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 SYNJ2BP’s ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.DOI: http://dx.doi.org/10.7554/eLife.24463.001

Novel Channels of the Outer Membrane of Mitochondria: Recent Discoveries Change Our View

“…A promising strategy to obtain such information in living cells is represented by proximity labeling of cytosol‐facing OMM proteins (see e.g., Ref. ). In this approach a monomeric peroxidase called APEX2 was genetically targeted to OMM.…”

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.…”

The cysteine‐free single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications

“…Two generations of APX engineering, namely APEX and APEX2, have led to successful proximity labeling under the reducing environment in mammalian cells. By fusing to various proteins of interest, APEX2 has opened up avenues for spatially resolved proteomic mapping in live cell sub‐compartments, such as ciliary plasma membrane, mitochondrial matrix, endoplasmic reticulum membrane, and nucleus …”

“…By fusing to various proteins of interest, APEX2 has opened up avenues for spatially resolved proteomic mapping in live cell sub-compartments, such as ciliary plasma membrane, mitochondrial matrix, endoplasmic reticulum membrane, and nucleus. [14][15][16][17][18][19] One prerequisite of a good tagging protein is to fold and express well when fusing with a protein of interest. We found that APEX2 fusions, however, often caused dramatically decreased expression in human cells.…”