GRASP55 restricts early-stage autophagy and regulates spatial organization of the early secretory network.

Jennifer Y Liu,Jayanta Debnath,Yu-Hsiu Tony Lin,Hector H Huang,Arun P Wiita,Jordan Ye,Andrew M Leidal

doi:10.1242/bio.058736

Jennifer Y Liu, Jayanta Debnath + Show 5 more

Open Access

https://doi.org/10.1242/bio.058736

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Journal: Biology open	Publication Date: Oct 12, 2021
Citations: 4	License type: CC BY 4.0

Affiliation: University of California, San Francisco

Abstract

ABSTRACTThere is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress-response pathway. Prior work has uncovered links between autophagy and the Golgi reassembly stacking protein of 55 kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)-Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.

Highlights

Autophagy is an evolutionarily conserved process by which cytoplasmic contents are delivered via double-membraned autophagosomes to lysosomes for degradation (Dikic and Elazar, 2018)
Loss of Golgi reassembly stacking protein of 55 kDa (GRASP55) enhances early-stage autophagy To investigate the role of GRASP55 in autophagy, we generated stable GRASP55 knockdown and CRISPR-Cas9 deleted HEK293T cells (Fig. S1A,B)
Upon culturing cells in amino acid-free media to induce autophagy, the number of LC3B puncta, a marker of autophagosomes, was significantly greater in GRASP55 knockdown cells compared to controls, both in the absence and presence of the lysosomal inhibitor bafilomycin A1 (BafA1) (Fig. 1A,B), suggesting that loss of GRASP55 enhanced the formation of autophagosomes rather than blocked autophagosometo-autolysosome maturation

Summary

Introduction

Autophagy is an evolutionarily conserved process by which cytoplasmic contents are delivered via double-membraned autophagosomes to lysosomes for degradation (Dikic and Elazar, 2018). Upon culturing cells in amino acid-free media to induce autophagy, the number of LC3B puncta, a marker of autophagosomes, was significantly greater in GRASP55 knockdown cells compared to controls, both in the absence and presence of the lysosomal inhibitor bafilomycin A1 (BafA1) (Fig. 1A,B), suggesting that loss of GRASP55 enhanced the formation of autophagosomes rather than blocked autophagosometo-autolysosome maturation.

Results

Conclusion