Endofin is required for HD-PTP and ESCRT-0 interdependent endosomal sorting of ubiquitinated transmembrane cargoes

Abstract

Summary Internalized and ubiquitinated signaling receptors are silenced by their intraluminal budding into multivesicular bodies aided by the endosomal sorting complexes required for transport (ESCRT) machinery. HD-PTP, an ESCRT protein, forms complexes with ESCRT-0, -I and -III proteins, and binds to Endofin, a FYVE-domain protein confined to endosomes with poorly understood roles. Using proximity biotinylation, we showed that Endofin forms a complex with ESCRT constituents and Endofin depletion increased integrin α5-and EGF-receptor plasma membrane density and stability by hampering their lysosomal delivery. This coincided with sustained receptor signaling and increased cell migration. Complementation of Endofin- or HD-PTP-depleted cells with wild-type Endofin or HD-PTP, but not with mutants harboring impaired Endofin/HD-PTP association or cytosolic Endofin, restored EGFR lysosomal delivery. Endofin also promoted Hrs indirect interaction with HD-PTP. Jointly, our results indicate that Endofin is required for HD-PTP and ESCRT-0 interdependent sorting of ubiquitinated transmembrane cargoes to ensure efficient receptor desensitization and lysosomal delivery.

Measuring EGFR plasma membrane density, stability, internalization, and recycling in alive adherent cells by cell surface ELISA

“…ESCRT downregulation disrupts cell surface receptor density, trafficking and homeostasis ( Apaja and Lukacs, 2014 ; Apaja et al., 2010 ; Kazan et al., 2021 ; Kharitidi et al., 2015 ). Here, we discuss how to measure receptor plasma membrane density at steady states (i.e., in the absence of receptor activation or inhibition) by cs-ELISA.…”

“…After starvation, one plate is stimulated with EGF (50 ng/mL prepared in 0.5% FBS-DMEM media) for 20 min at 37°C, whereas the second plate is left unstimulated. The 20 min EGF stimulation is sufficient to deliver EGFR towards the endo-lysosomal compartment, however upon ESCRT depletion EGFR trafficking is disrupted and its plasma membrane stability is affected ( Kazan et al., 2021 ). After EGF stimulation, the two plates are washed twice with PBS++ and placed on ice for 5–10 min to impede receptor internalization (keep PBS++ in wells).…”

“…After starvation, one plate is stimulated with EGF (50 ng/mL prepared in 0.5% FBS-DMEM media) for 20 min at 37°C, whereas the second plate is left unstimulated. The 20 min EGF stimulation is sufficient to deliver EGFR towards the endo-lysosomal compartment, however upon ESCRT depletion EGFR trafficking is disrupted and its plasma membrane stability is affected ( Kazan et al., 2021 ).…”

“…After starvation, one plate is stimulated with EGF (50 ng/mL prepared in 0.5% FBS-DMEM media) for 5 min at 37°C, whereas the second plate is left unstimulated. The 5 min EGF stimulation is sufficient to induce the rapid internalization of EGFR ( Kazan et al., 2021 ).…”

Unconventional Protein Secretion Dependent on Two Extracellular Vesicles: Exosomes and Ectosomes

“…Discoveries of a few years ago demonstrated the targeting and association of single proteins to the growing ILV lumen ( Chen et al, 2021 ). Details have been clarified by the identification of proteins, such as endofin and arrestin-domain containing protein (ARRDC1), in the assembly of cargos ( Ageta and Tsuchida, 2019 ; Kazan et al, 2021 ). Condensates of another protein of ample specificity, YBX1, have been shown to induce abundant liquid-liquid phase separations selectively recruiting a single miRNA, miR223 ( Liu H. et al, 2021 ).…”