STAM and Hrs Are Subunits of a Multivalent Ubiquitin-binding Complex on Early Endosomes

Bache, Kristi Cecilie Gronvold; Raiborg, Camilla; Mehlum, Anja; Stenmark, Harald

doi:10.1074/jbc.m210843200

Cited by 292 publications

(296 citation statements)

References 65 publications

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“…In contrast, we find that Hrs is not involved in the process. However, the functions of Hrs in lumenal vesicle formation (Lloyd et al, 2002;Bache et al, 2003b;Razi and Futter, 2006) are not clear and may well be indirect (Pons et al, 2008). Although Alix negatively regulates the formation of lumenal vesicles and EGF receptor sorting, our data show that Tsg101 is a necessary component of the budding reaction.…”

Section: Discussionmentioning

confidence: 70%

“…Evidence shows that this PtdIns3P-, Hrs-, and ESCRT-based sorting mechanism is coupled to the membrane invagination process. The number of intralumenal vesicles within endosomes is decreased after PtdIns 3-kinase inhibition (Reaves et al, 1996;Fernandez-Borja et al, 1999;Futter et al, 2001), and after Hrs knockout in Drosophila melanogaster (Lloyd et al, 2002) or knockdown in mammalian cells (Bache et al, 2003b;Razi and Futter, 2006). Presumably, Hrs, by initiating ES-CRT recruitment, is indirectly responsible for intralumenal vesicle formation (Bache et al, 2003a).…”

Section: Introductionmentioning

confidence: 99%

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In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

Falguières

Luyet

Bissig

et al. 2008

MBoC

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Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively. INTRODUCTIONIn eukaryotic cells, molecules of the plasma membrane, ligands and solutes are internalized into early endosomes, from where they can be recycled back to the plasma membrane, transported to the trans-Golgi network, or targeted to late endosomes and lysosomes (Gruenberg, 2001;Maxfield and McGraw, 2004;Mayor and Pagano, 2007). The latter pathway is followed in particular by ubiquitinated signaling receptors that need to be down-regulated. These receptors are incorporated into invaginations of the early endosomal membrane that form within their lumen (Hurley and Emr, 2006), thus giving rise to nascent multivesicular bodies (MVBs) or endosomal carrier vesicles (ECVs; Gruenberg and Stenmark, 2004;Piper and Katzmann, 2007), which function as intermediates between early and late endosomes. Eventually, intralumenal vesicles are delivered to lysosomes, where they are degraded together with their receptor cargo.Major progress has been made in our understanding of the molecular mechanisms that control the sorting of ubiquitinated receptors, in particular the epidermal growth factor (EGF) receptor (Hurley and Emr, 2006;Slagsvold et al., 2006;Piper and Katzmann, 2007;Williams and Urbe, 2007). These receptors interact via the ubiquitin moiety with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), which in turn binds clathrin and phosphatidyl-inositol-3-phosphate (PtdIns3P), thereby concentrating the receptor in early endosomal membrane domains (Raiborg et al., 2001;Urbe et al., 2003;Raiborg et al., ...

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Section: Discussionmentioning

confidence: 70%

Section: Introductionmentioning

confidence: 99%

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

Falguières

Luyet

Bissig

et al. 2008

MBoC

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“…It has also been shown that Hrs recruits STAM to the endosomal membrane, and that Hrs, STAM and Eps15 co-localize with ubiquitinated proteins in clathrin-positive microdomains on early endosomes (Asao et al, 1997;Bache et al, 2003). As both Hrs, STAM and Eps15 bind ubiquitinated proteins, they have been suggested to function together in the capture of ubiquitinated proteins for sorting into MVBs (Bache et al, 2003).…”

Section: Endosomal Sorting Of Ubiquitinated Cargomentioning

confidence: 99%

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Grøvdal

Stang

Sorkin

et al. 2004

Experimental Cell Research

160

143

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“…Depletion of Hrs or STAM using RNA interference (RNAi) or gene disruption inhibits RTK down-regulation in mammalian cells (Bache et al, 2003b;Hammond et al, 2003;Kanazawa et al, 2003;Lu et al, 2003) and in Drosophila (Lloyd et al, 2002). The budding yeast Saccharomyces cerevisiae also has orthologues of Hrs and STAM, which are Vps27 (vacuolar protein sorting 27) and Hse1 (Hbp/STAM/EAST 1), respectively.…”

Section: Introductionmentioning

confidence: 99%

“…Endosomal sorting of ubiquitinated RTKs is initiated by a complex of two Ub-binding proteins, hepatocyte growth factor-regulated substrate (Hrs) and signal-transducing adaptor molecule (STAM), which localizes on the cytoplasmic face of the early endosomal membrane (Komada et al, 1997;Bache et al, 2003b;Mizuno et al, 2003Mizuno et al, , 2004. Depletion of Hrs or STAM using RNA interference (RNAi) or gene disruption inhibits RTK down-regulation in mammalian cells (Bache et al, 2003b;Hammond et al, 2003;Kanazawa et al, 2003;Lu et al, 2003) and in Drosophila (Lloyd et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

Mizuno

Iura

Mukai

et al. 2005

MBoC

259

264

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Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This "receptor down-regulation" process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.

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STAM and Hrs Are Subunits of a Multivalent Ubiquitin-binding Complex on Early Endosomes

Cited by 292 publications

References 65 publications

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

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