Combinatorial selective ER-phagy remodels the ER during neurogenesis

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Combinatorial selective ER-phagy remodels the ER during neurogenesis

Melissa J. Hoyer^1,2, Cristina Capitanio^2,3,*, Ian R. Smith^1,*,#, Julia C. Paoli^1,2, Anna Bieber^2,3, Yizhi Jiang^1,2, Joao A. Paulo¹, Miguel A. Gonzalez-Lozano^1,2, Wolfgang Baumeister^2,3,4, Florian Wilfling^2,3,5, Brenda A. Schulman^2,3, J. Wade Harper^1,2 

¹Department of Cell Biology, Harvard Medical School, Boston MA 02115

²Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA

³Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany

⁴Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany

⁵Mechanisms of Cellular Quality Control, Max Planck Institute of Biophysics, 60438 Frankfurt a. M., Germany

*, equal contribution

# Current Address: Velia Therapeutics, San Diego, CA

Corresponding author: wade_harper@hms.harvard.edu

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis: a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodeling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of ER and the role of individual ER-phagy receptors is limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodeling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.