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 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.

Fig. 1. Labeling the mitochondrial matrix proteome in living HEK cells

(A) Labeling scheme. The APEX peroxidase was genetically targeted to the mitochondrial matrix via fusion to a 24-amino acid targeting peptide (5). Labeling was initiated by addition of biotin-phenol and H₂O₂ to live cells for 1 minute. Cells were then lysed, and biotinylated proteins recovered with streptavidin-coated beads, eluted, separated on a gel, and identified by mass spectrometry. The peroxidase-generated phenoxyl radical is short-lived and membrane-impermeant and hence covalently tags only neighboring and not distant endogenous proteins. (B) Confocal fluorescence imaging of biotinylated proteins (stained with neutravidin), after live labeling of HEK cells expressing mito-APEX as in (A). Controls were performed with either biotin-phenol or H₂O₂ omitted. (C) Super-resolution STORM images showing streptavidin and APEX (AF405/AF647) localization patterns at 22 nm resolution in U2OS cells. Samples were reacted as in (B). (D) Gel analysis of biotinylated mitochondrial matrix proteins, before (lanes 1–3) and after (lanes 4–6) streptavidin bead-enrichment. Samples were labeled as in (B). Substrates are biotinphenol and H₂O₂. Mammalian cells have four endogenously biotinylated proteins, three of which were observed in the negative control lanes (–3) of the streptavidin blot. (E) Electron microscopy (EM) of HEK cells expressing mito-APEX. EM contrast was generated by treating fixed cells with H₂O₂ and diaminobenzidine. APEX catalyzes the polymerization of diaminobenzidine into a local precipitate, which is subsequently stained with electron-dense OsO₄ (5). Dark contrast is apparent in the mitochondrial matrix, but not the intermembrane space.

Fig. 2. Specificity and depth of coverage of the mitochondrial matrix proteome

(A) Analysis of specificity. Left two columns show the fraction of proteins with prior mitochondrial annotation in the entire human proteome (column 1) and in our matrix proteome (column 2). Right two columns show the distribution of proteins with prior sub-mitochondrial localization information, for all mitochondrial proteins (column 3), and for our matrix proteome (column 4). See Table S6 for details. (B) Analysis of depth of coverage. Five groups of well-established mitochondrial matrix proteins (i–v) were crossed with our proteomic list. For each group, 80–91% of proteins were detected in our matrix proteome. See Table S7 for details. (C) Analysis of labeling specificity for protein complexes of the inner mitochondrial membrane. The subunits of Complexes I-IV and F₀-F₁ ATP synthase, for which structural information is available, are illustrated. Subunits detected in our proteome are shaded red; those not detected are shaded grey. Note that because structural information is not available for all 45 subunits of Complex I, some subunits that appear exposed here may not be exposed in the complete complex. (D) Same analysis as in (C), for proteins of the TOM/TIM/PAM protein import machinery that spans the outer and inner mitochondrial membranes. All proteins depicted in (C) and (D) are listed, with additional information, in Table S8.

Fig. 3. Sub-mitochondrial localization of the heme biosynthesis enzymes CPOX and PPOX

(A) Model showing the sub-mitochondrial localizations of the eight core enzymes that catalyze heme biosynthesis, according to previous literature (24). Four of these enzymes are detected in our matrix proteome and are colored red (with log₂(H/L) ratios from replicate 1 shown). Drawing adapted from (25). (B) Domain structures of PPOX and CPOX fusions to APEX, imaged by EM in (C) and (D), respectively. Additional EM images of PPOX-APEX are shown in Fig. S11B. (E) Our model for PPOX and CPOX localization, based on our EM data and previous literature (–22). The predicted membrane-binding region of PPOX (residues 92–209) is colored yellow (26). Hollow arrowheads point to predicted cleavage sites in CPOX. (F) Previous model showing the docking of a PPOX dimer and a FECH (ferrochelatase) dimer through the inner mitochondrial membrane (23). N- and C-terminal ends of PPOX are labeled. Our data contradict this model because the EM images in (C) show that the C-terminus and residue 205 of PPOX are in the matrix, not the IMS.