Autophagy captures the retromer-TBC1D5 complex to inhibit receptor recycling

ABSTRACT

Retromer prevents the destruction of numerous receptors by recycling them from endosomes to the trans-Golgi network or plasma membrane. This enables retromer to fine-tune the activity of many signaling pathways in parallel. However, the mechanism(s) by which retromer function adapts to environmental fluctuations such as nutrient withdrawal and how this affects the fate of its cargoes remains incompletely understood. Here, we reveal that macroautophagy/autophagy inhibition by MTORC1 controls the abundance of retromer⁺ endosomes under nutrient-replete conditions. Autophagy activation by chemical inhibition of MTOR or nutrient withdrawal does not affect retromer assembly or its interaction with the RAB7 GAP protein TBC1D5, but rather targets these endosomes for bulk destruction following their capture by phagophores. This process appears to be distinct from amphisome formation. TBC1D5 and its ability to bind to retromer, but not its C-terminal LC3-interacting region (LIR) or nutrient-regulated dephosphorylation, is critical for retromer to be captured by autophagosomes following MTOR inhibition. Consequently, endosomal recycling of its cargoes to the plasma membrane and trans-Golgi network is impaired, leading to their lysosomal turnover. These findings demonstrate a mechanistic link connecting nutrient abundance to receptor homeostasis.

Abbreviations: AMPK, 5’-AMP-activated protein kinase; APP, amyloid beta precursor protein; ATG, autophagy related; BafA, bafilomycin A₁; CQ, chloroquine; DMEM, Dulbecco’s minimum essential medium; DPBS, Dulbecco’s phosphate-buffered saline; EBSS, Earle’s balanced salt solution; FBS, fetal bovine serum; GAP, GTPase-activating protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; LANDO, LC3-associated endocytosis; LP, leupeptin and pepstatin; MTOR, mechanistic target of rapamycin kinase; MTORC1, MTOR complex 1; nutrient stress, withdrawal of amino acids and serum; PDZ, DLG4/PSD95, DLG1, and TJP1/zo-1; RPS6, ribosomal protein S6; RPS6KB1/S6K1, ribosomal protein S6 kinase B1; SLC2A1/GLUT1, solute carrier family 2 member 1; SORL1, sortillin related receptor 1; SORT1, sortillin 1; SNX, sorting nexin; TBC1D5, TBC1 domain family member 5; ULK1, unc-51 like autophagy activating kinase 1; WASH, WASH complex subunit.

KEYWORDS:

• Autophagy
• MTOR
• MTORC1
• retromer
• TBC1D5
• VPS35

Acknowledgements

We thank all laboratory members for insightful discussions and technical advice. We thank Rohan D. Teasdale (University of Queensland) for sharing knock-out cells; Yoon Lim (Centre for Cancer Biology) for assistance with optimising immunoprecipitations; Makoto Kamei (Centre for Cancer Biology & SAHMRI) for assistance with confocal microscopy. Randall Grose and Jarrod Goyne (SAHMRI) for assistance with flow cytometry. Christopher Proud and laboratory (SAHMRI) for sharing plasmids and reagents; Pirjo Apaja (SAHMRI) for sharing reagents. Plasmids from Addgene are indicated in the Materials and Methods section.

This investigation was supported by Lysosomal Health in Ageing at SAHMRI. JMC was supported by a Research Training Stipend and a Commonwealth Scholarship from the Australian Government, a Research Degree Excellence Grant from UniSA, and an EMCR Fellowship from The Hospital Research Foundation Group (2022-CF-EMCR-007). DD was supported by a National Health & Medical Research Council (NHMRC) Project Grant (1124490), an Australian Research Council Discovery Project (DP10100665) and a UniSA Support Package to SK. SK was supported by a NHMRC Senior Principal Research Fellowship (GNT1103006) and a L3 Investigator Grant (2007739).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2281126

Additional information

Funding

The work was supported by the Australian Government Australian Research Council [DP10100665]; National Health and Medical Research Council [GNT1103006]; National Health and Medical Research Council [2007739]; National Health and Medical Research Council [1124490]; University of South Australia; The Hospital Research Foundation Group [2022-CF-EMCR-007].