The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101

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.     

Cargo-dependent degradation of ESCRT-I as a feedback mechanism to modulate endosomal sorting

Ligand-mediated lysosomal degradation of growth factor receptors, mediated by the endosomal sorting complex required for transport (ESCRT) machinery, is a mechanism that attenuates the cellular response to growth factors. In this article, we present a novel regulatory mechanism that involves ligand-mediated degradation of a key component of the sorting machinery itself. We have investigated the endosomal localization of subunits of the four ESCRTs-Hrs (ESCRT-0), Tsg101 (ESCRT-I), EAP30/Vps22 (ESCRT-II) and charged multivesicular body protein 3/Vps24 (ESCRT-III). All the components were detected on the limiting membrane of multivesicular endosomes (MVEs). Surprisingly, however, Tsg101 and other ESCRT-I subunits were also detected within intraluminal vesicles (ILVs) of MVEs. Tsg101 was sequestered along with cargo during endosomal sorting into ILVs and further degraded in lysosomes. Importantly, ESCRT-mediated downregulation of two distinct cargoes, epidermal growth factor receptor (EGFR) and connexin43, mutually made cells refractory to degradation of the other cargo. Our observations indicate that the degradation of a key ESCRT component along with cargo represents a novel feedback control of endosomal sorting by preventing collateral degradation of cell surface receptors following stimulation of one specific pathway.     

Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release

The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55(gag), contains a proline-rich motif (PTAP) called the "late domain" in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of gag-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.     

Targeted deletion of the Tsg101 gene results in cell cycle arrest at G1/S and p53-independent cell death

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.     

ESCRT-I Function is Required for Tyrp1 Transport from Early Endosomes to the Melanosome Limiting Membrane

Melanosomes are lysosome-related organelles that coexist with lysosomes within melanocytes. The pathways by which melanosomal proteins are diverted from endocytic organelles toward melanosomes are incompletely defined. In melanocytes from mouse models of Hermansky-Pudlak syndrome that lack BLOC-1, melanosomal proteins such as tyrosinase-related protein 1 (Tyrp1) accumulate in early endosomes. Whether this accumulation represents an anomalous pathway or an arrested normal intermediate in melanosome protein trafficking is not clear. Here, we show that early endosomes are requisite intermediates in the trafficking of Tyrp1 from the Golgi to late stage melanosomes in normal melanocytic cells. Kinetic analyses show that very little newly synthesized Tyrp1 traverses the cell surface and that internalized Tyrp1 is inefficiently sorted to melanosomes. Nevertheless, nearly all Tyrp1 traverse early endosomes since it becomes trapped within enlarged, modified endosomes upon overexpression of Hrs. Although Tyrp1 localization is not affected by Hrs depletion, depletion of the ESCRT-I component, Tsg101, or inhibition of ESCRT function by dominant-negative approaches results in a dramatic redistribution of Tyrp1 to aberrant endosomal membranes that are largely distinct from those harboring traditional ESCRT-dependent, ubiquitylated cargoes such as MART-1. The lysosomal protein content of some of these membranes and the lack of Tyrp1 recycling to the plasma membrane in Tsg101-depleted cells suggests that ESCRT-I functions downstream of BLOC-1. Our data delineate a novel pathway for Tyrp1 trafficking and illustrate a requirement for ESCRT-I function in controlling protein sorting from vacuolar endosomes to the limiting membrane of a lysosome-related organelle.     

Functional ESCRT machinery is required for constitutive recycling of claudin-1 and maintenance of polarity in vertebrate epithelial cells

Genetic screens in Drosophila have identified regulators of endocytic trafficking as neoplastic tumor suppressor genes. For example, Drosophila endosomal sorting complex required for transport (ESCRT) mutants lose epithelial polarity and show increased cell proliferation, suggesting that ESCRT proteins could function as tumor suppressors. In this study, we show for the for the first time to our knowledge that ESCRT proteins are required to maintain polarity in mammalian epithelial cells. Inhibition of ESCRT function caused the tight junction protein claudin-1 to accumulate in intracellular vesicles. In contrast E-cadherin and occludin localization was unaffected. We investigated the cause of this accumulation and show that claudin-1 is constitutively recycled in kidney, colon, and lung epithelial cells, identifying claudin-1 recycling as a newly described feature of diverse epithelial cell types. This recycling requires ESCRT function, explaining the accumulation of intracellular claudin-1 when ESCRT function is inhibited. We further demonstrate that small interfering RNA knockdown of the ESCRT protein Tsg101 causes epithelial monolayers to lose their polarized organization and interferes with the establishment of a normal epithelial permeability barrier. ESCRT knockdown also reduces the formation of correctly polarized three-dimensional cysts. Thus, in mammalian epithelial cells, ESCRT function is required for claudin-1 trafficking and for epithelial cell polarity, supporting the hypothesis that ESCRT proteins function as tumor suppressors.     

Application of ring-closing metathesis macrocyclization to the development of Tsg101-binding antagonists

HIV-1 viral budding involves binding of the viral Gag^p6 protein to the ubiquitin E2 variant domain of the human tumor susceptibility gene 101 protein (Tsg101). Recognition of p6 by Tsg101 is mediated in part by a proline-rich motif that contains the sequence ‘Pro-Thr-Ala-Pro’ (‘PTAP’). Using the p6-derived 9-mer sequence ‘PEPTAPPEE’, we had previously improved peptide binding affinity by employing N-alkylglycine (‘peptoid’) residues. The current study applies ring-closing metathesis macrocyclization strategies to Tsg101-binding peptide–peptoid hybrids as an approach to stabilize binding conformations and to observe the effects of such macrocyclization on Tsg101-binding affinity and bioavailability.     

MiR-17-5p-mediated endoplasmic reticulum stress promotes acute myocardial ischemia injury through targeting Tsg101

Cardiovascular diseases are the leading cause of death globally, among which acute myocardial infarction (AMI) frequently occurs in the heart and proceeds from myocardium ischemia and endoplasmic reticulum (ER) stress-induced cell death. Numerous studies on miRNAs indicated their potential as diagnostic biomarkers and treatment targets for heart diseases. Our study investigated the role of miR-17-5p and its regulatory mechanisms during AMI. Echocardiography, MTT, flow cytometry assay, evaluation of caspase-3 and lactate dehydrogenase (LDH) activity were conducted to assess cell viability, apoptosis in an MI/R mice model, and an H₂O₂-induced H9c2 hypoxia cell model, respectively. The expression levels of ER stress response-related biomarkers were detected using qRT-PCR, IHC, and western blotting assays. The binding site of miR-17-5p on Tsg101 mRNA was determined by bioinformatic prediction and luciferase reporter assay. The expression levels of miR-17-5p were notably elevated in MI/R mice and hypoxia cell models, accompanied by enhanced cell apoptosis. Inhibition of miR-17-5p led to decreased apoptosis related to ER stress response in the hypoxia model, which could be counteracted by knockdown of Tsg101 (tumor susceptibility gene 101). Transfection with miR-17-5p mimics downregulated the expression of Tsg101 in H9c2 cells. Luciferase assay demonstrated the binding between miR-17-5p and Tsg101. Moreover, 4-PBA, the inhibitor of the ER stress response, abolished shTsg101 elevated apoptosis in hypoxic H9c2 cells. Our findings investigated the pro-apoptotic role of miR-17-5p during MI/R, disclosed the specific mechanism of miR-17-5p/Tsg101 regulatory axis in ER stress-induced myocardium injury and cardiomyocytes apoptosis, and presented a promising diagnostic biomarker and potential target for therapy of AMI.     

ESCRT machinery potentiates HIV-1 utilization of the PI(4,5)P(2)-PLC-IP3R-Ca(2+) signaling cascade

Human immunodeficiency virus type 1 (HIV-1) release efficiency is directed by late (L) domain motifs in the viral structural precursor polyprotein Gag, which serve as links to the ESCRT (endosomal sorting complex required for transport) machinery. Linkage is normally through binding of Tsg101, an ESCRT-1 component, to the P₇TAP motif in the p6 region of Gag. In its absence, budding is directed by binding of Alix, an ESCRT adaptor protein, to the LY₃₆PX_nL motif in Gag. We recently showed that budding requires activation of the inositol 1,4,5-triphosphate receptor (IP3R), a protein that “gates” Ca²⁺ release from intracellular stores, triggers Ca²⁺ cell influx and thereby functions as a major regulator of Ca²⁺ signaling. In the present study, we determined whether the L domain links Gag to Ca²⁺ signaling machinery. Depletion of IP3R and inactivation of phospholipase C (PLC) inhibited budding whether or not Tsg101 was bound to Gag. PLC hydrolysis of phosphatidylinositol-(4,5)-bisphosphate generates inositol (1,4,5)-triphosphate, the ligand that activates IP3R. However, with Tsg101 bound, Gag release was independent of Gq-mediated activation of PLC, and budding was readily enhanced by pharmacological stimulation of PLC. Moreover, IP3R was redistributed to the cell periphery and cytosolic Ca²⁺ was elevated, events indicative of induction of Ca²⁺ signaling. The results suggest that L domain function, ESCRT machinery and Ca²⁺ signaling are linked events in Gag release.     

Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes

Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.     

Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth

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. Here, we show that patches of tissue that are mutant for the Drosophila Tsg101 ortholog, erupted, cause dramatic overproliferation 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.     

Aspergillus nidulans ArfB plays a role in endocytosis and polarized growth

Filamentous fungi undergo polarized growth throughout most of their life cycles. The Spitzenkörper is an apical organelle composed primarily of vesicles that is unique to filamentous fungi and is likely to act as a vesicle supply center for tip growth. Vesicle assembly and trafficking are therefore important for hyphal growth. ADP ribosylation factors (Arfs), a group of small GTPase proteins, play an important role in nucleating vesicle assembly. Little is known about the role of Arfs in filamentous hyphal growth. We found that Aspergillus nidulans is predicted to encode six Arf family proteins. Analysis of protein sequence alignments suggests that A. nidulans ArfB shares similarity with ARF6 of Homo sapiens and Arf3p of Saccharomyces cerevisiae. An arfB null allele (arfB disrupted by a transposon [arfB::Tn]) was characterized by extended isotropic growth of germinating conidia followed by cell lysis or multiple, random germ tube emergence, consistent with a failure to establish polarity. The mutant germ tubes and hyphae that do form initially meander abnormally off of the axis of polarity and frequently exhibit dichotomous branching at cell apices, consistent with a defect in polarity maintenance. FM4-64 staining of the arfB::Tn strain revealed that another phenotypic characteristic seen for arfB::Tn is a reduction and delay in endocytosis. ArfB is myristoylated at its N terminus. Green fluorescent protein-tagged ArfB (ArfB::GFP) localizes to the plasma membrane and endomembranes and mutation (ArfB^G2A::GFP) of the N-terminal myristoylation motif disperses the protein to the cytoplasm rather than to the membranes. These results demonstrate that ArfB functions in endocytosis to play important roles in polarity establishment during isotropic growth and polarity maintenance during hyphal extension.     

Membrane-Elasticity Model of Coatless Vesicle Budding Induced by ESCRT Complexes

The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT) directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.     

Regulating polarity by directing traffic: Cdc42 prevents adherens junctions from Crumblin' aPart

The GTPase Cdc42 was among the original genes identified with roles in cell polarity, and interest in its cellular roles from yeast to humans remains high. Cdc42 is a well-known regulator of the actin cytoskeleton, but also plays important roles in vesicular trafficking. In this issue, Harris and Tepass (Harris, K.P, and U. Tepass. 2008. J. Cell. Biol. 183:1129–1143) provide new insights into how Cdc42 and Par proteins work together to modulate cell adhesion and polarity during embryonic morphogenesis by regulating the traffic of key cell junction proteins.     

Cell cycle-dependent modulations of fenestrin expression in Tetrahymena pyriformis

In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, several proteins display a specific asymmetric distribution suggesting their involvement in the generation and the maintenance of cell polarization. One of these proteins, a membrane skeleton protein called fenestrin, forms an antero-posterior gradient, and is accepted as a marker of cell polarity during different cellular processes, such as cell division or oral replacement. In conjugating cells, fenestrin forms an intracytoplasmic net which participates in pronuclear exchange. The function of fenestrin is still unknown. To better understand the role of fenestrin we characterized this protein in an amicronuclear Tetrahymena pyriformis. We show that in this ciliate not only does fenestrin localization change in a cell division-dependent manner, but its mRNA and protein level is also cell cycle-regulated. We determine that the two available anti-fenestrin antibodies, 3A7 and 9A7, recognize different pools of fenestrin isoforms, and that 9A7 is the more general. In addition, our results indicate that fenestrin is a phosphoprotein. We also show that the level of fenestrin in the amicronuclear T. pyriformis and the amicronuclear BI3840 strain of T. thermophila is several times lower than in micronuclear T. thermophila.     

Lethal (2) giant larvae regulates pleural mesothelial cell polarity in pleural fibrosis

Pleural fibrosis is barely reversible and the underlying mechanisms are poorly understood. Pleural mesothelial cells (PMCs) which have apical-basal polarity play a key role in pleural fibrosis. Loss of cell polarity is involved in the development of fibrotic diseases. Partition defective protein (PAR) complex is a key regulator of cell polarity. However, changes of PMC polarity and PAR complex in pleural fibrosis are still unknown. In this study, we observed that PMC polarity was lost in fibrotic pleura. Next we found increased Lethal (2) giant larvae (Lgl) bound with aPKC and PAR-6B competing against PAR-3A in PAR complex, which led to cell polarity loss. Then we demonstrated that Lgl1 siRNA prevented cell polarity loss in PMCs, and Lgl1 conditional knockout (ER-Cre^+/?Lgl1^flox/flox) attenuated pleural fibrosis in a mouse model. Our data indicated that Lgl1 regulates cell polarity of PMCs, inhibition of Lgl1 and maintenance of cell polarity in PMCs could be a potential therapeutic treatment approach for pleural fibrosis.