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

Author:

Hung Victoria1ORCID,Lam Stephanie S1ORCID,Udeshi Namrata D2,Svinkina Tanya2,Guzman Gaelen2,Mootha Vamsi K23ORCID,Carr Steven A2,Ting Alice Y12ORCID

Affiliation:

1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, United States

2. Broad Institute of MIT and Harvard, Cambridge, United States

3. Department of Molecular Biology, Howard Hughes Medical Institute, Massachusetts General Hospital, Harvard Medical School, Boston, United States

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.

Funder

National Institutes of Health

Howard Hughes Medical Institute

National Science Foundation

U.S. Department of Defense

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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