The epidermal growth factor receptor (EGFR) is targeted for lysosomal degradation by ubiquitin-mediated interactions with the ESCRTs (endosomal-sorting complexes required for transport) in multivesicular bodies (MVBs). We show that secretory carrier membrane protein, SCAMP3, localizes in part to early endosomes and negatively regulates EGFR degradation through processes that involve its ubiquitylation and interactions with ESCRTs. SCAMP3 is multimonoubiquitylated and is able to associate with Nedd4 HECT ubiquitin ligases and the ESCRT-I subunit Tsg101 via its PY and PSAP motifs, respectively. SCAMP3 also associates with the ESCRT-0 subunit Hrs. Depletion of SCAMP3 in HeLa cells by inhibitory RNA accelerated degradation of EGFR and EGF while inhibiting recycling. Conversely, overexpression enhanced EGFR recycling unless ubiquitylatable lysines, PY or PSAP motifs in SCAMP3 were mutated. Notably, dual depletions of SCAMP3 and ESCRT subunits suggest that SCAMP3 has a distinct function in parallel with the ESCRTs that regulates receptor degradation. This function may affect trafficking of receptors from prelysosomal compartments as SCAMP3 depletion appeared to sustain the incidence of EGFR-containing MVBs detected by immunoelectron microscopy. Together, our results suggest that SCAMP3, its modification with ubiquitin, and its interactions with ESCRTs coordinately regulate endosomal pathways and affect the efficiency of receptor down-regulation. INTRODUCTIONThe internalization of cell surface receptors and transporters coupled to degradation or recycling is critical for nutrient uptake and regulating cell signaling. The epidermal growth factor receptor (EGFR) has been studied extensively as a prototypical receptor that is targeted for lysosomal degradation after ligand-stimulated internalization. Disrupted degradation of the EGFR and other receptors has been linked to the pathogenesis of many diseases including several cancers. An essential element in the down-regulation process is ubiquitin conjugation to EGFR by Cbl, an E3 ubiquitin ligase (reviewed in Marmor and Yarden, 2004). It is thought that the ubiquitin enables successive passage of the receptor to the endosomal-sorting complexes required for transport: ESCRT-0, I, II, and III. These complexes are evolutionarily conserved and specialize in targeting and packaging of membrane proteins into intraluminal vesicles (ILVs) upstream of lysosomal degradation. The ESCRT-0 complex is composed of Hrs, STAM, and Eps15b and is enriched in flat clathrin patches on early endosomes via interactions involving Hrs' FYVE and coiled-coiled domains (Raiborg et al., 2001a(Raiborg et al., , 2002Bache et al., 2003b;Roxrud et al., 2008). Ubiquitin-interacting motifs (UIMs) in ESCRT-0 subunits bind ubiquitylated ligands such as EGFR; interaction of Hrs with the UEV domain of ESCRT-I protein Tsg101 is thought to facilitate passage of the receptor to ESCRT-I (Polo et al., 2002;Bache et al., 2003a;Lu et al., 2003). ESCRT-I in turn recruits ESCRT-II, which may function in sequential liga...
Traffic at the trans-Golgi network (TGN) and endosomes is regulated by glucose via an unknown mechanism that depends on protein kinase A (PKA). TGN–endosomal clathrin adaptors exhibit specific responses to glucose starvation that likely are coordinated with other cell behaviors regulated by PKA.
Localization of endosomal clathrin adaptors is regulated by glucose availability via an unknown mechanism. Studies in intact and permeabilized cells show that clathrin adaptor localization is precisely tuned by cellular ATP concentration. These data provide evidence for how membrane traffic is coordinated with overall proliferation rates.
Background: There are multiple interacting clathrin adaptors at the trans-Golgi network and endosomes in yeast. Results: Autoregulation of one adaptor, Gga2, alters the temporal delay between recruitment of Gga2 and a second adaptor, Ent5, to organelles. Conclusion:The interaction between Gga2 and Ent5 is regulated by an autoregulatory sequence. Significance: This autoregulatory mechanism may ensure accurate membrane traffic in vivo.
Background Information. In the yeast Saccharomyces cerevisiae, acute glucose starvation induces rapid endocytosis followed by vacuolar degradation of many plasma membrane proteins. This process is essential for cell viability, but the regulatory mechanisms that control it remain poorly understood. Under normal growth conditions, a major regulatory decision for endocytic cargo occurs at the trans-Golgi network (TGN) where proteins can recycle back to the plasma membrane or can be recognized by TGN-localised clathrin adaptors that direct them towards the vacuole. However, glucose starvation reduces recycling and alters the localization and post-translational modification of TGN-localised clathrin adaptors. This raises the possibility that during glucose starvation endocytosed proteins are routed to the vacuole by a novel mechanism that bypasses the TGN or does not require TGN-localised clathrin adaptors. Results. Here, we investigate the role of TGN-localised clathrin adaptors in the traffic of several amino acid permeases, including Can1, during glucose starvation. We find that Can1 transits through the TGN after endocytosis in both starved and normal conditions. Can1 and other amino acid permeases require TGN-localised clathrin adaptors for maximal delivery to the vacuole. Furthermore, these permeases are actively sorted to the vacuole, because ectopically forced de-ubiquitination at the TGN results in the recycling of the Tat1 permase in starved cells. Finally, we report that the Mup1 permease requires the clathrin adaptor Gga2 for vacuolar delivery. In contrast, the clathrin adaptor protein complex AP-1 plays a minor role, potentially in retaining permeases in the TGN, but it is otherwise dispensable for vacuolar delivery. Conclusions and significance. This work elucidates one membrane trafficking pathway needed for yeast to respond to acute glucose starvation. It also reveals the functions of TGNlocalised clathrin adaptors in this process. Our results indicate that the same machinery is needed for vacuolar protein sorting at the GN in glucose starved cells as is needed in the presence of glucose. In addition, our findings provide further support for the model that the TGN is a transit point for many endocytosed proteins, and that Gga2 and AP-1 function in distinct pathways at the TGN.
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