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.
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- "Identifying the trafficking proteins that associate with different vesicle populations is an essential step in understanding the mechanisms that regulate vesicle trafficking. To accomplish this task, specific vesicle populations can be enriched by subcellular fractionation, immuno-isolation, or fluorescence sorting, and then the proteins present can be identified (Franzusoff et al., 1992; Takamori et al., 2006; Duclos et al., 2011; Zhang et al., 2011; Rhee et al., 2013). Two-color fluorescence microscopy is often used to confirm that a candidate protein binds the relevant vesicle population in vivo, in the appropriate biological context. "
ABSTRACT: Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin-binding domain-tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations. © 2015 Bentley et al.The Journal of Cell Biology 01/2015; 208(3). DOI:10.1083/jcb.201408056 · 9.69 Impact Factor
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- "Because cells respond to different fuel sources by utilizing different bioenergetic programs (Stanley et al., 2014), defining these bioenergetic contributions in the context of multiple fuel sources provides added biological relevance. Previous studies have identified the contributions of individual metabolic genes to cancer cell survival (Ros et al., 2012) or tumor formation (Possemato et al., 2011), identified drugs that are effective in distinct bioenergetic programs (Gohil et al., 2010), mapped proteomic components of mitochondria (Pagliarini et al., 2008; Rensvold et al., 2013; Rhee et al., 2013), or derived computational models of central carbon metabolism (Greenberg et al., 2011; Noor et al., 2010; Shlomi et al., 2011). In this study, we developed a high-throughput method in order to identify critical components regulating cellular ATP levels in specific metabolic programs and performed a functional RNAi screen to characterize cellular bioenergetics under glycolytic and oxidative phosphorylation (OXPHOS) conditions. "
ABSTRACT: Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels. We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity. Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.Cell Reports 04/2014; 7(3). DOI:10.1016/j.celrep.2014.03.065 · 8.36 Impact Factor
- "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). "
Dataset: J Physiol Marchi 2014