Architecture Mapping of the Inner Mitochondrial Membrane Proteome by Chemical Tools in Live Cells

Abstract

The inner mitochondrial membrane (IMM) proteome plays a central role in maintaining mitochondrial physiology and cellular metabolism. Various important biochemical reactions such as oxidative phosphorylation, metabolite production, and mitochondrial biogenesis are conducted by the IMM proteome, and mitochondria-targeted therapeutics have been developed for IMM proteins, which is deeply related for various human metabolic diseases including cancer and neurodegenerative diseases. However, the membrane topology of the IMM proteome remains largely unclear because of the lack of methods to evaluate it in live cells in a high-throughput manner. In this article, we reveal the in vivo topological direction of 135 IMM proteins, using an in situ-generated radical probe with genetically targeted peroxidase (APEX). Owing to the short lifetime of phenoxyl radicals generated in situ by submitochondrial targeted APEX and the impermeability of the IMM to small molecules, the solvent-exposed tyrosine residues of both the matrix and intermembrane space (IMS) sides of IMM proteins were exclusively labeled with the radical probe in live cells by Matrix-APEX and IMS-APEX, respectively and identified by mass spectrometry. From this analysis, we confirmed 58 IMM protein topologies and we could determine the topological direction of 77 IMM proteins whose topology at the IMM has not been fully characterized. We also found several IMM proteins (e.g., LETM1 and OXA1) whose topological information should be revised on the basis of our results. Overall, our identification of structural information on the mitochondrial inner-membrane proteome can provide valuable insights for the architecture and connectome of the IMM proteome in live cells.

BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation

“…Using heavy biotin-phenol in the APEX system coupled to BioSITe is another potential of the method as APEX requires a very short labeling time (<1 min). 2,26 …”

“…The signature ions, m / z 227.08, m / z 480.19 and m / z 497.22, correspond to fragmented biotin, biotin-phenol modified tyrosine immonium ions with loss of NH 3 , and biotin-phenol modified tyrosine immonium ions, respectively (Figure 4B). 2,26 …”

“…26 In this study, desthio-biotin-phenol-containing peptides were enriched and used to define the IMS proteome. When we compared proteins and peptides identified by all three methods (in-gel digestion based methods by Hung et al, desthiobiotin-phenol-based study by Lee et al, and BioSITe), BioSITe yielded more labeled peptides and proteins (Supplementary Figure 3A,B).…”

“…30 Biotinylation of the IMS side in NDUFB8 was not identified by desthiobiotin-phenol-based analysis. 26 Finally, in the case of a known mitochondrial protein, coiled-coil domain containing protein 51 (CCDC51), whose topology has not yet been well characterized, BioSITe led to the identification of a novel site at Y275, which is positioned between the two putative trans-membrane domains, thereby establishing its topology in the inner membrane of the mitochondria. 31 As labeled by IMS-APEX2, we hypothesized that the region containing Y275 is located in the IMS.…”

Small Molecule Interactome Mapping by Photo‐Affinity Labeling (SIM‐PAL) to Identify Binding Sites of Small Molecules on a Proteome‐Wide Scale

“…Enrichment and recovery of the conjugated peptides allows for structural characterization of the profiled interactions. Methods for conjugated peptide recovery include the use of biotin analogs with weaker binding, enzymatically cleavable handles, and the acid‐cleavable handle used in this method (Lee et al., ; Lemeer, Zörgiebel, Ruprecht, Kohl, & Kuster, ; Speers & Cravatt, ; Szychowski et al., ). The acid‐cleavable handle is readily cleaved upon exposure to low pH, conditions that are highly compatible with MS. Further enabling identification of the small molecule–conjugated peptide, SIM‐PAL uses a set of isotopes embedded into the CuAAC handle that are transferred to the small molecule–conjugated peptides, which provides independent verification of small molecule–conjugated peptides by MS.…”