Mitochondrial translocase blocking ATP5MG-tFT fusion protein interactome in human embryonic kidney (HEK) 293 cells

The experiment uses HEK-293 Flp-In T-Rex cells to express fusion protein that combines ATP synthase subunit ATP5MG with a tandem fluorescent protein timer (tFT). The tFT tag combines mCherry and super folder GFP (sfGFP) fluorescent proteins. An exceptionally stable structure of sfGFP interferes with its import into mitochondria. Thus, ATP5MG-tFT fusion stalls during translocation into mitochondria. We identify the proteome of cells after 24 hours of fusion protein expression. As a control, we use cells that express tFT fusion not directed to mitochondria (ILE-Deg-tFT) or cells that did not express fusion protein. Next, we use chemical crosslinking and GFP-Trap-based immunoprecipitation to define interaction partner proteins of ATP5MG-tFT and ILE-Deg-tFT fusions.