A cell death assay for assessing the mitochondrial targeting of proteins (373.3)

Abstract

Some of the 800 mitochondrial diseases are linked with aberrant protein localization. We devised a high‐throughput cell survival assay for assessing mitochondrial localization of proteins. Cells are transfected with plasmids coding for fusion constructs of mitochondrial proteins and the peptide KLAKLAKKLAKLAK (KLAK), which disrupts the mitochondrial membrane potential and causes cell death; cell survival of KLAK fusion proteins and empty KLAK plasmids (control) is assessed using the MTT assay (ΔMTT). KLAK fusion constructs of proteins that localize in mitochondrial membrane (acetyl‐CoA carboxylase 2, ACC2, carnitine palmitoyltransferase 1, and uncoupling protein 1), or are of uncertain mitochondrial localization (holocarboxylase synthetase), produced ΔMTT values between 0.211 and 0.411. The assay has a wide dynamic range (Z‐Prime = 0.58) and a low coefficient of variation (4.8%). The assay is also useful to investigate effects of bioactive compounds on the mitochondrial localization of proteins, evidenced by the observation that chlorogenic acid and gallic acid increased cell survival by 131% and 78%, respectively, in HEK293 transfected with ACC2‐KLAK, presumably due to a loss of mitochondrial ACC2. We conclude that our assay identifies both mitochondrial proteins and compounds that alter mitochondrial localization.Grant Funding Source: Supported by ARD Hatch, NIFA, and NIH.