Article

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: 31.48). 01/2013; DOI: 10.1126/science.1230593
Source: PubMed

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, while 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. The 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. The specificity of live-cell peroxidase-mediated proteomic mapping combined with its ease of use offers biologists a powerful tool for understanding the molecular composition of living cells.

1 Bookmark
 · 
82 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism. Copyright © 2014 Elsevier Inc. All rights reserved.
    Cell 12/2014; 159(7):1615-1625. DOI:10.1016/j.cell.2014.11.046 · 33.12 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Defects in autophagy have been linked to a wide range of medical illnesses, including cancer as well as infectious, neurodegenerative, inflammatory, and metabolic diseases. These observations have led to the hypothesis that autophagy inducers may prevent or treat certain clinical conditions. Lifestyle and nutritional factors, such as exercise and caloric restriction, may exert their known health benefits through the autophagy pathway. Several currently available FDA-approved drugs have been shown to enhance autophagy, and this autophagy-enhancing action may be repurposed for use in novel clinical indications. The development of new drugs that are designed to be more selective inducers of autophagy function in target organs is expected to maximize clinical benefits while minimizing toxicity. This Review summarizes the rationale and current approaches for developing autophagy inducers in medicine, the factors to be considered in defining disease targets for such therapy, and the potential benefits of such treatment for human health.
    Journal of Clinical Investigation 01/2015; 125(1):14-24. DOI:10.1172/JCI73938 · 13.77 Impact Factor
  • [Show abstract] [Hide abstract]
    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.