Proteomic analysis of detergent‐resistant microdomains obtained from mitochondria and mitochondria‐associated ER membranes

The mitochondria‐associated membrane (MAM) is a portion of the endoplasmic reticulum (ER) tethered by protein filaments to the outer mitochondrial membrane (OMM). The MAM is important because it facilitates crosstalk between the ER and mitochondria and regulates Ca2+ signaling, protein folding, and lipid transport. Recently, detergent‐resistant microdomains (DRMs) of the ER and mitochondria have been shown to regulate apoptotic signaling events. However, the protein content of the DRMs from these two regions is unknown. The objective of this study was to determine the protein content of a combined MAM/mitochondria DRM sample using a high throughput mass spectrometry‐based method. We successfully (1) isolated DRMs from MAM/mitochondria using the non‐ionic detergent Triton X‐114, (2) digested DRM proteins using gel‐assisted technology, and (3) identified 107 biologically‐relevant proteins across 4 replicates. These proteins include those known to either localize in the OMM at regions in contact with the MAM (the voltage dependent anion selective channel) or those that concentrate in the MAM such as chaperones (calnexin and calreticulin) and oxidoreductases (pyruvate and malate dehydrogenases). Our study provides data to support the postulate that protein activities at the MAM/mitochondria interface are dependent, in part, on the presence of DRMs. This work was supported by NIH R03‐DA027182.