Search

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. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is a highly conserved lysosomal degradative pathway important for maintaining cellular homeostasis. Much of our current knowledge of autophagy is focused on the initiation steps in this process. Recently, an understanding of later steps, particularly lysosomal fusion leading to autolysosome formation and the subsequent role of lysosomal enzymes in degradation and recycling, is becoming evident. Autophagy can function in both cell survival and cell death, however, the mechanisms that distinguish adaptive/survival autophagy from autophagy-dependent cell death remain to be established. Here, using proteomic analysis of Drosophila larval midguts during degradation, we identify a group of proteins with peptidase activity, suggesting a role in autophagy-dependent cell death. We show that Cp1/cathepsin L-deficient larval midgut cells accumulate aberrant autophagic vesicles due to a block in autophagic flux, yet later stages of midgut degradation are not compromised. The accumulation of large aberrant autolysosomes in the absence of Cp1 appears to be the consequence of decreased degradative capacity as they contain undigested cytoplasmic material, rather than a defect in autophagosome-lysosome fusion. Finally, we find that other cathepsins may also contribute to proper autolysosomal degradation in Drosophila larval midgut cells. Our findings provide evidence that cathepsins play an essential role in the autolysosome to maintain basal autophagy flux by balancing autophagosome production and turnover. Abbreviations: 26-29-p: 26-29kD-proteinase; ADCD: autophagy-dependent cell death; Atg8a: Autophagy-related protein 8a; Cp1/cathepsin L: Cysteine proteinase-1; CtsB: Cathepsin B; cathD: cathepsin D; CtsF: Cathepsin F; GFP: green fluorescent protein; LAMP1: lysosomal-associated membrane protein 1; Mitf: microphthalmia associated transcription factor; PCA: principal component analysis; RNAi: RNA interference; RPF: relative to puparium formation [ABSTRACT FROM AUTHOR]