ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells

Lewis, Samantha C.; Uchiyama, Lauren F.; Nunnari, Jodi

doi:10.1126/science.aaf5549

Cited by 550 publications

(497 citation statements)

References 52 publications

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“…Other organellar membranes are also visible, such as the ER. Consistent with other studies (Kornmann et al, 2009; Lewis et al, 2016), preliminary analysis of our EM data from HEK 293T cells suggests that a substantially larger fraction of nucleoids co-localize with mitochondria-ER contact sites than would be expected if the nucleoids were randomly distributed throughout the matrix (Figure S1G). …”

Section: Discussionsupporting

confidence: 91%

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

Han

Udeshi

Deerinck

et al. 2017

Cell Chemical Biology

114

120

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SUMMARY A recurring challenge in cell biology is to define the molecular components of macromolecular complexes of interest. The predominant method, immunoprecipitation, recovers only strong interaction partners that survive cell lysis and repeated detergent washes. We explored peroxidase-catalyzed proximity biotinylation, APEX, as an alternative, focusing on the mitochondrial nucleoid, the dynamic macromolecular complex that houses the mitochondrial genome. Via 1-min live-cell biotinylation followed by quantitative, ratiometric mass spectrometry, we enriched 37 nucleoid proteins, seven of which had never previously been associated with the nucleoid. The specificity of our dataset was very high, and we validated three hits by follow-up studies. For one novel nucleoid-associated protein, FASTKD1, we discovered a role in downregulation of mitochondrial complex I via specific repression of ND3 mRNA. Our study demonstrates that APEX is a powerful tool for mapping macromolecular complexes in living cells, and can identify proteins and pathways that have been missed by traditional approaches.

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Section: Discussionsupporting

confidence: 91%

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

Han

Udeshi

Deerinck

et al. 2017

Cell Chemical Biology

114

120

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“…The microtubules are of vital importance in this network and are in contact with the sarcoplasmic/ER and mitochondria (FIG. 2b); loss of contact results in Ca 2+ overload in the organelles, unfolded protein response in the ER, and mitochondrial dysfunction [50][51][52][53] . Contact between organelles and traf ficking through the cells is mediated by acetylated microtubules 52 .…”

Section: Key Pointsmentioning

confidence: 99%

Proteostasis in cardiac health and disease

Henning

Brundel²

2017

Nat Rev Cardiol

144

126

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The worldwide increase in life expectancy gives rise to an increasing prevalence of cardiac diseases, which remain the leading cause of disability and death in the developed world [1][2][3] . Currently, the mechanisms under lying how ageing contributes to the initiation or acceler ation of cardiac diseases are essentially unresolved. Prevailing theories of ageing centre on gradual derail ment of cellular protein homeostasis -proteostasis -either by design (genetically) or by 'wear and tear' (environmentally) 3 . Central to the role of proteostasis derailment as a major factor in ageing is the observation that protein function declines over time.Proteins are the most versatile and complex macro molecules and are involved in the proper functioning of every biological process 4 . After synthesis as a poly peptide chain from the genetic code, proper function of a protein relies heavily on its correct folding into a 3D native state and on various post translational modifi cations, mostly involving the addition of chem ical groups (including phosphate, acetate, and carbo hydrate). Protein maturation, transport, and ultimately breakdown is monitored and supported by various classes of proteins, collectively called 'protein quality control' (PQC) [3][4][5] . Balanced proteostasis, therefore, depends on proper PQC and is crucial for cellular and organismal health 6 . The intricate network making up the PQC involves numerous proteins, of which the main players are the chaperones and their regula tors 4,7-9 (assisting in folding and refolding of proteins) and the pathways that clear irreversibly misfolded and aggregation prone proteins (the ubiquitin-proteasome system [UPS] and autophagy systems) 9-11 . With ageing, the gradual accumulation of misfolded or damaged pro teins, together with concomitant failure of PQC, results in proteotoxic stress and compromised defence against reactive oxygen species (ROS) 10 , a process that is likely to be accelerated in various age related diseases includ ing neurodegenerative diseases 12,13 , type 2 diabetes mel litus 14 , cancer 15 , and cardiac diseases [16][17][18][19][20] . In addition to the accumulation of misfolded proteins, ageing is accompanied by an imbalance in the proteome, as indi cated by lowered expression of chaperone, proteaso mal, ribosomal, and mitochondrial proteins, whereas proteins related to oxidative stress are upregulated 21,22 . Although mild impairment of proteostasis extends lifespan in Caenorhabditis elegans, referred to as hormesis, sustained impairment is accompanied by loss of PQC to neutralize this effect 3,5 . Importantly, evidence indicates that the amount of soluble, aggregate prone protein oligomers, rather than the abundance of pro tein aggregates, correlates with disease severity 23,24 . Therefore, protein aggregates, which contain both misfolded proteins and elevated levels of chaper ones, constitute an adequate PQC response, aimed at sequestering potentially harmful protein oligomers, rather than embodying the ultimate cellular threat 25 . This ...

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“…Different forms of membrane contacts display a wide variety of functions depending on the organelles involved and the types of membrane contacts. For instance, the endoplasmic reticulum and mitochondrial contacts (EMC) 3 are the hub for Ca 2ϩ exchange, phospholipid exchange, autophagy, mitochondrial biogenesis, and ER stress response (5)(6)(7)(8)(9)(10). At ER-plasma membrane junctions, in addition to Ca 2ϩ and lipid transport, cell signaling regulation and ER shaping occurs (11)(12)(13).…”

mentioning

confidence: 99%

Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum–mitochondrial contacts

Cho

Adelmant

Lim

et al. 2017

Journal of Biological Chemistry

100

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Edited by John M. DenuTo maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites coupled by protein tethers. In particular, endoplasmic reticulum (ER)-mitochondrial contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca 2؉ and lipids), intracellular signaling, apoptosis, and autophagy. The significance of EMCs has been highlighted by reports indicating that EMC dysregulation is linked to neurodegenerative diseases. Therefore, obtaining a better understanding of the physical and functional components of EMCs should provide new insights into the pathogenesis of several neurodegenerative diseases. Here, we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method. We identified 405 proteins with a 2.0-fold cutoff ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified reticulon 1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication.

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ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells

Cited by 550 publications

References 52 publications

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

Proteostasis in cardiac health and disease

Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum–mitochondrial contacts

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