Proteomic Identification of the SLC25A46 Interactome in Transgenic Mice Expressing SLC25A46-FLAG

Abstract

The outer mitochondrial membrane protein SLC25A46 has been recently identified as a novel genetic cause of a wide spectrum of neurological diseases. The aim of the present work was to elucidate the physiological role of SLC25A46 through the identification of its interactome with immunoprecipitation and proteomic analysis in whole cell extracts from the cerebellum, cerebrum, heart, and thymus of transgenic mice expressing ubiquitously SLC25A46-FLAG. Our analysis identified 371 novel putative interactors of SLC25A46 and confirmed 17 known ones. A total of 79 co-immunoprecipitated proteins were common in two or more tissues, mainly participating in mitochondrial activities such as oxidative phosphorylation (OXPHOS) and ATP production, active transport of ions or molecules, and the metabolism. Tissue-specific co-immunoprecipitated proteins were enriched for synapse annotated proteins in the cerebellum and cerebrum for metabolic processes in the heart and for nuclear processes and proteasome in the thymus. Our proteomic approach confirmed known mitochondrial interactors of SLC25A46 including MICOS complex subunits and also OPA1 and VDACs, while we identified novel interactors including the ADP/ATP translocases SLC25A4 and SLC25A5, subunits of the OXPHOS complexes and F1Fo-ATP synthase, and components of the mitochondria–ER contact sites. Our results show that SLC25A46 interacts with a large number of proteins and protein complexes involved in the mitochondria architecture, energy production, and flux and also in inter-organellar contacts.

Repurposing of Amisulpride, a known antipsychotic drug, to target synovial fibroblasts activation in arthritis

“…A brief description of the protocol followed is provided in the supplementary material and methods. The samples digestion, run and initial analysis were processed as previously described (91) . A summary of the process followed is described in the supplementary material and methods.…”

“…The samples digestion, run and initial analysis were processed as previously described (91). A summary of the process followed is described in the supplementary material and methods.…”

Development of a Method Combining Peptidiscs and Proteomics to Identify, Stabilize, and Purify a Detergent-Sensitive Membrane Protein Assembly

“…coli membrane protein open-reading frames (ORFs) have been systematically SPA-tagged. A similar public library is also available to characterize the Saccharomyces cerevisiae membrane proteome. , Other recent work reports the high-throughput construction of new strains and cell lines to map protein interaction networks in mitochondria and mouse brain tissues using AP/MS. ,, Given the widespread availability of gene-editing technology, we anticipate that our peptidisc-AP/MS approach will be easily expandable to precisely characterize membrane protein interactomes across multiple different organisms and tissue types.…”

Development of a Method Combining Peptidiscs and Proteomics to Identify, Stabilize and Purify a Detergent-Sensitive Membrane Protein Assembly

“…A similar public library is also available to characterize the S. cerevisiae membrane proteome ( 1, 2 ). Other recent work reports the high throughput construction of new strains and cell lines to map protein interaction networks in mitochondria and mouse brain tissue using AP/MS ( 13, 39, 40 ). Given the widespread availability of geneediting technology, we anticipate that our Peptidisc-AP/MS approach will be easily expandable to precisely characterize membrane protein interactomes across multiple different organisms and tissue types.…”