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

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, while 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. The 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. The specificity of live-cell peroxidase-mediated proteomic mapping combined with its ease of use offers biologists a powerful tool for understanding the molecular composition of living cells.

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

“…NSUN3 was suggested to localise to mitochondria (Rhee et al , 2013); however, the target spectrum and biological function of the protein have remained unknown. To confirm the mitochondrial localisation of NSUN3, we generated a HEK293 cell line stably expressing NSUN3‐GFP from a tetracycline‐inducible promoter.…”

Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

“…ucsd.edu. 1 To whom correspondence may be addressed. Email: clchen@genetics.med.harvard.edu or perrimon@receptor.med.harvard.edu.…”

“…Recently, a method based on an engineered ascorbate peroxidase (APEX) has been developed and shown to function in cultured mammalian cells for proteomic mapping (1). Upon activation, the APEX enzyme turns a biotin-phenol substrate into a highly reactive radical that covalently tags neighboring proteins on electron-rich amino acids such as tyrosine.…”

Regulation of heme biosynthesis and transport in metazoa

“…The enzyme receiving CPgenIII, coproporphyrinogen oxidase (CPOX), is anchored on the mitochondrial matrix with the active site in the intermembrane space [62]. ABCB6 was proposed to be responsible for transporting CPgenIII across the mitochondrial outer membrane [63].…”

“…The product of CPOX, protoporphyrinogen IX (PPgenIX), is then converted into PPIX by protoporphyrinogen oxidase (PPOX), which forms a complex with the final enzyme FECH in the mitochondrial matrix [62,65,66]. A recent report showed that PPgenIX may be transported to PPOX by transmembrane protein 14C (TMEM14C) [67].…”