Article

Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging

Department of Chemistry, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
Science (Impact Factor: 31.48). 01/2013; 339(6125). DOI: 10.1126/science.1230593
Source: PubMed

ABSTRACT Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living
cells, but only for a handful of recombinant proteins at a time, whereas the latter can detect thousands of endogenous proteins
simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially
and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable
peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach
to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling
was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space
(IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were
reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated
proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular
composition of living cells.

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    • "The topology of MCUR1 has been solved using a proteinase K-based biochemical assay, which showed that its Nand C-terminal residues are projected into the IMS (Mallilankaraman et al. 2012a). Thus, a large portion of the protein (ß250 aa) should span into the matrix, explaining the classification of MCUR1 as a mitomatrix protein (Rhee et al. 2013). "
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