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Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation

ABSTRACT: The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BPs ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000091659 | MassIVE | Fri Apr 07 05:56:00 BST 2023

REPOSITORIES: MassIVE

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Project description:SEPN1 is a type II protein of the endoplasmic reticulum (ER) whose loss of function gives rise to a collection of debilitating autosomal recessive myopathies gathered under the umbrella term of SEPN1-related myopathy (RM). At the moment, SEPN1-RM lacks an effective pharmacological treatment; thus, the medical management of the disease is only supportive. The potentially fatal diaphragmatic weakness leading to respiratory insufficiency in patients still ambulant is the main reason for medical concerns. Thus, studies on SEPN1-RM pathogenesis are necessary to implement a targeted pharmacological therapy aimed at relieving the general muscle weakness and the more worrisome diaphragmatic dysfunction. Here, we show a physical and functional interaction between SEPN1 and the ER stress mediator ERO1 alpha (henceforth, ERO1). Both SEPN1 and ERO1 are involved in the redox regulation of proteins into the ER, although in an opposite way SEPN1 imposes a less oxidant ER poise while ERO1 a more oxidant one, conceivable with a reductase function of SEPN1 and an oxidase one of ERO1. Furthermore, both are mainly localized in a region of the ER in close contact with mitochondria termed mitochondria-associated membranes (MAMs) and their loss impacts oppositely on the short-range MAMs, thereby impinging ER-mitochondria Ca2+ dynamics, OXPHOS, and bioenergetics. We find that ERO1 depletion restores the impaired short-range MAMs due to SEPN1 loss together with mitochondrial ATP. ERO1 knockout in a mouse background of SEPN1 loss blunts ER stress and the consequent Unfolded Protein Response (UPR) while it rescues the diaphragmatic weakness by improving ER/mitochondria contacts, calcium dynamics, and OXPHOS. Importantly, treatments of SEPN1 knock out mice with the chemical chaperone tauroursodeoxycholic acid (TUDCA) mimic the results of ERO1 loss improving calcium dynamics, OXPHOS, ER/mitochondria contacts, thereby rescuing diaphragmatic weakness as well. In addition, TUDCA-treated SEPN1-RM patients-derived myoblasts show a dynamic dose-dependent increase in ATP levels under ER stress conditions suggesting improved mitochondrial function. Thus, our findings point to the ERO1 axis in the pathogenesis of SEPN1-RM thereby impinging on MAMs and mitochondria bioenergetics and recall for the efficacy of a pharmacology therapy with ad hoc ER stress/ERO1 inhibitors for SEPN1-RM.

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Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation

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