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

Department of Chemistry, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
Science (Impact Factor: 33.61). 01/2013; 339(6125). DOI: 10.1126/science.1230593
Source: PubMed


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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    • "The answer to the above proposed questions is challenging, because, as described above, there exist a diversity of import routes for mitochondrial translocation and, perhaps more importantly, because we have still an incomplete knowledge of the mitochondrial proteome [19]. It is unclear how many proteins exist in the organelle, where they are localized and what are their specific mechanisms of import. "
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    ABSTRACT: The eukaryotic cell, with its organelle organization, represents a challenge for protein traffic. Contrary to what occurs in the endoplasmic reticulum, mitochondrial protein import is proposed to occur posttranslational, as proteins are synthesized in cytoplasmic ribosome and only then imported to the organelle. Because the diameter of the Tom and Tim pores is too narrow for the passage of a folded protein, it is assumed that polypeptides must be already in an unfolded, import competent, state for organelle entry. However, it has been suggested that mitochondria might be able to actively unfold proteins itself at the outer membrane. Here we discuss the influence of cytoplasmatic protein folding on mitochondrial import. Despite the contribution of active mitochondrial unfolding to protein import is not excluded, this mechanism is in consistent with a number of experimental evidences. Accordingly, other alternative models for mitochondrial import are here discussed. Understanding the molecular constraints regulating this process is of crucial importance, since its failure can lead to a number of pathological situations.
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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. "
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    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.
    Preview · Article · Jan 2015 · The Journal of Cell Biology
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    • "Thus, we conclude that a pool of mitochondria-associated ARL2 is in the matrix, though we cannot exclude the possibility that some ARL2 is also in the IMS. Indeed, we believe there is a pool of ARL2 in the IMS, based upon our earlier sub-mitochondria fractionation data [13] and more recent techniques that combine proteomics with spatially restricted protein tagging [15]; each of which concluded that ARL2 is in the IMS. Because ARL2 is present in mitochondria in amounts that preclude a stoichiometric binding to any of the complexes of the electron transport chain or transporters and is not found in any stable complexes, it is likely that ARL2 actions in the matrix are catalytic or regulatory and transient in nature, rather than as a stoichiometric component of a complex [62]. "
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    ABSTRACT: ARF-like 2 (ARL2) is a member of the ARF family and RAS superfamily of regulatory GTPases, predicted to be present in the last eukaryotic common ancestor, and essential in a number of model genetic systems. Though best studied as a regulator of tubulin folding, we previously demonstrated that ARL2 partially localizes to mitochondria. Here, we show that ARL2 is essential to a number of mitochondrial functions, including mitochondrial morphology, motility, and maintenance of ATP levels. We compare phenotypes resulting from ARL2 depletion and expression of dominant negative mutants and use these to demonstrate that the mitochondrial roles of ARL2 are distinct from its roles in tubulin folding. Testing of current models for ARL2 actions at mitochondria failed to support them. Rather, we found that knockdown of the ARL2 GTPase activating protein (GAP) ELMOD2 phenocopies two of three phenotypes of ARL2 siRNA, making it a likely effector for these actions. These results add new layers of complexity to ARL2 signaling, highlighting the need to deconvolve these different cell functions. We hypothesize that ARL2 plays essential roles inside mitochondria along with other cellular functions, at least in part to provide coupling of regulation between these essential cell processes.
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