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PubMed2004

A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor.

Ligand-stimulated growth factor receptors are rapidly internalized and transported to early endosomes. Unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to the same organelle. At early endosomes, stimulated receptors are sorted for the lysosomal degradation pathway, whereas unstimulated receptors are mostly recycled back to the cell surface. To investigate the role of Hrs, an early endosomal protein, in this sorting process, we overexpressed Hrs in HeLa cells and examined the intracellular trafficking of epidermal growth factor receptor (EGFR) in EGF-stimulated and unstimulated cells. Overexpression of Hrs inhibited the trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. On the other hand, overexpression of Hrs mutants with a deletion or a point mutation within the FYVE domain did not inhibit the trafficking. These results suggest that Hrs regulates the sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to reside in a microdomain of early endosomes, plays an essential role in the function of Hrs.

Endosomal Sorting Complexes Required for Transport↗

PubMed2002

Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes.

After endocytosis, some membrane proteins recycle from early endosomes to the plasma membrane whereas others are transported to late endosomes and lysosomes for degradation. Conjugation with the small polypeptide ubiquitin is a signal for lysosomal sorting. Here we show that the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, is involved in the endosomal sorting of ubiquitinated membrane proteins. Hrs contains a clathrin-binding domain, and by electron microscopy we show that Hrs localizes to flat clathrin lattices on early endosomes. We demonstrate that Hrs binds directly to ubiquitin by way of a ubiquitin-interacting motif (UIM), and that ubiquitinated proteins localize specifically to Hrs- and clathrin-containing microdomains. Whereas endocytosed transferrin receptors fail to colocalize with Hrs and rapidly recycle to the cell surface, transferrin receptors that are fused to ubiquitin interact with Hrs, localize to Hrs- and clathrin-containing microdomains and are sorted to the degradative pathway. Overexpression of Hrs strongly and specifically inhibits recycling of ubiquitinated transferrin receptors by a mechanism that requires a functional UIM. We conclude that Hrs sorts ubiquitinated membrane proteins into clathrin-coated microdomains of early endosomes, thereby preventing their recycling to the cell surface.

Animals↗

PubMed2005

Hrs, a mammalian master molecule in vesicular transport and protein sorting, suppresses the degradation of ESCRT proteins signal transducing adaptor molecule 1 and 2.

The degradation and sorting of cytoplasmic and cell-surface proteins are crucial steps in the control of cellular functions. We previously identified three mammalian Vps (vacuolar protein sorting) proteins, Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and signal transducing adaptor molecule (STAM) 1 and -2, which are tyrosine-phosphorylated upon cytokine/growth factor stimulation. Hrs and the STAMs each contain a ubiquitin-interacting motif and through formation of a complex are involved in the vesicle transport of early endosomes. To explore the mechanism and cellular function of this complex in mammalian cells, we established an Hrs-defective fibroblastoid cell line (hrs(-/-)); embryos with this genotype died in utero. In the hrs(-/-) cells only trace amounts of STAM1 and STAM2 were detected. Introduction of wild-type Hrs or an Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) fully restored STAM1 and STAM2 expression, whereas mutants with no STAM binding ability (Hrs-dC2, Hrs-dM) failed to express the STAMs. This regulated control of STAM expression by Hrs was independent of transcription. Interestingly, STAM1 degradation was mediated by proteasomes and was partially dependent on the ubiquitin-interacting motif of STAM1. Revertant Hrs expression in hrs(-/-) cells not only led to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also restored STAM1 levels in early endosomes and eliminated the enlarged endosome phenotype caused by the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins.

Adaptor Proteins, Signal Transducing↗

PubMed2004

ALIX-ing phospholipids with endosome biogenesis.

Endosomes, which comprise a diverse population of membrane vesicles and tubules, sort proteins and lipids to various cellular destinations. The organization and functions of these pleiomorphic cellular organelles have been extensively studied. Matsuo et al.1 now provide new exciting evidence on the role of lysobiphosphatidic acid (LBPA), a resident phospholipid of internal vesicles of the late endosome, in the control of membrane invagination and endosome biogenesis. In vivo, LBPA functions are controlled by the adaptor protein Alix and depend on pH gradients along the endosomal compartments.

Animals↗

PubMed2005

SIMPLE interacts with NEDD4 and TSG101: evidence for a role in lysosomal sorting and implications for Charcot-Marie-Tooth disease.

Mutation of the SIMPLE gene (small integral membrane protein of the lysosome/late endosome) is the molecular basis of Charcot-Marie-Tooth disease type 1C (CMT1C), a demyelinating peripheral neuropathy. Although the precise function of SIMPLE is unknown, prior reports suggest it localizes to the lysosome/late endosome. Furthermore, murine Simple interacts with Nedd4 (neural precursor cell expressed, developmentally downregulated 4), an E3 ubiquitin ligase that is important for regulating lysosomal degradation of plasma membrane proteins. To bring insights into the biochemical function of human SIMPLE, we confirmed that human SIMPLE interacts with NEDD4 and also report a novel interaction with tumor susceptibility gene 101 (TSG101), a class E vacuolar sorting protein. TSG101 is known to function downstream of NEDD4, sorting ubiquitinated substrates into multivesicular bodies (MVBs), which then deliver their cargo into the lysosomal lumen for degradation. Given the interaction with NEDD4 and TSG101, and the localization of SIMPLE along the lysosomal degradation pathway, we hypothesize that SIMPLE plays a role in the lysosomal sorting of plasma membrane proteins. We examine three CMT1C-associated SIMPLE mutations and show that they do not affect the interaction with NEDD4 or TSG101, nor do they lead to altered subcellular localization.

B-Lymphocytes↗

PubMed1999

TOM1 genes map to human chromosome 22q13.1 and mouse chromosome 8C1 and encode proteins similar to the endosomal proteins HGS and STAM.

The avian tom1 (target of myb 1) gene has been previously characterized from v-myb-transformed cells. We report here cloning of the human and mouse tom1 orthologs. Both genes are expressed ubiquitously, with the highest levels in skeletal muscle, brain, and intestines, as assessed by Northern blot and mRNA in situ hybridization. The N-terminal domain of the TOM1 protein shares similarity with HGS (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), which are associated with vesicular trafficking at the endosome. A putative coiled-coil domain was also detected in the central part of the TOM1 protein. This domain structure suggests that TOM1 is another member of a family of genes implicated in the trafficking regulation of growth-factor-receptor complexes that are destined for degradation in the lysosome. We also show that a human paralog of TOM1 (TOM1-like gene 1) exists. Furthermore, we provide a transcription map over a 190-kb contig of the TOM1 region. This map includes its distal neighbors HMOX1 and MCM5 and two proximal novel genes, one of which is a HMG-box-containing gene (HMG2L1), and the other of unknown function. Using a genomic PAC clone, we demonstrate that the mouse Tom1 and Hmox1 genes are part of an as yet undescribed syntenic group between mouse chromosome 8C1 and human chromosome 22q13.1.

Adaptor Proteins, Signal Transducing↗

PubMed2005

HRS inhibits EGF receptor signaling in the RT4 rat schwannoma cell line.

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is required for trafficking of cell surface receptors to the lysosome. Previously, we identified HRS as a protein that interacts with the neurofibromatosis 2 tumor suppressor schwannomin. In the present study, we established modified RT4 schwannoma cell lines that inducibly express HRS and constitutively express epidermal growth factor receptor (EGFR) fused to the green fluorescent protein. We demonstrated that HRS expression reduced EGFR abundance and EGF-mediated Stat3 activation. HRS expression also targeted EGFR to late endosomes. Schwannomin inhibited EGF-mediated Stat3 activation, consistent with HRS and schwannomin interacting in the same signaling pathway. Paradoxically, past studies have shown that HRS overexpression blocked EGFR trafficking to the late endosome and EGFR downregulation contrary to predictions of HRS function in HRS knockout studies. This study is the first to show that HRS can reduce the abundance of total and active EGFR and may reflect cell type-specific HRS function.

Animals↗

PubMed2006

RILP interacts with the VPS22 component of the ESCRT-II complex.

The Rab-interacting lysosomal protein (RILP) has been identified as an effector for the small GTPases Rab7 and Rab34. It has been demonstrated that Rab7 and RILP are key proteins for the biogenesis of lysosomes and phagolysosomes. Indeed, expression of dominant negative mutants of Rab7 or of the C-terminal half of RILP impairs biogenesis and function of these organelles. In this study we have isolated, using the yeast two-hybrid system, the EAP30/SNF8/VPS22 subunit of the ESCRT-II complex as a RILP interacting protein. We demonstrated that VPS22 interacts with the N-terminal half of RILP. The interaction data obtained with the two-hybrid system were confirmed by co-immunoprecipitation. In addition, confocal immunofluorescence revealed colocalization of GFP-RILP and HA-VPS22. These data suggest that RILP could have a role in the biogenesis of multivesicular bodies.

Adaptor Proteins, Signal Transducing↗

PubMed2006

ESCRT-I core and ESCRT-II GLUE domain structures reveal role for GLUE in linking to ESCRT-I and membranes.

ESCRT complexes form the main machinery driving protein sorting from endosomes to lysosomes. Currently, the picture regarding assembly of ESCRTs on endosomes is incomplete. The structure of the conserved heterotrimeric ESCRT-I core presented here shows a fan-like arrangement of three helical hairpins, each corresponding to a different subunit. Vps23/Tsg101 is the central hairpin sandwiched between the other subunits, explaining the critical role of its "steadiness box" in the stability of ESCRT-I. We show that yeast ESCRT-I links directly to ESCRT-II, through a tight interaction of Vps28 (ESCRT-I) with the yeast-specific zinc-finger insertion within the GLUE domain of Vps36 (ESCRT-II). The crystal structure of the GLUE domain missing this insertion reveals it is a split PH domain, with a noncanonical lipid binding pocket that binds PtdIns3P. The simultaneous and reinforcing interactions of ESCRT-II GLUE domain with membranes, ESCRT-I, and ubiquitin are critical for ubiquitinated cargo progression from early to late endosomes.

Amino Acid Sequence↗

PubMed2005

Novel function of POSH, a JNK scaffold, as an E3 ubiquitin ligase for the Hrs stability on early endosomes.

POSH (plenty of SH3s) acts as a scaffold that links activated Rac1 and downstream c-Jun N-terminal kinase (JNK) signaling modules. However, it is unknown whether it's functional domain-mediated roles including the interesting RING-finger domain or its cellular function. Here, we provide evidence that subcellular localization of POSH is regulated by a particular domain of the protein and POSH was colocalized with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) on early endosomes via interaction of Hrs with POSH's two rear SH3 domains. Moreover, the RING domain of POSH specifically regulates the stability of Hrs, but not of JNK1, via a ubiquitin-proteasomal degradation pathway. Finally, we demonstrate that JNK1 does not interact with Hrs under the conditions of POSH interacted with Hrs, but instead reduces the POSH-catalyzed ubiquitination of Hrs and their reciprocal interaction. Together, these data suggest that POSH has a distinct role as a specific E3 ubiquitin ligase for Hrs on early endosomes, and there exists a relationship between its separate activities as a scaffold and as an E3.

Animals↗

PubMed2004

Regulation of notch endosomal sorting and signaling by Drosophila Nedd4 family proteins.

The Notch receptor mediates a short-range signal that regulates many cell fate decisions. The misregulation of Notch has been linked to cancer and to developmental disorders. Upon binding to its ligands, Delta (Dl) or Serrate (Ser), the Notch ectodomain is shed by the action of an ADAM protease. The Notch intracellular domain is subsequently released proteolytically from the membrane by Presenilin and translocates to the nucleus to activate the transcription factor, Suppressor of Hairless. We show in Drosophila that Notch signaling is limited by the activity of two Nedd4 family HECT domain proteins, Suppressor of deltex [Su(dx)] and DNedd4. We rule out models by which Su(dx) downregulates Notch through modulating Deltex or by limiting the adherens junction accumulation of Notch. Instead, we show that Su(dx) regulates the postendocytic sorting of Notch within the early endosome to an Hrs- and ubiquitin-enriched subdomain en route to the late endosome. We propose a model in which endocytic sorting of Notch mediates a decision between its activation and downregulation. Such intersections between trafficking routes may provide key points at which other signals can modulate Notch activity in both normal development and in the pathological misactivation of Notch.

Animals↗

PubMed2006

Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery.

AMSH is an endosomal ubiquitin isopeptidase that can limit EGF receptor downregulation . It directly binds to the SH3 domain of STAM, which is constitutively associated with Hrs, a component of clathrin-coated structures on endosomes. This clathrin coat has been implicated in the recruitment of ubiquitinated growth factor receptors prior to their incorporation into internal vesicles of the multivesicular body (MVB) , through the concerted action of ESCRT complexes I, II, and III . We now show that AMSH is embedded within a network of interactions with components of the MVB-sorting machinery. AMSH and STAM, like Hrs , both bind directly to clathrin. AMSH also interacts with mVps24/CHMP3, a component of ESCRT III complex, and this interaction is reinforced through simultaneous STAM binding. We have explored the effect of interacting components on the in vitro enzymatic activity of AMSH. The enzyme shows specificity for K63- over K48-linked polyubiquitin chains in vitro and is markedly stimulated by coincubation with STAM, indicating that activation of AMSH is coupled to its association with the MVB-sorting machinery. Other interacting factors do not directly stimulate AMSH but may serve to orient the enzyme with respect to substrates on the endosomal membrane.

Adaptor Proteins, Signal Transducing↗

PubMed2004

ESCRT-II, an endosome-associated complex required for protein sorting: crystal structure and interactions with ESCRT-III and membranes.

ESCRT-I, -II, and -III protein complexes are sequentially recruited to endosomal membranes, where they orchestrate protein sorting and MVB biogenesis. In addition, they play a critical role in retrovirus budding. Structural understanding of ESCRT interaction networks is largely lacking. The 3.6 A structure of the yeast ESCRT-II core presented here reveals a trilobal complex containing two copies of Vps25, one copy of Vps22, and the C-terminal region of Vps36. Unexpectedly, the entire ESCRT-II core consists of eight repeats of a common building block, a "winged helix" domain. Two PPXY-motifs from Vps25 are involved in contacts with Vps22 and Vps36, and their mutation leads to ESCRT-II disruption. We show that purified ESCRT-II binds directly to the Vps20 component of ESCRT-III. Surprisingly, this binding does not require the protruding N-terminal coiled-coil of Vps22. Vps25 is the chief subunit responsible for Vps20 recruitment. This interaction dramatically increases binding of both components to lipid vesicles in vitro.

Amino Acid Motifs↗

PubMed2005

Structural basis for endosomal targeting by the Bro1 domain.

Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs.

Animals↗

PubMed2006

The conserved c2 domain protein lethal (2) giant discs regulates protein trafficking in Drosophila.

Drosophila sensory organ precursor (SOP) cells undergo several rounds of asymmetric cell division to generate the four different cell types that make up external sensory organs. Establishment of different fates among daughter cells of the SOP relies on differential regulation of the Notch pathway. Here, we identify the protein Lethal (2) giant discs (Lgd) as a critical regulator of Notch signaling in the SOP lineage. We show that lgd encodes a conserved C2 domain protein that binds to phospholipids present on early endosomes. When Lgd function is compromised, Notch and other transmembrane proteins accumulate in enlarged early endosomal compartments. These enlarged endosomes are positive for Rab5 and Hrs, a protein involved in trafficking into the degradative pathway. Our experiments suggest that Lgd is a critical regulator of endocytosis that is not present in yeast and acts in the degradative pathway after Hrs.

Animals↗

PubMed2006

The Drosophila Notch inhibitor and tumor suppressor gene lethal (2) giant discs encodes a conserved regulator of endosomal trafficking.

Notch signaling is involved in many developmental and pathological processes, and its activity must be precisely controlled in order to prevent aberrant development and disease. We have previously shown that the tumor suppressor gene lethal (2) giant discs (lgd) is required to prevent ectopic activation of Notch in developmental processes in Drosophila. Here we show that lgd is required in all imaginal disc cells to suppress the activity of the Notch pathway. lgd encodes a member of a poorly characterized protein family present in all animals, which includes a member that is involved in an inheritable form of mental retardation in humans. Our analysis reveals that Lgd is required for endosomal trafficking of Notch and other proteins. In the absence of Lgd, Notch is activated in a ligand-independent manner in probably all imaginal disc cells in an endosomal compartment downstream of the block in hrs mutants.

Animals↗

PubMed2006

The endosome-associated protein Hrs is hexameric and controls cargo sorting as a "master molecule".

The structure of the endosomal-associated protein, Hrs, has been determined with cryo-electron microscopy. Hrs interacts with a number of proteins, including SNAP-25 and STAM1, forming a complex that binds ubiquitin moieties. Analytical ultracentrifugation studies revealed that Hrs exists as a hexamer. The symmetry and the structure of the hexameric form of Hrs were determined with the single-particle reconstruction method. Hrs comprises three antiparallel dimers with a central core and distinct caps on either end. Crystal structures of VHS and FYVE domains fit into the Hrs end caps in the EM density map. Thus, the location of domains that interact with the endosomal membrane, the VHS, FYVE, and C-terminal domains, facilitates the anchorage of Hrs to the membrane, initiating the functional processes of Hrs on the endosome. Based on our model, the Hrs hexamer interacts with the membrane and acts as a "master molecule" that presents multiple sites for protein binding.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Endocytosis: the DUB version.

Dynamic modification of endosomal cargo proteins, such as the epidermal growth factor receptor, by ubiquitin can regulate their sorting into the lumen of multivesicular bodies through interactions with a complex protein network incorporating the endosomal sorting complexes required for transport (ESCRTs). Two deubiquitinating enzymes, AMSH and UBPY, interact with ESCRT protein components but exert opposite effects upon the rate of epidermal growth factor receptor downregulation. This might reflect their distinct specificities for different types of polyubiquitin chain linkage. We propose that AMSH might rescue ubiquitinated cargo from lysosomal degradation through disassembly of K63-linked polyubiquitin chains. UBPY function is essential for effective downregulation but is likely to be multifaceted, encompassing activity against both K63-linked and K48-linked polyubiquitin chains and including regulation of the stability of ESCRT-associated proteins such as STAM, by reversing their ubiquitination.

Endocytosis↗