Defining the protein and lipid constituents of tubular recycling endosomes

Abstract

Once internalized, receptors reach the sorting endosome and are either targeted for degradation or recycled to the plasma membrane, a process mediated at least in part by tubular recycling endosomes (TREs). TREs may be efficient for sorting owing to the ratio of large surface membrane area to luminal volume; following receptor segregation, TRE fission likely releases receptor-laden tubules and vesicles for recycling. Despite the importance of TRE networks for recycling, these unique structures remain poorly understood, and unresolved questions relate to their lipid and protein composition and biogenesis. Our previous studies have depicted the endocytic protein MICAL-L1 as an essential TRE constituent, and newer studies show a similar localization for the GTP-binding protein Rab10. We demonstrate that TREs are enriched in both phosphatidic acid (PA) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), supporting the idea of MICAL-L1 recruitment by PA and Rab10 recruitment via PI(4,5)P2. Using siRNA knock-down, we demonstrate that Rab10-marked TREs remain prominent in cells upon MICAL-L1 or Syndapin2 depletion. However, depletion of Rab10 or its interaction partner, EHBP1, led to loss of MICAL-L1-marked TREs. We next used phospholipase D inhibitors to decrease PA synthesis, acutely disrupt TREs, and enable monitoring of TRE regeneration after inhibitor washout. Rab10 depletion prevented TRE regeneration, whereas MICAL-L1 knock-down did not. It is surprising that EHBP1 depletion did not affect TRE regeneration under these conditions. Overall, our study supports a primary role for Rab10 and the requirement for PA and PI(4,5)P2 in TRE biogenesis and regeneration, with Rab10 likely linking the sorting endosome to motor proteins and the microtubule network.

Endosomal Phosphatidylinositol-3-Phosphate-Associated Functions Are Dispensable for Establishment of the Cytomegalovirus Pre-Assembly Compartment but Essential for the Virus Growth

“…The switch from Rab5-/PI(3)P-positive membranes towards the tubular subdomains involves replacing Rab5 with other GTPases. Among the eight Rab subfamily members that localize at tubular endosomes, Rab10 and Rab22a are essential for tubulation of endosomal membranes that sort clathrin-independent cargo [ 69 , 70 ], whereas Rab11a plays a key role in the segregation of clathrin-dependent cargo (i.e., transferrin receptor) [ 22 , 71 ]. These small GTPases are associated with membranes with lipid composition other than PI(3)P [ 70 , 72 ], indicating that their recruitment and activation are associated with the PI conversion.…”

“…Among the eight Rab subfamily members that localize at tubular endosomes, Rab10 and Rab22a are essential for tubulation of endosomal membranes that sort clathrin-independent cargo [ 69 , 70 ], whereas Rab11a plays a key role in the segregation of clathrin-dependent cargo (i.e., transferrin receptor) [ 22 , 71 ]. These small GTPases are associated with membranes with lipid composition other than PI(3)P [ 70 , 72 ], indicating that their recruitment and activation are associated with the PI conversion. Although the conversion mechanism of Rab5a-to-Rab11a is not fully elucidated, recent studies suggest that activation of Rab11a at endosomes requires localized production of PI(3)P by the class-II PI3K [ 22 ] and recycling cargo sorting depends on the background PI(3)P levels at EEs generated by the Vps34 activity [ 71 ].…”

A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C driven phosphoinositide conversion

“…However, the membrane identity of cargo vesicles directly prior to plasma membrane delivery is poorly defined. Importantly, phosphatidylinositol-4 phosphate (PI(4)P) is established on vesicles originating from both the Golgi and recycling endosomes (30)(31)(32). Furthermore, the delivery of cell polarity proteins to the plasma membrane, such as ß-integrin and e-cadherin, is dependent on the direct recruitment of phosphatidylinositol 4-phosphate 5kinase type-1 gamma (PIP5K1C), which converts PI(4)P into PI(4,5)P2 (25,33).…”

“…Furthermore, the delivery of cell polarity proteins to the plasma membrane, such as ß-integrin and e-cadherin, is dependent on the direct recruitment of phosphatidylinositol 4-phosphate 5kinase type-1 gamma (PIP5K1C), which converts PI(4)P into PI(4,5)P2 (25,33). This dependence implies that the phosphoinositide composition of some cargo vesicles is converted to PI (4,5)P2 along the route to the plasma membrane (32). Indeed, this outcome has been directly observed during the polarized trafficking of Par proteins during oogenesis in Drosophila (34).…”

Endosomal recycling tubule scission and integrin recycling involve the membrane curvature-supporting protein LITAF

“…The ERC is functionally interconnected with another recycling compartment, the tubular recycling endosome (TRE) ( Cullen and Steinberg, 2018 ; Dhawan et al, 2020 ; Naslavsky et al, 2006 ). The TRE is controlled by ARF6 ( Radhakrishna and Donaldson, 1997 ) and Rab10 ( Etoh and Fukuda, 2019 ; Farmer et al, 2021 ).…”