Both clathrin-positive and -negative coats are involved in endosomal sorting of the EGF receptor

Sorting of endocytosed EGF receptor (EGFR) to internal vesicles of multivesicular bodies (MVBs) depends on sustained activation and ubiquitination of the EGFR. Ubiquitination of EGFR is mediated by the ubiquitin ligase Cbl, being recruited to the EGFR both directly and indirectly through association with Grb2. Endosomal sorting of ubiquitinated proteins further depends on interaction with ubiquitin binding adaptors like Hrs. Hrs localizes to flat, clathrin-coated domains on the limiting membrane of endosomes. In the present study, we have investigated the localization of EGFR, Cbl and Grb2 with respect to coated and non-coated domains of the endosomal membrane and to vesicles within MVBs. Both EGFR, Grb2, and Cbl were concentrated in coated domains of the limiting membrane before translocation to inner vesicles of MVBs. While almost all Hrs was in clathrin-positive coats, EGFR and Grb2 in coated domains only partially colocalized with Hrs and clathrin. The extent of colocalization of EGFR and Grb2 with Hrs and clathrin varied with time of incubation with EGF. These results demonstrate that both clathrin-positive and clathrin-negative electron dense coats exist on endosomes and are involved in endosomal sorting of the EGFR.     

Structural characterization of the ATPase reaction cycle of endosomal AAA protein Vps4

The multivesicular body (MVB) pathway functions in multiple cellular processes including cell surface receptor down-regulation and viral budding from host cells. An important step in the MVB pathway is the correct sorting of cargo molecules, which requires the assembly and disassembly of endosomal sorting complexes required for transport (ESCRTs) on the endosomal membrane. Disassembly of the ESCRTs is catalyzed by ATPase associated with various cellular activities (AAA) protein Vps4. Vps4 contains a single AAA domain and undergoes ATP-dependent quaternary structural change to disassemble the ESCRTs. Structural and biochemical analyses of the Vps4 ATPase reaction cycle are reported here. Crystal structures of Saccharomyces cerevisiae Vps4 in both the nucleotide-free form and the ADP-bound form provide the first structural view illustrating how nucleotide binding might induce conformational changes within Vps4 that lead to oligomerization and binding to its substrate ESCRT-III subunits. In contrast to previous models, characterization of the Vps4 structure now supports a model where the ground state of Vps4 in the ATPase reaction cycle is predominantly a monomer and the activated state is a dodecamer. Comparison with a previously reported human VPS4B structure suggests that Vps4 functions in the MVB pathway via a highly conserved mechanism supported by similar protein-protein interactions during its ATPase reaction cycle.     

SCF acts in endosomal sorting of the GH receptor

The ubiquitin ligase SCF^TrCP is required for internalisation of the growth hormone receptor (GHR) and acts via a direct interaction with the ubiquitin-dependent endocytosis motif. Details of how the ligase communicates its information to the clathrin-mediated internalisation machinery are unknown. For the EGF receptor, c-Cbl acts both at the cell surface and in endosomes. We hypothesised that SCF^TrCP is required for GHR degradation at both sites. This was tested by truncating GHR after a di-leucine-based internalisation motif (GHR349). This receptor enters the cells via the adapter complex AP2. We show that TrCP acts in an early stage of cargo selection: both TrCP silencing and mutation of the ubiquitin-dependent endocytosis motif force the GHR to recycle between endosomes and the plasma membrane, together with the transferrin receptor. Depletion of Tsg101 (ESCRT-I) has the same effect, while silencing of Hrs (ESCRT-0) prevents GH recycling. GH passes through late endosomal vesicles, marked by Lamp1. Coexpressing GHR and EGFR demonstrates that both receptors use the same route to the lysosomes. We show for the first time that SCF^TrCP is involved in cargo-specific sorting at endosomes and that Tsg101 rather than Hrs might direct the cargo into the ESCRT machinery.     

CIN85 regulates ubiquitination and degradative endosomal sorting of the EGF receptor

CIN85 has been demonstrated to interact with a number of proteins involved in endocytosis and intracellular sorting. However, the exact functional role of CIN85 in endocytosis remains unclear. We have investigated whether CIN85 plays a role in EGF-induced EGF receptor (EGFR) internalization, as previously suggested, or whether CIN85 is rather involved in endosomal sorting of the EGFR. When over-expressing a dominant negative interfering CIN85 mutant consisting of three SH3 domains only, we found that internalization of EGF was inhibited. However, when knocking down CIN85 by RNAi, the EGF–EGFR uptake appeared similar to in control cells. Furthermore, in CIN85 depleted cells, EGF-induced ubiquitination of the EGFR was decreased, and degradation of EGF–EGFR complexes was delayed. Our data further demonstrated that depletion of CIN85 increased the recycling of EGF, suggesting that CIN85 plays a role in endosomal sorting of the ubiquitinated EGFR. Our data also demonstrated that CIN85 was constitutively associated with Hrs, and this strengthens the hypothesis of a functional role of CIN85 in endosomal EGFR sorting.     

Proteasomal inhibition alters the trafficking of the neurotrophin receptor TrkA

Neurotrophin receptors of the Trk family promote neuronal survival. The signal transduction of Trk receptors is regulated by endosomal trafficking. Monoubiquitination of receptor tyrosine kinases is an established signal for sorting of internalized receptors to late endosomes. The NGF receptor TrkA is sorted to late endosomes and undergoes ubiquitination, indicating a so far undefined regulatory role of proteasomal activity in the trafficking of TrkA. Surprisingly, we found that proteasomal inhibition alters the trafficking of TrkA from the late endosomal sorting pathway to the recycling pathway. Many neurodegenerative diseases are associated with impaired proteasomal activity. Thus, our study suggests that missorting of neurotrophic receptors might contribute to neuronal death in those neurodegenerative diseases that are known to be associated with impaired proteasomal function.     

The ESCRT‐III protein CeVPS‐32 is enriched in domains distinct from CeVPS‐27 and CeVPS‐23 at the endosomal membrane of epithelial cells

Background information. Within the endocytic pathway, the ESCRT (endosomal sorting complex required for transport) machinery is essential for the biogenesis of MVBs (multivesicular bodies). In yeast, ESCRTs are recruited at the endosomal membrane and are involved in cargo sorting into intralumenal vesicles of the MVBs. Results. In the present study, we characterize the ESCRT‐III protein CeVPS‐32 (Caenorhabditis elegans vacuolar protein sorting 32) and its interactions with CeVPS‐27, CeVPS‐23 and CeVPS‐4. In contrast with other CevpsE (class E vps) genes, depletion of Cevps‐32 is embryonic lethal with severe defects in the remodelling of epithelial cell shape during organogenesis. Furthermore, Cevps‐32 animals display an accumulation of enlarged early endosomes in epithelial cells and an accumulation of autophagosomes. The CeVPS‐32 protein is enriched in epithelial tissues and in residual bodies during spermatid maturation. We show that CeVPS‐32 and CeVPS‐27/Hrs (hepatocyte‐growth‐factor‐regulated tyrosine kinase substrate) are enriched in distinct subdomains at the endosomal membrane. CeVPS‐27‐positive subdomains are also enriched for the ESCRT‐I protein CeVPS‐23/TSG101 (tumour susceptibility gene 101). The formation of CeVPS‐27 subdomains is not affected by the depletion of CeVPS‐23, CeVPS‐32 or the ATPase CeVPS‐4. Conclusion. Our results suggest that the formation of membrane subdomains is essential for the maturation of endosomes.     

Molecular assemblies and membrane domains in multivesicular endosome dynamics

Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo “back-fusion” with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.     

Intracellular trafficking of Notch receptors and ligands

The Notch pathway represents a highly conserved signaling network, which regulates the formation and maintenance of various organ systems along development and during adulthood. Direct cell-cell contacts between ligand- and receptor-expressing cells underlie activation of the Notch pathway. Notch signaling requires endocytosis in both signal emitting and receiving cells. Recent findings on the roles of a number of modulators show that they act either on the maintenance of an active receptor at the membrane, or on the production of active ligand, or on signal transduction after activation.     

Cryo-EM structure of dodecameric Vps4p and its 2:1 complex with Vta1p

The type I AAA (ATPase associated with a variety of cellular activities) ATPase Vps4 and its co-factor Vta1p/LIP5 function in membrane remodeling events that accompany cytokinesis, multivesicular body biogenesis, and retrovirus budding, apparently by driving disassembly and recycling of membrane-associated ESCRT (endosomal sorting complex required for transport)-III complexes. Here, we present electron cryomicroscopy reconstructions of dodecameric yeast Vps4p complexes with and without their microtubule interacting and transport (MIT) N-terminal domains and Vta1p co-factors. The ATPase domains of Vps4p form a bowl-like structure composed of stacked hexameric rings. The two rings adopt dramatically different conformations, with the “upper” ring forming an open assembly that defines the sides of the bowl and the lower ring forming a closed assembly that forms the bottom of the bowl. The N-terminal MIT domains of the upper ring localize on the symmetry axis above the cavity of the bowl, and the binding of six extended Vta1p monomers causes additional density to appear both above and below the bowl. The structures suggest models in which Vps4p MIT and Vta1p domains engage ESCRT-III substrates above the bowl and help transfer them into the bowl to be pumped through the center of the dodecameric assembly.     

Coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding

We develop coarse-grained models and effective energy functions for simulating thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (K_d > 1 μM) and apply them to binding of Vps27 to membrane-tethered ubiquitin. Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair potentials and Debye-Hückel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. In simulations of membrane-attached protein complexes, interactions between amino acids and the membrane are described by residue-dependent short-range potentials and long-range electrostatics. We parameterize the energy functions by fitting the osmotic second virial coefficient of lysozyme and the binding affinity of the ubiquitin-CUE complex. For validation, extensive replica-exchange Monte Carlo simulations are performed of various protein complexes. Binding affinities for these complexes are in good agreement with the experimental data. The simulated structures are clustered on the basis of distance matrices between two proteins and ranked according to cluster population. In ∼ 70% of the complexes, the distance root-mean-square is less than 5 Å from the experimental structures. In ∼ 90% of the complexes, the binding interfaces on both proteins are predicted correctly, and in all other cases at least one interface is correct. Transient and nonspecifically bound structures are also observed. With the validated model, we simulate the interaction between the Vps27 multiprotein complex and a membrane-tethered ubiquitin. Ubiquitin is found to bind preferentially to the two UIM domains of Vps27, but transient interactions between ubiquitin and the VHS and FYVE domains are observed as well. These specific and nonspecific interactions are found to be positively cooperative, resulting in a substantial enhancement of the overall binding affinity beyond the ∼ 300 μM of the specific domains. We also find that the interactions between ubiquitin and Vps27 are highly dynamic, with conformational rearrangements enabling binding of Vps27 to diverse targets as part of the multivesicular-body protein-sorting pathway.     

Competitive binding of UBPY and ubiquitin to the STAM2 SH3 domain revealed by NMR

To date, the signal transducing adaptor molecule 2 (STAM2) was shown to harbour two ubiquitin binding domains (UBDs) known as the VHS and UIM domains, while the SH3 domain of STAM2 was reported to interact with deubiquitinating enzymes (DUBs) like UBPY and AMSH. In the present study, NMR evidences the interaction of the STAM2 SH3 domain with ubiquitin, demonstrating that SH3 constitutes the third UBD of STAM2. Furthermore, we show that a UBPY-derived peptide can outcompete ubiquitin for SH3 binding and vice versa. These results suggest that the SH3 domain of STAM2 plays versatile roles in the context of ubiquitin mediated receptor sorting.     

A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.     

Spatially Distinct Pools of TORC1 Balance Protein Homeostasis

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Sprouty2 Regulates PI(4,5)P2/Ca Signaling and HIV-1 Gag Release

We reported recently that activation of the inositol 1,4,5-triphosphate receptor (IP3R) is required for efficient HIV-1 Gag trafficking and viral particle release. IP3R activation requires phospholipase C (PLC)-catalyzed hydrolysis of PI(4,5)P₂ to IP3 and diacylglycerol. We show that Sprouty2 (Spry2), which binds PI(4,5)P₂ and PLCγ, interfered with PI(4,5)P₂ in a manner similar to that of U73122, an inhibitor of PI(4,5)P₂ hydrolysis, suggesting that Spry2 negatively regulates IP3R by preventing formation of its activating ligand, IP3. Mutation to Asp of R252, a crucial determinant of PI(4,5)P₂ binding in the C-terminal domain of Spry2, prevented the interference, indicating that binding to the phospholipid is required. By contrast, deletion of the PLCγ binding region or mutation of a critical Tyr residue in the region did not prevent the interference but Spry2-PI(4,5)P₂ colocalization was not detected, suggesting that PLC binding is required for their stable association. Like U73122, Spry2 over-expression inhibited wild type Gag release as virus-like particles. Disrupting either binding determinant relieved the inhibition. IP3R-mediated Ca²⁺signaling, in turn, was found to influence Spry2 subcellular distribution and ERK, a Spry2 regulator. Our findings suggest that Spry2 influences IP3R function through control of PI(4,5)P₂ and IP3R influences Spry2 function by controlling its distribution and ERK activation.     

Solution structure of UIM and interaction of tandem ubiquitin binding domains in STAM1 with ubiquitin

STAM1 and Hrs are the components of ESCRT-0 complex for lysosomal degradation of membrane proteins is composed of STAM1 Hrs and has multiple ubiquitin binding domains. Here, the solution structure of STAM1 UIM, one of the ubiquitin binding motif, was determined by NMR spectroscopy. The structure of UIM adopts an α-helix with amphipathic nature. The central hydrophobic residues in UIM provides the binding surface for ubiquitin binding and are flanked with positively and negatively charged residues on both sides. The docking model of STAM1 UIM-ubiquitin complex is suggested. In NMR and ITC experiments with the specifically designed mutant proteins, we investigated the ubiquitin interaction of tandem ubiquitin binding domains from STAM1. The ubiquitin binding affinity of the VHS domain and UIM in STAM1 was 52.4 and 94.9 μM, and 1.5 and 2.2 fold increased, respectively, than the value obtained from the isolated domain or peptide. The binding affinities here would be more physiologically relevant and provide more precise understanding in ESCRT pathway of lysosomal degradation.     

The two faces of PTP1B in cancer

PTP1B is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and is a promising drug target for type 2 diabetes and obesity. Accumulating evidence also indicates that PTP1B is involved in cancer, but contrasting findings suggest that it can exert both tumor suppressing and tumor promoting effects depending on the substrate involved and the cellular context. In this review, we will discuss the diverse mechanisms by which PTP1B may influence tumorigenesis as well as recent in vivo data on the impact of PTP1B deficiency in murine cancer models. Together, these results highlight not only the great potential of PTP1B inhibitors in cancer therapy but also the need for a better understanding of PTP1B function prior to use of these compounds in human patients.     

The Molecular Architecture of HIV

Assembly of human immunodeficiency virus type 1 is driven by oligomerization of the Gag polyprotein at the plasma membrane of an infected cell, leading to membrane envelopment and budding of an immature virus particle. Proteolytic cleavage of Gag at five positions subsequently causes a dramatic rearrangement of the interior virion organization to form an infectious particle. Within the mature virus, the genome is encased within a conical capsid core. Here, we describe the molecular architecture of the virus assembly site, the immature virus, the maturation intermediates and the mature virus core and highlight recent advances in our understanding of these processes from electron microscopy and X-ray crystallography studies.     

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.     

The Role of Cellular Factors in Promoting HIV Budding

Human immunodeficiency virus type 1 (HIV-1) becomes enveloped while budding through the plasma membrane, and the release of nascent virions requires a membrane fission event that separates the viral envelope from the cell surface. To facilitate this crucial step in its life cycle, HIV-1 exploits a complex cellular membrane remodeling and fission machinery known as the endosomal sorting complex required for transport (ESCRT) pathway. HIV-1 Gag directly interacts with early-acting components of this pathway, which ultimately triggers the assembly of the ESCRT-III membrane fission complex at viral budding sites. Surprisingly, HIV-1 requires only a subset of ESCRT-III components, indicating that the membrane fission reaction that occurs during HIV-1 budding differs in crucial aspects from topologically related cellular abscission events.     

Dual degradation mechanisms ensure disposal of NHE6 mutant protein associated with neurological disease

Clinical features characterizing Angelman syndrome, previously shown to be caused by disruption of UBE3A, were recently also described in neurologically disabled patients with mutations in SLC9A6, which encodes the Na⁺/H⁺ exchanger NHE6. In the present work we have focused on NHE6Δ255-256, the protein product of a specific 6-bp patient deletion in SLC9A6. To resolve the molecular mechanism causing the cellular dysfunction associated with this mutant, we have characterized its intracellular behaviour in comparison to wild type NHE6. Our study demonstrates that NHE6Δ255-256 is much less stable than the wild type protein. Whereas wild type NHE6 is transported to the plasma membrane and early endosomes and remains stable, NHE6Δ255-256 is degraded via two independent pathways mediated by proteasomes and lysosomes, respectively. Depletion of NHE6 had no detectable effect on endosomal pH, but co-depletion of NHE6 and the closely related NHE9 caused enhanced acidification of early endosomes. Our results suggest that NHE6 participates in regulation of endosomal pH and provides a cellular basis for understanding the loss of NHE6 function leading to a neurological phenotype resembling Angelman syndrome.