Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum–mitochondrial contacts

Abstract

Edited by John M. DenuTo maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites coupled by protein tethers. In particular, endoplasmic reticulum (ER)-mitochondrial contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca 2؉ and lipids), intracellular signaling, apoptosis, and autophagy. The significance of EMCs has been highlighted by reports indicating that EMC dysregulation is linked to neurodegenerative diseases. Therefore, obtaining a better understanding of the physical and functional components of EMCs should provide new insights into the pathogenesis of several neurodegenerative diseases. Here, we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method. We identified 405 proteins with a 2.0-fold cutoff ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified reticulon 1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication.

The axonal endoplasmic reticulum: One organelle—many functions in development, maintenance, and plasticity

“…Importantly, MAMs are particularly abundant in axons, suggesting a major interdependence of axonal function and mitochondrial dynamics (Bird, 1978;McGraw et al, 1980;Westrum and Gray, 1986;Wu et al, 2017). ER tethers include reticulon 1C, that interacts with components of MAMs regulating lipid exchange (Reali et al, 2015), and reticulon 1A, that promotes the formation of MAMs (Cho et al, 2017).…”

Proximity labeling of protein complexes and cell type-specific organellar proteomes in Arabidopsis enabled by TurboID

“…mitochondrial matrix and intermembrane space (Rhee et al 2013;Hung et al 2016)) as well as for local proteomes (e.g. inside cilia (Mick et al 2015), at synaptic clefts , at endoplasmic reticulum-mitochondria contact sites (Cho et al 2017;Hung et al 2017), demonstrate the usefulness and versatility of these techniques. Although the potential utility for PL in plants is equally tremendous, its adaptation in plants is proving challenging.…”

Mitochondrial-nuclear heme trafficking is regulated by GTPases that control mitochondrial dynamics

“…Scenario “3” and “4”-based mechanisms are motivated by studies on lipid trafficking, albeit there is scant experimental evidence for their respective roles in trafficking heme, a lipid-like metabolite 2 . Notably, the putative heme trafficking factor PGRMC1 has recently been identified as part of the mammalian ER-mitochondrial tethering complex 41 , thus direct interactions between mitochondria and ER may facilitate heme trafficking through out the cell, including to the nucleus. Since mitochondrial fission and fusion factors have been physically and functionally linked to mitochondrial-ER contact sites 25 , we speculate that Dnm1/DRP1 and Mgm1/OPA1 may also act to regulate heme trafficking via the ER.…”