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

Abstract

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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Regulation of the EGF Transcriptional Response by Endocytic Sorting

“…[ALG-2 (apoptosis-linked gene 2) interacting protein X], which inhibits formation of intralumenal vesicles (10,46). Knockdown of all candidates was nearly complete at protein level (Fig.…”

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