The interaction of SFC1, succinate and TIM23 in yeast mitochondrial protein import and dual targeting

Previous studies in our lab discovered that addition of succinate or depletion of the glyoxylate pathway, alters the distribution of fumarase and aconitase, increasing the levels of the mitochondrial versus the cytosolic isoform. Sfc1 is a mitochondrial succinate-fumarate carrier that transports succinate into and fumarate out of the mitochondria. Sfc1 is in fact, a component of the glyoxylate shunt and thus a Sfc1 knockout strain (ΔSfc1) cannot grow on ethanol and/or acetate as a sole carbon source. Moreover, ΔSfc1 changes the subcellular distribution of fumarase resulting in lower levels of fumarase in the cytosol and higher levels in the mitochondria . The objective of this study is to understand the role of succinate and Sfc1 in mitochondrial protein import and dual targeting in yeast. The results obtained from pulse chase experiments suggested that succinate and ΔSfc1 facilitate aconitase (Aco1) import into mitochondria while overexpression of Sfc1 inhibits Aco1 import. Our model is that Sfc1 and the Tim23 complex can physically interact, which in turn regulates protein import. The model predicts that in the absence of succinate or when Sfc1 is over-expressed, Tim23 is attached to Sfc1 resulting in lower translocation of proteins. To test this hypothesis, protein-protein interactions between Tim23 and Sfc1 were analyzed by immunoprecipitation, Bimolecular Fluorescence Complementation techniques and TurboID in vivo. The results revealed direct interaction between Tim23 and Sfc1 and addition of succinate induces the dissociation between Tim23 and Sfc1. These observations indicate that succinate and Sfc1 may regulate yeast mitochondrial protein import as a way to maintain proteostasis.