Rabenosyn-5 defines the fate of the transferrin receptor following clathrin-mediated endocytosis

Abstract

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.endosomal sorting | lysosomal degradation | super resolution | receptor trafficking | patterned illumination T he endocytic pathway is known principally for its role in the uptake of receptors and ligands (1-4) and increasingly is being recognized as critical in other aspects of cell physiology. For example, the control of cellular growth through mammalian target of rapamycin signaling involves associations with late endosomal membranes (5, 6), and microRNA regulatory mechanisms are associated with endosomal/lysosomal membranes (7-9). Thus, it is necessary to understand how the distinct membrane compartments of the endocytic pathway form, traffic internalized ligands, and serve as scaffolds for the assembly of regulatory complexes.In the classical view of the endocytic pathway (3, 10-13), internalizing cargoes are incorporated into vesicles formed by clathrin-mediated or other mechanisms of endocytosis at the plasma membrane. These vesicles fuse with a homogenous population of early endosomes from which cargo then is sorted to different destinations. However, recent results demonstrate that genetic networks involved in the early trafficking of the transferrin (Tf) and EGF receptors (TfR and EGFR, respectively) differ from each other substantially (14) and find that EGF and Tf populate different subsets of vesicles almost immediately after addition to live cells at physiological temperature (15). Further evidence for early endosome heterogeneity is found in studies in which endosomes display different motilities and different phosphoinositide effector complements (16-18). These results suggest that different populations of e...

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“…Recent work highlighted initial sorting of transferrin receptor (TfR) traffic to require the divalent Rab5/Rab4 effector rabenosyn-5 (42). Early studies have demonstrated depletion of rabenosyn-5 to delay receptor recycling (43) and more recently redirect traffic of TfR from the normal pathway of rapid recycling to targeting to lysosomes for degradation (42). Bivalent Rab4 and Rab5 effector proteins, such as rabenosyn-5 and rabaptins, act cooperatively to link Rab5-and Rab4-containing microdomains on early endosomes (44).…”