Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum mitochondrial contacts [Neurobiology]

To maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites (MCS) coupled by protein tethers. In particular, endoplasmic reticulum (ER)-mitochondria contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca2+ and lipids), intracellular signaling, apoptosis and autophagy. The significance of EMCs have 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 cut-off ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified RETICULON1A (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.

from # All Medicine by Alexandros G. Sfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2wfNkcS
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