Biology subjects

Yoo, C.-M.

Publications and source records attributed to Yoo, C.-M..

4 recordsLinked to original sources

bioRxiv2026

Spatial Metabolomics by Desthiobiotin Ligase (DESTNI) in Live Cells

Proximity labeling has transformed spatial proteomics by enabling compartment-resolved mapping of protein environments in living cells, yet its extension to small-molecule metabolites has not been demonstrated, probably due to limitations in labeling chemistry and labeled metabolites identification. Here, we introduce DESTNI, an engineered desthiobiotin (DTB) ligase derived from TurboID through directed evolution, and establish a platform for spatially resolved profiling of amine-containing metabolites. A directed evolution strategy based on a yeast display system yielded DESTNI with efficient DTB-dependent reactivity, enabling robust and compartment-specific proximity labeling across diverse subcellular environments. To identify the DTB-modified amino metabolome, we developed an integrated analytical framework combining DTB-modified amino metabolite standards, in vitro DESTNI profiling, and in silico MS/MS prediction, enabling systematic annotation of DTB-modified amino metabolites. To extend this chemistry to metabolites, we combined synthetic DTB-conjugated metabolite reference standards, in vitro DESTNI-reactive metabolite discovery, and machine-learning prediction of DTB-derivatized metabolites and oligopeptides. Organelle-targeted DESTNI recovered reproducible compartment-enriched amino metabolite signatures, including mitochondrial matrix-enriched glycine, 5-aminolevulinic acid, ornithine and spermidine adducts, as well as nuclear-enriched {gamma}-aminobutyric acid and 5-aminovaleric acid adducts. Together, this work establishes DESTNI as a proximity labeling platform that bridges spatial proteomics and metabolomics and provides a general strategy for mapping subcellular biochemical environments in living cells.

biochemistry↗

bioRxiv2026

Water oxidation-driven histidine dioxidation enables probe-free proximity labeling

Photocatalytic proximity labeling (photo-PL) has emerged as a powerful tool for spatial proteomics in subcellular compartments. However, many photo-PL toolboxes rely on singlet oxygen (1O2) to generate highly unstable endoperoxide intermediates that must be trapped immediately by high concentrations of exogenous probes. This constraint can bias spatial proteome coverage, particularly in dynamic and heterogeneous compartments such as endosomes and exosomes, where probe accessibility is intrinsically nonuniform. Here, we develop IDM, an organic photocatalyst that generates hydroxyl (*OH) and superoxide (O *-) radicals via water oxidation instead of 1O , enabling a synergistic dual-radical mechanism for probe-free proximal protein mapping in live cells (PF-Map). The resulting radicals dioxidize proximal histidine residues into a persistent dioxidized state (His-2O) that is thermodynamically stabilized as lactam tautomers, which remain electrophilic and chemically addressable after cell lysis. By decoupling histidine oxidation from live{square}cell probe capture, PF{square}Map can minimize spatial bias arising from heterogeneous probe distribution. Applying PF{square}Map to intracellular vesicle trafficking, we find that both PF{square}Map and a probe{square}dependent workflow (PD{square}Map) robustly identify exosome markers, whereas PF{square}Map additionally reveals a hidden vesicle trafficking-related subproteome that PD-Map underestimated. Together, we establish a minimally biased photocatalytic strategy for spatial protein mapping in complex biological systems. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/731337v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@13da43dorg.highwire.dtl.DTLVardef@1e9cb7corg.highwire.dtl.DTLVardef@2e7557org.highwire.dtl.DTLVardef@19e16c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

The mitochondrial cohibitin complex facilitates the biogenesis of inner membrane proteins

The inner membrane of mitochondria contains many membrane-embedded carrier proteins of the SLC25 family to facilitate the exchange of metabolites between the cytosol and mitochondria. These carriers use a specific import route for their biogenesis that relies on the TIM22 complex as an inner membrane translocase. The molecular details of carrier biogenesis are not well understood. Using an improved, desthiobiotin-based proximity labeling approach called Destni in living yeast cells, we identified the mitochondrial protein Aim11 as an interactor of newly imported carrier proteins. Aim11 forms a 160 kDa complex together with Iai11, Gep7 and Mtc1 in the mitochondrial inner membrane that we named the comrade-of-prohibitin (cohibitin) complex owing to its genetic interaction with prohibitins. Deletion of Aim11 impairs the import of carrier proteins into the inner membrane and renders cells hypersensitive to carrier overexpression. Our data suggest that the cohibitin complex plays a quality control function that supports the TIM22-mediated insertion of carrier proteins into the inner membrane of mitochondria. SummaryKizmaz et al. identified Aim11 as a novel quality control factor that facilitates the insertion of carrier proteins into the inner membrane of mitochondria. Aim11 is part of the membrane-embedded cohibitin complex which cooperates with prohibitins in inner membrane protein biogenesis.

biochemistry↗

bioRxiv2021

In vivo mitochondrial matrix proteome profiling reveals RTN4IP1/OPA10 as an antioxidant NADPH oxidoreductase

Targeting proximity labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice expressing a mitochondrial matrix-targeted ascorbate peroxidase (MAX-Tg) to analyze tissue-specific matrix proteomes. Desthiobiotin-phenol labeling of muscle tissues from MAX-Tg mice allowed for efficient profiling of mitochondrial-localized proteins in these tissues. Comparative analysis of matrix proteomes from MAX-Tg muscle tissues revealed differential enrichment of mitochondrial proteins related to energy production in between different muscle groups. Reticulon 4 interacting protein 1 (RTN4IP1), also known as Optic Atrophy-10 (OPA10), was highly enriched in the cardiac and soleus muscles and was found to localize to the mitochondrial matrix via a strong mitochondrial targeting sequence at its N-terminus. Protein structure analysis revealed that RTN4IP1 is an NADPH oxidoreductase with structural homology to bacterial quinone oxidoreductase. Enzymatic activity assays, interactome analysis, and metabolite profiling confirmed a function for RTN4IP1 in coenzyme Q (CoQ) biosynthesis. Rtn4ip1-knockout C2C12 cells had reduced CoQ9 levels, were vulnerable to oxidative stress, and had decreased oxygen consumption rates and ATP production. Collectively, RTN4IP1 is a mitochondrial antioxidant NADPH oxidoreductase supporting oxidative phosphorylation activity in muscle tissue.

cell biology↗