After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.
The cell's genetic program is modulated by extracellular signals that activate cell surface receptors and, in turn, intracellular effectors, to regulate transcription. For cells to function normally, these signals must be turned off to avoid permanent activation—a situation often associated with cancer. For many receptors, signaling is repressed, or down-regulated, in a process that first internalizes and then degrades the receptors. After receptors are removed from the cell surface into structures called early endosomes, they are selectively incorporated within vesicles that form inside the endosome. During this process, endosomal membranes are pulled away from the cytoplasm towards the endosome lumen, against the flow of intracellular membrane traffic, eventually resulting in the formation of a “multivesicular body” (vesicles within vesicles). The common view is that these intralumenal vesicles are then delivered to lysosomes, where they are degraded along with their receptor cargo. We have investigated the mechanisms responsible for the biogenesis of intralumenal vesicles in multivesicular bodies. We find that the small protein SNX3, which binds the signaling lipid phosphatidyl inositol-3-phosphate, is necessary for the formation of intralumenal vesicles, but is not involved in the degradation of the cell surface receptor for EGF. Conversely, we find that Hrs, which also binds phosphatidyl inositol-3-phosphate and mediates receptor sorting into intralumenal vesicles, is essential for lysosomal targeting but dispensable for multivesicular body biogenesis. Phosphatidyl inositol-3-phosphate thus controls the complementary functions of Hrs and SNX3 in the sorting of signaling receptors and multivesicular body biogenesis.
SNX3 plays a direct role in the formation of intralumenal vesicles of multivesicular bodies (MVBs) but is not involved in EGF receptor degradation, whereas Hrs is essential for lysosomal targeting but dispensable for MVB biogenesis. Hence, intralumenal vesicle formation in MVB biogenesis can be uncoupled from lysosomal targeting.