires complex membrane rearrangements. At the site of these rearrangements, syncytial membrane, cytoplasm, and vesicles accumulate in the cystic bulge. In wild-type testis, an actin-dependent process drives the cystic bulge away from the 64 spermatid nuclei toward the distal end. The loss of synchrony in the movement of the cystic bulge is the first detectable defect during spermatogenesis in dVps28 mutant testis. Because dah mutants have no phenotype in males, these results indicate an independent connection between dVps28 function and the actin cytoskeleton (Sevrioukov, 2005 and references therein).

Actin acts at many stages in the endocytic pathway. In yeast, the initial internalization step requires actin polymerization, and in mammalian cells, early endosomes move on the tip of actin tails. Additionally, late endocytic organelles require the actin cytoskeleton for fusion in yeast and mammalian cells. Furthermore, a screen of the yeast genome for mutations interfering with protein sorting to the vacuole identified several regulators of the actin cytoskeleton (Sevrioukov, 2005 and references therein).

Importantly, several of these mutants identified on the basis of a vacuolar sorting phenotype also exhibited defects in the organization of the actin cytoskeleton. For example, aberrant actin patches and a reduction of actin cables were observed in yeast lacking Vps36p, one of the subunits of the ESCRT-II complex. It will be interesting to see whether a similar functional connection between the actin cytoskeleton and the ESCRT-I complex may underlie the enigmatic phenotypes of Tsg101 mutations in mice that cause cell cycle arrest and early embryonic lethality (Sevrioukov, 2005 and references therein).


DEVELOPMENTAL BIOLOGY / EFFECTS OF MUTATION

The eyelessFLP/FRT system has been used to screen the Drosophila genome for genes that restrict tissue growth during eye development. Clones of eye cells homozygous for randomly induced mutations appear 'white' due to the absence of the mini-white+ (m-w+) gene, and wild-type 'twin spots', which carry two copies of m-w+, appear 'red'. Mutations that increased the relative representation of mutant over wild-type tissue were retained, as were those that increased the overall size of the eye. Several mutants had obviously enlarged eyes composed largely of wild-type ('red') tissue, suggesting that the mutations act non-cell-autonomously to deregulate organ growth. One such gene was named erupted (ept) (Moberg, 2005).

Most ept homozygotes die at or prior to the first larval instar, although rare corpses of mutant L2 larvae are sometimes observed. Eyes and heads of adult flies mosaic for the ept2 allele are dramatically enlarged and misshapen when compared to those of the wild-type flies. Significantly, these eyes are largely 'red' indicating that they are composed of mostly wild-type cells. The eye-antennal discs of ept2 mosaic third instar larvae are also enlarged, and they are composed of mostly wild-type cells. Thus, ept mutant cells appear compromised in their ability to contribute to adult eye tissue, but they seem to be able to stimulate the overgrowth of adjacent wild-type cells during larval stages of eye development (Moberg, 2005).

Adult retinal sections show that ept mosaic eyes have a disorganized cellular architecture, and they confirm that most cells are genetically wild-type, as indicated by the presence of pigment granules. Many ommatidia have missing or extra photoreceptors, and there is evidence of fusion events between adjacent clusters. ept mutant clones in the adult eye are small and have few recognizable cells. Rare ept mutant cells appear as 'bloated' cell bodies with lightly staining rhabdomeres. Thus, the Drosophila ept locus has two functions: a cell-autonomous role in cell viability and morphology, and a nonautonomous role in restricting eye size (Moberg, 2005).

The erupted gene was localized by complementation to the ~18 kb region of overlap of two deficiencies, Df(3L)Exel9002 and Df(3L)Exel9004. This region contains two predicted genes: CG9712, the Drosophila ortholog of the mammalian gene Tsg101, and CG9669, a small gene of unknown function. Mutations were not detected in the coding region of either gene in ept2, and no large-scale chromosomal rearrangements of the region were found. However, Southern blot analysis with a Tsg101-specific probe detects an ~8 kb increase in the size of restriction fragments derived from the 5' portion of the Tsg101 locus. PCR and polymorphism analysis indicate that sequences within the 5' end are present in ept2. However, attempts to amplify the entire 5' region from ept2 DNA were unsuccessful, despite the fact that this region is readily amplified from FRT80B DNA. These data indicate that ept2 contains a lesion that disrupts the continuity of sequences within the 5' portion of Tsg101. Since the probe used in Southern analysis flanks this apparent breakpoint, and detects a single larger fragment in both BamH1 and Nhe1 digests, it seems likely that the ept2 allele contains an ~8 kb insertion within the Tsg101 genomic locus. To confirm the identity of ept, a new lethal ept allele, eptP26, was generated by mobilization of a viable P element insertion located ~30 base pairs upstream of the Tsg101 transcriptional start site. eptP26 fails to complement the lethality of ept2, and it displays cellular and organismal phenotypes in the developing eye that are very similar to ept2. The strength of these phenotypes suggests that the eptP26 allele is weaker than ept2. Analysis of eptP26 revealed a duplication of the P element inserted into codon 163 (proline) of Tsg101 exon 5. To further confirm that ept alleles inactivate Tsg101, a full-length Tsg101 cDNA under the control of the heat-inducible hsp70 promoter (hs-Tsg101) was used to rescue the lethality of ept2/eptP26 trans-heterozygotes to viability. By these genetic data, it is concluded that ept mutations disrupt the function of the Tsg101 gene, and that Tsg101 is required for normal eye development. Consistent with this, RNA in situ analysis reveals that Tsg101 is expressed at low levels in the developing eye and antennal discs (Moberg, 2005).

Inactivation of mammalian Tsg101 and yeast Vps23p blocks the transit of certain cell surface receptors through the MVB pathway, leading to accumulation of ubiquitinated receptors in endosomes (Babst, 2000; Li, 1999). When eye discs carrying clones of ept mutant cells were stained with an anti-ubiquitin (Ub) antibody, little staining was observed in wild-type portions of the discs, but strong staining is observed in mutant cells and appears as bright 'puncta', suggesting that Ub accumulates in a specific intracellular compartment. This phenotype is consistent with a role for Tsg101 in the routing of cell surface proteins into the MVB pathway in Drosophila cells similar to that previously shown for mammalian Tsg101 and yeast Vps23p (Moberg, 2005).

Defects in the trafficking of membrane-associated proteins can affect many aspects of cell biology, including cell shape and polarity. Therefore the localization of two cell polarity markers was tested in ept mutant cells: Crumbs (Crb), a marker of the zonula adherens (ZA), and Discs large (Dlg), a protein that localizes to septate junctions and the basolateral membrane domain. Crb localization is significantly altered in ept mutant eye disc cells. An optical section through the middle of the disc epithelium reveals that Crb protein accumulates in a subapical domain in ept cells. A lateral section confirms that Crb in ept cells is not localized to the apical surface, as it is in adjacent wild-type cells. In contrast, ept cells display the normal localization of Dlg. The Dlg-positive lateral profile of ept cells indicates that they are more rounded than adjacent wild-type cells. Costaining for both Dlg and Crb in ept clones confirms these observations, and it reveals that, while some Crb protein localizes properly to the apical domain in ept cells, a significant amount of Crb is detected in nonnuclear, subapical aggregates, which show limited overlap with Ub-positive epitopes. Thus, loss of Tsg101 function in epithelial cells appears to cell-autonomously compromise cell shape coincident with defects in the compartmentalization of the cell polarity protein Crb (Moberg, 2005).

The Notch receptor has two properties that implicate it in a pathway by which ept mutations non-cell-autonomously promote tissue growth. (1) The restricted activation of Notch in cells along the dorsoventral (D/V) boundary of the eye imaginal disc is required for growth of the entire eye. (2) Ub-dependent endocytosis plays an important role in regulating Notch activity in vivo. In mammalian cells, ubiquitination and endocytosis contributes to Notch1 activation, and, in Drosophila, there is evidence to suggest that the ubiquitin ligase Deltex may be required for endocytosis-dependent Notch activation. Further, alleles of the endosomal sorting gene Hrs, the homolog of yeast Vps27, affect Notch localization in imaginal disc cells, indicating that Notch is a physiological target of the MVB pathway (Moberg, 2005).

In light of these observations, ept mosaic eye discs were stained with an antibody specific to the Notch cytoplasmic domain (anti-Ncyto). Notch protein is detected in wild-type eye discs most prominently in a stripe of cells within the morphogenetic furrow (MF) and is concentrated at the apical cell surface. In contrast, ept cells contain elevated levels of Notch. This increase occurs in ept clones throughout the eye disc, but it is most apparent in clones that lie within or posterior to the MF. Moreover, the Notch in ept cells accumulates in nonnuclear, intracellular puncta that also stain positive for Ub, and for the endosomal protein Hrs. Together, these data indicate that ept mutations block the routing of ubiquitinated cell surface proteins, among them Notch, in an Hrs-positive endosomal compartment (Moberg, 2005).

Notch is normally processed in cells by a series of cleavage events required for receptor maturation and presentation at the cell surface, and for ligand-stimulated activation of the Notch pathway. Because ubiquitination and endocytosis have been shown to affect Notch cleavage, attempts were made to determine if ept mutations also affect Notch processing. Eye-antennal discs composed of ept mutant cells [ept/M(3)] or FRT80B control cells (FRT80B/M(3)) were generated by the eyFLP/Minute technique (Moberg, 2004). Immunoblot of tissue extracts with the anti-Ncyto antibody confirms that Notch levels are increased considerably in eye-antennal discs composed of ept mutant cells, and shows that ept mutant cells are enriched in a ~120 kDa form of Notch. The molecular identity of this fragment has not been determined, but its size appears similar to certain processed forms of Notch. Indeed, while no one form of Notch predominates in wild-type cells, this species appears to be the most abundant Notch species in ept cells (Moberg, 2005).

To examine Notch activation, clones of ept mutant cells were generated in the presence of the Notch-inducible transgene E(spl)mβ-CD2, a Suppressor of Hairless (Su(H))-dependent transcriptional reporter that has been used to detect equatorial Notch activation in the developing eye. Posterior to the MF, CD2 expression is detected in the interommatidial cells, and outlines a single cell from each photoreceptor cluster in a mirror-image pattern along the equator. Thus, in addition to equatorial activation, the reporter detects Notch activation in postmitotic interommatidial cells, and in the R3-R4 cell fate choice. In ept mutant clones, reporter activity is strongly elevated. The degree of activation exceeds that observed in wild-type eye discs, and it does not appear to depend upon the location of ept cells within the disc, occurring on either side of the MF and in the antennal disc. Some ept cells within a single optical section appear not to activate the Notch reporter. However, in most of these cases, CD2, which localizes to cell membranes, can be detected in a focal plane slightly offset from that of the nuclear green fluorescent protein (GFP). Thus, these data show that defects in Notch regulation in ept cells are accompanied by ectopic and excessive activation of the Notch pathway (Moberg, 2005).

The requirement for Notch in eye disc growth has been linked to its ability to induce expression of the eyegone (eyg) gene at the D/V boundary of the eye disc. eyg encodes a Pax6-like transcription factor (Eyg) required for disc growth, and, like Notch, ectopic expression of eyg is able to induce growth nonautonomously. Consistent with its effect on Notch, it was found that ept mutant cells express elevated levels of Eyg compared to surrounding wild-type cells. Thus, Eyg may function downstream of Notch within ept cells to promote the growth of surrounding cells in a manner similar to its normal growth-promoting role at the D/V boundary (Moberg, 2005).

Recent work suggests that the unpaired (upd) gene may be an important growth regulatory target of Notch. upd encodes the secreted ligand (Upd) of the Domeless (Dome) receptor, which signals through the JAK-STAT pathway. JAK-STAT signaling is implicated in many processes during Drosophila development, including the control of cell proliferation, cell motility, stem cell renewal, and planar cell polarity. upd is required for normal growth of the eye, and ectopic expression of upd in the larval eye nonautonomously promotes cell proliferation and produces enlarged and misshapen eyes (Bach, 2003) similar to those observed in ept mosaics. Significantly, Notch is both necessary and sufficient to activate upd transcription (Chao, 2004) along the posterior margin of the eye disc (Moberg, 2005).

When ept mosaic eye discs were stained with an anti-Upd antiserum, a dramatic increase was observed in the level of Upd protein in ept mutant cells compared to adjacent wild type cells. Consistent with a transcriptional link between Notch and upd, Upd protein accumulation appears coincident with expression of the Notch reporter, and ept mosaic eye-antennal discs contain clones of cells expressing very high levels of upd mRNA. Together, these observations suggest that Notch, perhaps acting via Eyg, promotes ectopic upd expression in ept mutant cells (Moberg, 2005).

Clonal overexpression of upd induces localized tissue outgrowths and deregulates the division of surrounding cells (Chao, 2004; Tsai, 2004). This mitogenic activity is linked to induction of cyclin D (Tsai, 2004), and to accelerated progression through the G1 phase of the cell cycle (Bach, 2003). ept mutant clones can produce phenotypes quite similar to clonal overexpression of upd. In one example of an ept clone, lower half of the disc appeared morphologically normal, while the other half, despite being composed largely of wild-type cells, was misshapen and enlarged. This localized effect correlated with proximity to a large ept mutant clone expressing Upd. Similar hyperplastic growth was associated with clones of upd-expressing cells in the antennal disc. The patterns of BrdU incorporation in ept mosaic eye discs are disorganized, and the number of BrdU-labeled nuclei increases in proximity to Upd-expressing ept mutant cells in the eye and antenna. This aberrant cell proliferation occurs in GFP-positive wild-type cells. Hence, the growth-promoting activity of ept mutations is likely mediated by a diffusible extracellular signal like Upd (Moberg, 2005).

Receipt of the Upd signal via Domeless initiates a signaling cascade that activates a transcription factor encoded by the stat92E gene. stat92E encodes the Drosophila ortholog of the mammalian signal transducers and activators of transcription (STAT) family of transcriptional regulators, which function in diverse processes such as immunity and oncogenesis, and is the only member of this gene family in Drosophila. Heterozygosity for a stat92E loss-of-function allele (stat92E06346) strongly suppresses the nonautonomous eye overgrowth associated with mosaicism for ept mutations, such that ept-mosaic;stat92E06346/+ eyes are comparable in size to control FRT80B mosaic eyes. Thus, nonautonomous overgrowth elicited by ept mutations is sensitive to the genetic dosage of the Upd-responsive transcription factor stat92E. In light of the effect on Upd, these data strongly indicate that the growth-promoting activity of ept mutant cells requires Upd-dependent activation of the JAK-STAT pathway in adjacent tissue (Moberg, 2005).

ept mutant clones in mosaic eye discs are small and survive poorly into adulthood. It is possible that this is the result of cell competition, a process by which slow-growing cells in the vicinity of wild-type cells are eliminated. If so, then the poor survival of ept cells might be rescued by eliminating competing cells. Therefore the growth characteristics were examined of ept/M(3) discs, which are composed almost entirely of cells lacking Tsg101 function. ept/M(3) animals reach the larval 'wandering' stage 4 days later than control larvae, and, when they do, they are enlarged. A small fraction of these animals pupate and die before becoming pharate adults. The remainder die as giant larvae containing high levels of Upd (Moberg, 2005).

Allowing ept mutant cells to grow in epithelia lacking wild-type cells also uncovers a context-dependent cell-autonomous overgrowth phenotype. Rather than surviving poorly as they do in mosaic discs, ept/M(3) eye discs overgrow into large masses that lack normal disc morphology. These masses are composed of folded and convoluted sheets of cells fused together, and they often include a distended sac-like structure. The overgrowth phenotypes of ept/M(3) animals and discs do not reflect an increased rate of growth: control L3 larvae are the same size as ept/M(3) larvae of the same temporal age, and the ept/M(3) eye discs, while mispatterned, are not obviously increased in size. Thus, the ept/M(3) masses are the result of an extended larval phase, and a failure of the disc to stop growing when it reaches the appropriate size. Thus, cells lacking Tsg101 may be unable to respond to signals that normally sense and restrict organ size (Moberg, 2005).


EVOLUTIONARY HOMOLOGS

Initial identification and characterization of TSG101

Using a novel strategy that enables the isolation of previously unknown genes encoding selectable recessive phenotypes, a gene (tsg101) was identified whose homozygous functional disruption produces cell transformation. Antisense RNA from a transactivated promoter introduced randomly into transcribed genes throughout the genome of mouse 3T3 fibroblasts was used to knock out alleles of chromosomal genes adjacent to promoter inserts, generating clones that grew in 0.5% agar and formed metastatic tumors in nude mice. Removal of the transactivator restored normal growth. The protein encoded by tsg101 cDNA encodes a coiled-coil domain that interacts with stathmin, a cytosolic phosphoprotein implicated previously in tumorigenesis. Overexpression of tsg101 antisense transcripts in naive 3T3 cells results in cell transformation and increased stathmin-specific mRNA (Li, 1996).

TSG101 homolog functions yeast

Mutations gef1, stp22, STP26, and STP27 in Saccharomyces cerevisiae were identified as suppressors of the temperature-sensitive alpha-factor receptor (mutation ste2-3) and arginine permease (mutation can1ts). These suppressors inhibit the elimination of misfolded receptors (synthesized at 34 degrees C) as well as damaged surface receptors (shifted from 22 to 34 degrees C). The stp22 mutation (allelic to vps23 and the STP26 mutation also caused missorting of carboxypeptidase Y, and ste2-3 was suppressed by mutations vps1, vps8, vps10, and vps28 but not by mutation vps3. In the stp22 mutant, both the mutant and the wild-type receptors (tagged with GFP) accumulated within an endosome-like compartment and were excluded from the vacuole. GFP-tagged Stp22p also accumulated in this compartment. Upon reaching the vacuole, cytoplasmic domains of both mutant and wild-type receptors appeared within the vacuolar lumen. Stp22p and Gef1p are similar to tumor susceptibility protein TSG101 and voltage-gated chloride channel, respectively. These results identify potential elements of plasma membrane quality control and indicate that cytoplasmic domains of membrane proteins are translocated into the vacuolar lumen (Li, 1999).

TSG101 functions in late endosomal trafficking

The mammalian tumor susceptibility gene tsg101 encodes the homologue of Vps23p, a class E Vps protein essential for normal membrane trafficking in the late endosome/multivesicular body of yeast. Both proteins assemble into large (approximately 350 kDa) cytosolic protein complexes; the yeast complex contains another class E Vps protein, Vps28p. tsg101 mutant cells exhibit defects in sorting and proteolytic maturation of the lysosomal hydrolase cathepsin D, as well as in the steady-state distribution of the mannose-6-phosphate receptor. Additionally, endocytosed EGF receptors that are normally sorted to the lysosome are instead rapidly recycled back to the cell surface in tsg101 mutant cells. It is proposed that tsg101 mutant cells are defective in the delivery of cargo proteins to late endosomal compartments. One consequence of this endosomal trafficking defect is the delayed down-regulation/degradation of activated cell surface receptors, resulting in prolonged signaling. This may contribute to the tumorigenic phenotype exhibited by the tsg101 mutant fibroblasts (Babst, 2000).

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. The mammalian orthologue of Vps28p has been identified 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 have 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 translocates to the surface of these vacuoles. It is concluded that TSG101 and its interacting components are directly involved in endosomal sorting (Bishop, 2001).

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. TSG101 interacts with HRS, an early endosomal protein, and 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 prevents 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. These 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 (Lu, 2003).

Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway. The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins. Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway. Activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27. ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27. A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23. It is proposed that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin. Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes (Katzmann, 2003).

ALG-2 (apoptosis-linked gene 2) is a Ca2+-binding protein that belongs to the PEF (penta-EF-hand) protein family. Alix (ALG-2-interacting protein X)/AIP1 (ALG-2-interacting protein 1), one of its binding partners, interacts with TSG101 and CHMP4 (charged multivesicular body protein 4), which are components of ESCRT-I (endosomal sorting complex required for transport I) and ESCRT-III respectively. In the present study, the association between ALG-2 and ESCRT-I was investigated. By a GST (glutathione S-transferase) pull-down assay using HEK-293T (human embryonic kidney 293T) cell lysates, endogenous TSG101 and two other exogenously expressed ESCRT-I components [hVps28 (human vacuolar protein sorting 28) and hVps37A] associate with GST-ALG-2 in the presence of Ca2+. By the yeast two-hybrid assay, however, a positive interaction was observed with only TSG101 among the three ESCRT-I components, suggesting that ALG-2 associates with hVps28 and hVps37A indirectly through TSG101. Using various deletion mutants of TSG101, the central PRR (proline-rich region) was found to be sufficient for interaction with ALG-2 by the GST-pull-down assay. Direct binding of ALG-2 to the TSG101 PRR was demonstrated by an overlay assay using biotin-labelled ALG-2 as a probe. In immunofluorescence microscopic analysis of HeLa cells that overexpress a GFP (green fluorescent protein)-fused ATPase-defective dominant-negative form of SKD1/Vps4B (GFP-SKD1(E235Q)), ALG-2 exhibits a punctate distribution at the perinuclear area and co-localizes with GFP-SKD1(E235Q) to aberrant endosomes. This punctate distribution of ALG-2 is markedly diminished by treatment of HeLa cells with a membrane-permeant Ca2+ chelator. Moreover, a Ca2+-binding-defective mutant of ALG-2 does not co-localize with GFP-SKD1(E235Q). These findings suggest that ALG-2 may function as a Ca2+-dependent accessory protein of the endosomal sorting machinery by interacting directly with TSG101 as well as with Alix (Katoh, 2005).

Effect of Tsg101 mutation

Functional inactivation of the tumor susceptibility gene tsg101 in NIH 3T3 fibroblasts results in cellular transformation and the ability to form metastatic tumors in nude mice. The N-terminal region of tsg101 protein is structurally similar to the catalytic domain of ubiquitin-conjugating enzymes, suggesting a potential role of tsg101 in ubiquitin-mediated protein degradation. The C-terminal domain of TSG101 can function as a repressor of transcription. To investigate the physiological function of tsg101, a null mutation of the mouse gene was generated by gene targeting. Homozygous tsg101-/- embryos fail to develop past day 6.5 of embryogenesis (E6.5), are reduced in size, and do not form mesoderm. Mutant embryos show a decrease in cellular proliferation in vivo and in vitro but no increase in apoptosis. Although levels of p53 transcripts are not affected in tsg101-/- embryos, p53 protein accumulates dramatically, implying altered posttranscriptional control of p53. In addition, transcription of the p53 effector, cyclin-dependent kinase inhibitor p21(WAF-1/CIP-1), was increased 5- to 10-fold, whereas activation of MDM2 transcription secondary to p53 elevation was not observed. Introduction of a p53 null mutation into tsg101-/- embryos rescues the gastrulation defect and prolongs survival until E8.5. These results demonstrate that tsg101 is essential for the proliferative burst before the onset of gastrulation and establish a functional connection between tsg101 and the p53 pathway in vivo (Ruland, 2001).

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, primary mouse embryonic fibroblasts were derived from Tsg101 conditional knockout mice. Expression of Cre recombinase from a retroviral vector efficiently down-regulates Tsg101. The deletion of Tsg101 causes growth arrest and cell death but does not result in increased proliferation and cellular transformation. Inactivation of p53 has no influence on the deleterious phenotype, but Tsg101-/- cells are 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 results in growth arrest at the G(1)/S transition through inactivation of cyclin-dependent kinase 2. As a consequence, DNA replication is not initiated in Tsg101-deficient cells. These 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 (Krempler, 2002).

Tumor susceptibility gene 101 (Tsg101) was identified in a random mutagenesis screen for potential tumor suppressors in NIH 3T3 cells. Altered transcripts of this gene have been detected in sporadic breast cancers and many other human malignancies. However, the involvement of this gene in neoplastic transformation and tumorigenesis is still elusive. Using gene targeting, genetically engineered mice were generated with a floxed allele of Tsg101. Essential functions of this gene were investigated in vivo and whether the loss of function of Tsg101 results in tumorigenesis was examined. Conventional knockout mice were generated through Cre-mediated excision of the first coding exon in the germ line of mouse mammary tumor virus (MMTV)-Cre transgenic mice. The complete ablation of Tsg101 in the developing embryo resulted in death around implantation. In contrast, mammary gland-specific knockout mice developed normally but were unable to nurse their young as a result of impaired mammogenesis during late pregnancy. Neither heterozygous null mutants nor somatic knockout mice developed mammary tumors after a latency of 2 years. The Cre-mediated deletion of Tsg101 in primary cells demonstrates that this gene is essential for the growth, proliferation, and survival of mammary epithelial cells. In summary, these results suggest that Tsg101 is required for normal cell function of embryonic and adult tissues but that this gene is not a tumor suppressor for sporadic forms of breast cancer (Wagner, 2003).

Cells conditionally deficient in Tsg101 arrest at the G(1)/S cell cycle checkpoint and die. A series of Tsg101 conditional knock-out cell lines were created that lack p53, p21(Cip1), or p19(Arf) to determine the involvement of the Mdm2-p53 circuit as a regulator for G(1)/S progression and cell death. 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 results in G(1)/S progression, suggesting that the p53-dependent G(1) arrest in the Tsg101 knock-out is mediated by p21(Cip1). The Cre-mediated excision of Tsg101 in immortalized fibroblasts that lack p19(Arf) seem not to alter the ability of Mdm2 to sequester p53, and the p21-mediated G(1) arrest is not restored. Based on these findings, it is proposed 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. Finally, the deletion of Tsg101 from primary tumor cells that express mutant p53 and that lack p21(Cip1) expression results in cell death, suggesting that additional transforming mutations during tumorigenesis do not affect the important role of Tsg101 for cell survival (Carstens, 2004).

The early endosome comprises morphologically distinct regions specialised in sorting cargo receptors. A central question is whether receptors move through a predetermined structural pathway, or whether cargo selection contributes to the generation of endosome morphology and membrane flux. Here, depletion of tumour susceptibility gene 101 impairs the selection of epidermal growth factor receptor away from recycling receptors within the limiting membrane of the early endosome. Consequently, epidermal growth factor receptor sorting to internal vesicles of the multivesicular body and cargo recycling to the cell surface or Golgi complex are inhibited. These defects are accompanied by disruption of bulk flow transport to the lysosome and profound structural rearrangement of the early endosome. The pattern of tubular and vacuolar domains is replaced by enlarged vacuoles, many of which are folded into multicisternal structures resembling the 'Class E' compartments that define several Saccharomyces cerevisiae vacuolar protein sorting mutants. The cisternae are interleaved by a fine matrix but lack other surface elaborations, most notably clathrin (Doyotte, 2005).

TSG101 recognizes ubiquitin and acts in the removal of endosomal protein-ubiquitin conjugates

There is increasing evidence that ubiquitination of receptors provides an important endosomal sorting signal. Mammalian class E vacuolar protein-sorting (vps) proteins recognize ubiquitin. Both tumor susceptibility gene 101 (TSG101)/human VPS (hVPS)28 and hepatocyte growth factor receptor substrate (Hrs) cytosolic complexes bind ubiquitin-agarose. TSG101 and hVPS28 are localized to endosomes that contain internalized EGF receptor and label strongly for ubiquitinated proteins. Microinjection of anti-hVPS28 specifically retards EGF degradation and leads to endosomal accumulation of ubiquitin-protein conjugates. Likewise, depletion of TSG101 impairs EGF trafficking and causes dramatic relocalization of ubiquitin to endocytic compartments. Similar defects are found in cells overexpressing Hrs, further emphasizing the links between class E protein function, receptor trafficking, and endosomal ubiquitination (Bishop, 2002).

The UEV domain of the TSG101 protein functions in both HIV-1 budding and the vacuolar protein sorting (VPS) pathway, where it binds ubiquitylated proteins as they are sorted into vesicles that bud into late endosomal compartments called multivesicular bodies (MVBs). TSG101 UEV-ubiquitin interactions are therefore important for delivery of both substrates and hydrolytic enzymes to lysosomes, which receive proteins via fusion with MVBs. This study reports the crystal structure of the TSG101 UEV domain in complex with ubiquitin at 2.0 Å resolution. TSG101 UEV contacts the Ile44 surface and an adjacent loop of ubiquitin through a highly solvated interface. Mutations that disrupt the interface inhibit MVB sorting, and the structure also explains how the TSG101 UEV can independently bind its ubiquitin and Pro-Thr/Ser-Ala-Pro peptide ligands. Remarkably, comparison with mapping data from other UEV and related E2 proteins indicates that although the different E2/UEV domains share the same structure and have conserved ubiquitin binding activity, they bind through very different interfaces (Sundquist, 2004).

Tal is a Tsg101-specific E3 ubiquitin ligase

The tumor suppressor gene 101 (tsg101) regulates vesicular trafficking processes in yeast and mammals. A novel protein, Tal (Tsg101-associated ligase), is described whose RING finger is necessary for multiple monoubiquitylation of Tsg101. Bivalent binding of Tsg101 to a tandem tetrapeptide motif (PTAP) and to a central region of Tal is essential for Tal-mediated ubiquitylation of Tsg101. By studying endocytosis of the epidermal growth factor receptor and egress of the human immunodeficiency virus, it is concluded that Tal regulates a Tsg101-associated complex responsible for the sorting of cargo into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane (Amit, 2004).

Stabilization of the unliganded glucocorticoid receptor by TSG101

The glucocorticoid receptor (GR) has been shown to undergo hormone-dependent down-regulation via transcriptional, post-transcriptional, and posttranslational mechanisms. However, the mechanisms involved in modulating GR levels in the absence of hormone remain enigmatic. This study demonstrates that TSG101, a previously identified GR-interacting protein, stabilizes the hypophosphorylated form of GR in the absence of ligand. A non-phosphorylated version of GR (S203A/S211A) shows enhanced interaction with TSG101 as compared with the wild type GR, suggesting that TSG101 interacts more favorably with GR when it is not phosphorylated. A significant accumulation of GR S203A/S211A protein is detected in the absence of ligand when TSG101 is overexpressed, whereas no increase in the wild type phosphorylated GR or phosphomimetic GR S203E/S211E was observed in mammalian cells. In contrast, down-regulation of TSG101 expression by siRNA renders the hypophosphorylated form of GR unstable. TSG101 stabilizes GR by impeding its degradation by the proteasome and extending receptor half-life. Thus, in absence of a ligand, TSG101 binds GR and protects the non-phosphorylated receptor from degradation (Ismaili, 2005).

Tsg101 is essential for viral budding

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies (Garrus, 2001).

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the 'late domain') binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. This study reports that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding (Pornillos, 2003).

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 interferes 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, the effect on RSV Gag release of co-expression was tested 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 interferes with HIV-1 budding, also inhibits RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacts in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacts with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 does not prevent interaction; however, three-dimensional spatial imaging suggests that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 is not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interferes with VLP release minimally; however, a significant fraction of the released VLPs is tethered to Tsg101. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. It is concluded that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101-Nedd4 interaction (Medina, 2005).

Retrovirus budding is greatly stimulated by the presence of Gag sequences known as late or L domains. The L domain of human immunodeficiency virus type 1 (HIV-1) maps to a highly conserved Pro-Thr-Ala-Pro (PTAP) sequence in the p6 domain of Gag. The p6 PTAP motif interacts with the cellular endosomal sorting protein TSG101. Consistent with a role for TSG101 in virus release, overexpressing the N-terminal, Gag-binding domain of TSG101 (TSG-5') suppresses HIV-1 budding by blocking L domain function. To elucidate the role of TSG101 in HIV-1 budding, the significance of the binding between Gag and TSG-5' on the inhibition of HIV-1 release was evaluated. A mutation in TSG-5' that disrupts the Gag/TSG101 interaction suppresses the ability of TSG-5' to inhibit HIV-1 release. The effect of overexpressing a panel of truncated TSG101 derivatives and full-length TSG101 (TSG-F) on virus budding was evaluated. Overexpressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding. Furthermore, overexpression of the C-terminal portion of TSG101 (TSG-3') potently inhibits budding of not only HIV-1 but also murine leukemia virus. Confocal microscopy data indicate that TSG-F and TSG-3' overexpression induces an aberrant endosome phenotype; this defect is dependent upon the C-terminal, Vps-28-binding domain of TSG101. It is proposed that TSG-5' suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas overexpressing TSG-F or TSG-3' globally inhibits virus release by disrupting the cellular endosomal sorting machinery. These results highlight the importance of TSG101 and the endosomal sorting pathway in virus budding and suggest that inhibitors can be developed that, like TSG-5', target HIV-1 without disrupting endosomal sorting (Goila-Gaur, 2003).

Efficient human immunodeficiency virus type 1 (HIV-1) budding requires an interaction between the PTAP late domain in the viral p6(Gag) protein and the cellular protein TSG101. In yeast, Vps23p/TSG101 binds both Vps28p and Vps37p to form the soluble ESCRT-I complex, which functions in sorting ubiquitylated protein cargoes into multivesicular bodies. Human cells also contain ESCRT-I, but the VPS37 component(s) have not been identified. Bioinformatics and yeast two-hybrid screening methods were therefore used to identify four novel human proteins (VPS37A-D) that share weak but significant sequence similarity with yeast Vps37p and to demonstrate that VPS37A and VPS37B bind TSG101. Detailed studies produced four lines of evidence that human VPS37B is a Vps37p ortholog. (1) TSG101 binds to several different sites on VPS37B, including a putative coiled-coil region and a PTAP motif. (2) TSG101 and VPS28 co-immunoprecipitate with VPS37B-FLAG, and the three proteins comigrate together in soluble complexes of the correct size for human ESCRT-I (approximately 350 kDa). (3) Like TGS101, VPS37B becomes trapped on aberrant endosomal compartments in the presence of VPS4A proteins lacking ATPase activity. (4) Finally, VPS37B can recruit TSG101/ESCRT-I activity and thereby rescue the budding of both mutant Gag particles and HIV-1 viruses lacking native late domains. Further studies of ESCRT-I revealed that TSG101 mutations that inhibit PTAP or VPS28 binding block HIV-1 budding. Taken together, these experiments define new components of the human ESCRT-I complex and characterize several TSG101 protein/protein interactions required for HIV-1 budding and infectivity (Stuchell, 2004).

Retroviral late domains (L domains) are short amino acid sequences in the Gag protein that facilitate the process of budding. L domains act by recruiting the ESCRT complexes, which normally function in the formation of multivesicular bodies. The PTAP late domain of human immunodeficiency virus (HIV) is believed to specifically recruit this machinery by binding the ESCRT protein TSG101. Expression of a C-terminal fragment of TSG101 (TSG-3') blocks the budding of both PTAP-dependent and PPPY-dependent retroviruses. TSG-3' expression leads to the formation of large spherical entities that are called TICS (TSG-3'-induced cellular structures) in the cytoplasm. Rous sarcoma virus (RSV) and murine leukemia virus (MLV) Gag proteins are selectively recruited to these structures, but HIV type 1 Gag is completely excluded. Experiments with various HIV and RSV vector constructs as well as HIV and RSV chimeras suggest that recruitment to the TICS is late domain independent and does not involve recognition of any single amino acid sequence. TICS appear to have no limiting membrane and do not colocalize with markers for any membranous cellular compartment. Wild-type TSG101 is also recruited to TICS, but most other ESCRT proteins are excluded. These structures are similar in nature to aggresomes, colocalize with the aggresome marker GFP-250, and are highly enriched in ubiquitin but in other ways do not fully meet the description of aggresomes. It is concluded that the block to retroviral budding by TSG-3' may be the result of its sequestration of Gag, depletion of free TSG101, or depletion of free ubiquitin (Johnson, 2005).

The release of Bluetongue virus (BTV) and other members of the Orbivirus genus from infected host cells occurs predominantly by cell lysis, and in some cases, by budding from the plasma membrane. Two nonstructural proteins, NS3 and NS3A, have been implicated in this process. Both proteins bind to human Tsg101 and its ortholog from Drosophila melanogaster with similar strengths in vitro. This interaction is mediated by a conserved PSAP motif in NS3 and appears to play a role in virus release. The depletion of Tsg101 with small interfering RNA inhibits the release of BTV and African horse sickness virus, a related orbivirus, from HeLa cells up to fivefold and threefold, respectively. Like most other viral proteins which recruit Tsg101, NS3 also harbors a PPXY late-domain motif that allows NS3 to bind NEDD4-like ubiquitin ligases in vitro. However, the late-domain motifs in NS3 do not function as effectively in facilitating the release of mini Gag virus-like particles from 293T cells as the late domains from human immunodeficiency virus type 1, human T-cell leukemia virus, and Ebola virus. A mutagenesis study showed that the arginine residue in the PPRY motif is responsible for the low activity of the NS3 late-domain motifs. These data suggest that the BTV late-domain motifs either recruit an antagonist that interferes with budding or fail to recruit an agonist which is different from NEDD4 (Wirblich, 2006).


REFERENCES

Search PubMed for articles about Drosophila tumor suppressor protein 101

Amit, I., et al. (2004). Tal, a Tsg101-specific E3 ubiquitin ligase, regulates receptor endocytosis and retrovirus budding. Genes Dev. 18(14): 1737-52. 15256501

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Biological Overview

date revised: 12 January 2018

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