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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Apr 25, 2017
- Source ID
- 10.7554/elife.24463
Entities
People
- Alice Y Ting
- Gaelen Guzman
- Namrata D Udeshi
- Stephanie S Lam
- Steven A Carr
- Tanya Svinkina
- Vamsi Mootha
- Victoria Hung
Organizations
- Broad Institute
- Harvard Medical School
- Howard Hughes Medical Institute
- Massachusetts Institute of Technology
- National Institutes of Health
- National Science Foundation
- United States Department of Defense