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PubMed2004

mVps24p functions in EGF receptor sorting/trafficking from the early endosome.

Yeast Vps24p (vacuolar protein sorting) is part of a protein complex suggested to function in sorting/trafficking during endocytosis. We have characterized a mammalian homolog of the yeast protein, mVps24p, and examined its role in epidermal growth factor receptor trafficking. Endogenous mVps24p was distributed in both cytosol and in puncta and partially colocalized with markers for the trans-Golgi network. Adventitious expression of hrs or a mVps4p mutant deficient in ATPase activity caused a redistribution of both mVps24p and the M6PR to the resultant clustered/enlarged early endosomes. Expression of an mVps24p N-terminal fragment, that interacts with phosphatidylinositol 3,5-bisphosphate but not with mVps4p, produces enlarged early endosomes. More importantly, the mVps24p N-terminal fragment resulted in not only enhanced recycling, but also decreased degradation of the EGF receptor. These findings are consistent with a model in which mVps24p has a role in trafficking from the early endosome.

Amino Acid Sequence↗
PubMed2006

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.

Cells, Cultured↗
PubMed2006

Rhophilin-2 is targeted to late-endosomal structures of the vesicular machinery in the presence of activated RhoB.

Rhophilin-2 or p76(RBE), a protein whose expression is induced by the cyclic AMP pathway in thyrocytes, contains several protein-protein interaction domains including HR-1, Bro1 and PDZ domains, and is a partner of RhoB in its GTP-bound form (Eur J Biochem, 269(24): 6241-9, 2002). We here define its subcellular localization and dissect the significance of its domains. By subcellular fractionation and colocalization experiments, rhophilin-2 is recruited to subcellular organelles by activated RhoB-GTP. As for its yeast homologue, Npi3/Bro1p, the Bro1 domain of rhophilin-2 is necessary to its recruitment to the vesicular structures, which are not labeled for EEA1 nor Lamp1, but well with the late endosome marker CD63.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I.

The multivesicular body (MVB) pathway is responsible for both the biosynthetic delivery of lysosomal hydrolases and the downregulation of numerous activated cell surface receptors which are degraded in the lysosome. We demonstrate that ubiquitination serves as a signal for sorting into the MVB pathway. In addition, we characterize a 350 kDa complex, ESCRT-I (composed of Vps23, Vps28, and Vps37), that recognizes ubiquitinated MVB cargo and whose function is required for sorting into MVB vesicles. This recognition event depends on a conserved UBC-like domain in Vps23. We propose that ESCRT-I represents a conserved component of the endosomal sorting machinery that functions in both yeast and mammalian cells to couple ubiquitin modification to protein sorting and receptor downregulation in the MVB pathway.

Amino Acid Sequence↗
PubMed2002

Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila.

Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. However, factors that regulate endosomal sorting of TKRs are largely unknown. Here, we demonstrate that Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) is one such factor. Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active epidermal growth factor (EGF) and Torso TKRs, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling.

Amino Acid Motifs↗
PubMed2003

The protein network of HIV budding.

HIV release requires TSG101, a cellular factor that sorts proteins into vesicles that bud into multivesicular bodies (MVB). To test whether other proteins involved in MVB biogenesis (the class E proteins) also participate in HIV release, we identified 22 candidate human class E proteins. These proteins were connected into a coherent network by 43 different protein-protein interactions, with AIP1 playing a key role in linking complexes that act early (TSG101/ESCRT-I) and late (CHMP4/ESCRT-III) in the pathway. AIP1 also binds the HIV-1 p6(Gag) and EIAV p9(Gag) proteins, indicating that it can function directly in virus budding. Human class E proteins were found in HIV-1 particles, and dominant-negative mutants of late-acting human class E proteins arrested HIV-1 budding through plasmal and endosomal membranes. These studies define a protein network required for human MVB biogenesis and indicate that the entire network participates in the release of HIV and probably many other viruses.

Animals↗
PubMed2003

Protein sorting into multivesicular endosomes.

Multivesicular endosomes are important as compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes. Work during the past year has shed light on the molecular mechanisms of protein sorting into multivesicular endosomes and yielded information about the machinery involved in multivesicular endosome formation. Monoubiquitination functions as a signal for sorting transmembrane proteins into intraluminal vesicles of multivesicular endosomes and subsequent delivery to lysosomes. A molecular machinery that contains the ubiquitin-binding protein Hrs/Vps27 appears to be central in this sorting process. Three conserved multisubunit complexes, ESCRT-I, -II and -III, are essential for both sorting and multivesicular endosomes formation. Enveloped RNA viruses such as HIV can redirect these complexes from multivesicular endosomes to the plasma membrane to facilitate viral budding.

Animals↗
PubMed2001

Resistance is futile: assimilation of cellular machinery by HIV-1.

HIV-1 budding appears to require Vps4 and Tsg101-two proteins that have links to endosomal sorting machinery. A picture emerges wherein divergent viruses recruit endosomal proteins like Tsg101 to gain access to ubiquitin processes that play a crucial role during viral budding.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting.

The sorting of transmembrane proteins (e.g., cell surface receptors) into the multivesicular body (MVB) pathway to the lysosomal/vacuolar lumen requires the function of the ESCRT protein complexes. The soluble coiled-coil-containing proteins Vps2, Vps20, Vps24, and Snf7 are recruited from the cytoplasm to endosomal membranes where they oligomerize into a protein complex, ESCRT-III. ESCRT-III contains two functionally distinct subcomplexes. The Vps20-Snf7 subcomplex binds to the endosomal membrane, in part via the myristoyl group of Vps20. The Vps2-Vps24 subcomplex binds to the Vps20-Snf7 complex and thereby serves to recruit additional cofactors to this site of protein sorting. We provide evidence for a role for ESCRT-III in sorting and/or concentration of MVB cargoes.

Animals↗
PubMed2003

The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4.

Ubiquitination of the chemokine receptor CXCR4 serves as a targeting signal for lysosomal degradation, but the mechanisms mediating ubiquitination and lysosomal sorting remain poorly understood. Here we report that the Nedd4-like E3 ubiquitin ligase AIP4 mediates ubiquitination of CXCR4 at the plasma membrane, and of the ubiquitin binding protein Hrs on endosomes. CXCR4 activation promotes CXCR4 colocalization with AIP4 and Hrs within the same region of endosomes. Endosomal sorting of CXCR4 is dependent on Hrs as well as the AAA ATPase Vps4, the latter involved in regulating the ubiquitination status of both CXCR4 and Hrs. We propose a model whereby AIP4, Hrs, and Vps4 coordinate a cascade of ubiquitination and deubiquitination events that sort CXCR4 to the degradative pathway.

Adenosine Triphosphatases↗
PubMed2000

Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function 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 and we show that 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. We propose 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.

3T3 Cells↗
PubMed2006

Regulation of ubiquitin-binding proteins by monoubiquitination.

Proteins containing ubiquitin-binding domains (UBDs) interact with ubiquitinated targets and regulate diverse biological processes, including endocytosis, signal transduction, transcription and DNA repair. Many of the UBD-containing proteins are also themselves monoubiquitinated, but the functional role and the mechanisms that underlie this modification are less well understood. Here, we demonstrate that monoubiquitination of the endocytic proteins Sts1, Sts2, Eps15 and Hrs results in intramolecular interactions between ubiquitin and their UBDs, thereby preventing them from binding in trans to ubiquitinated targets. Permanent monoubiquitination of these proteins, mimicked by the fusion of ubiquitin to their carboxyl termini, impairs their ability to regulate trafficking of ubiquitinated receptors. Moreover, we mapped the in vivo monoubiquitination site in Sts2 and demonstrated that its mutation enhances the Sts2-mediated effects of epidermal-growth-factor-receptor downregulation. We propose that monoubiquitination of ubiquitin-binding proteins inhibits their capacity to bind to and control the functions of ubiquitinated targets in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting.

Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it has shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. We describe a complex of Vps27p and Hse1p that localizes to endosomal compartments and is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is as a sorting receptor for ubiquitinated membrane proteins destined for degradation.

Amino Acid Sequence↗
PubMed2003

HIV's great escape.

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Calcium-Binding Proteins↗
PubMed2001

HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress.

Retroviral Gag proteins encode sequences, termed late domains, which facilitate the final stages of particle budding from the plasma membrane. We report here that interactions between Tsg101, a factor involved in endosomal protein sorting, and short peptide motifs in the HIV-1 Gag late domain and Ebola virus matrix (EbVp40) proteins are essential for efficient egress of HIV-1 virions and Ebola virus-like particles. EbVp40 recruits Tsg101 to sites of particle assembly and a short, EbVp40-derived Tsg101-binding peptide sequence can functionally substitute for the HIV-1 Gag late domain. Notably, recruitment of Tsg101 to assembling virions restores budding competence to a late-domain-defective HIV-1 in the complete absence of viral late domain. These studies define an essential virus-host interaction that is conserved in two unrelated viruses. Because the Tsg101 is recruited by small, conserved viral sequence motifs, agents that mimic these structures are potential inhibitors of the replication of these lethal human pathogens.

Amino Acid Motifs↗
PubMed2006

Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting.

Hrs has an essential role in sorting of monoubiquitinated receptors to multivesicular bodies for lysosomal degradation, through recognition of ubiquitinated receptors by its ubiquitin-interacting motif (UIM). Here, we present the structure of a complex of Hrs-UIM and ubiquitin at 1.7-A resolution. Hrs-UIM forms a single alpha-helix, which binds two ubiquitin molecules, one on either side. These two ubiquitin molecules are related by pseudo two-fold screw symmetry along the helical axis of the UIM, corresponding to a shift by two residues on the UIM helix. Both ubiquitin molecules interact with the UIM in the same manner, using the Ile44 surface, with equal binding affinities. Mutational experiments show that both binding sites of Hrs-UIM are required for efficient degradative protein sorting. Hrs-UIM belongs to a new subclass of double-sided UIMs, in contrast to its yeast homolog Vps27p, which has two tandem single-sided UIMs.

Amino Acid Motifs↗