Correction: Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation.
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ABSTRACT:
SUBMITTER: Hung V
PROVIDER: S-EPMC6682411 | biostudies-literature | 2019 Aug
REPOSITORIES: biostudies-literature
Publications
Correction: Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation.
Hung Victoria V Lam Stephanie S SS Udeshi Namrata D ND Svinkina Tanya T Guzman Gaelen G Mootha Vamsi K VK Carr Steven A SA Ting Alice Y AY
eLife 20190805
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Project description:The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP's ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.
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Project description:The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BPs ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.
2023-04-07 | MSV000091659 | MassIVE
Project description:Despite ongoing efforts to control malaria infection, progress in lowering the number of deaths and infections appears to have stalled. The continued high incidence of malaria infection and mortality is in part due to emergence of parasites resistant to frontline antimalarials. This highlights the need for continued identification of novel protein drug targets. Mitochondrial functions in Plasmodium falciparum, the deadliest species of human malaria parasite, are targets of validated antimalarials including atovaquone and proguanil (Malarone). Thus, there has been great interest in identifying other essential mitochondrial proteins as candidates for novel drug targets. Garnering an increased understanding of the proteomic landscape inside the P. falciparum mitochondrion will also allow us to learn about the basic biology housed within this unique organelle. We employed a proximity biotinylation technique and mass spectrometry to identify novel P. falciparum proteins putatively targeted to the mitochondrion. We fused the leader sequence of a mitochondrially targeted chaperone, Hsp60, to the promiscuous biotin ligase TurboID. Through these experiments, we generated a list of 122 "putative mitochondrial" proteins. To verify whether these proteins were indeed mitochondrial, we chose five candidate proteins of interest for localization studies using ectopic expression and tagging of each full-length protein. This allowed us to localize four candidate proteins of unknown function to the mitochondrion, three of which have previously been assessed to be essential. We suggest that phenotypic characterization of these and other proteins from this list of 122 could be fruitful in understanding the basic mitochondrial biology of these parasites and aid antimalarial drug discovery efforts.
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Project description:The cytosol-facing organelle outer membrane (OM) is a layer that separates from but also communicates with cellular compartments for the import and exchange of proteins, metabolites and signaling molecules. OM resident proteins are also the targets of cellular surveillance systems by which the cell controls organelle homeostasis for promoting cell survival under stress. However, discovery of OM proteins and their dynamically interacting partners by traditional approaches can be very challenging. In this study, we developed an outer membrane proximity labeling system (OMPL) using biotin ligase mediated proximity biotinylation to map the proximity proteome of the cytosol-facing mitochondria, chloroplast and peroxisome outer membranes in living Arabidopsis cells. The power of this optimized system is highlighted by the discovery of the cytosolic factors and OM receptor candidates that are potentially involved in protein local translation and translocation, membrane contact sites and organelle quality control. Moreover, this OMPL plants generated in this study can be used for rapid and specific isolation of intact mitochondria and peroxisome. Our findings provide ample ground for further investigations on important questions in organelle biology.
2024-01-31 | PXD039752 | JPOST Repository
Project description:Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.
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Project description:Characterizing the proteome composition of organelles and subcellular regions of living cells can facilitate the understanding of cellular organization as well as protein interactome networks. Proximity labeling-based methods coupled with mass spectrometry (MS) offer a high-throughput approach for systematic analysis of spatially restricted proteomes. Proximity labeling utilizes enzymes that generate reactive radicals to covalently tag neighboring proteins with biotin. The biotinylated endogenous proteins can then be isolated for further analysis by MS. To analyze protein-protein interactions or identify components that localize to discrete subcellular compartments, spatial expression is achieved by fusing the enzyme to specific proteins or signal peptides that target to particular subcellular regions. Although these technologies have only been introduced recently, they have already provided deep insights into a wide range of biological processes. Here, we describe and compare current methods of proximity labeling as well as their applications. As each method has its own unique features, the goal of this review is to describe how different proximity labeling methods can be used to answer different biological questions. WIREs Dev Biol 2017, 6:e272. doi: 10.1002/wdev.272 For further resources related to this article, please visit the WIREs website.
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