Molecular Characterization of Quality Control Factors at the Protein Entry Gate of Mitochondria

Abstract

Mitochondria perform crucial functions for cell survival. In order to fulfill these functions, mitochondria contain about 1,000-1,300 proteins. Approximately, 99% of the mitochondrial proteins are synthesized as precursors on cytosolic ribosomes and imported into mitochondria via sophisticated import machineries. The translocase of the outer membrane complex (TOM complex) forms an entry gate for most mitochondrial precursors. Defects in protein import via the TOM complex lead to the accumulation of mitochondrial precursor proteins, which in turn cause massive proteotoxic stress. To ensure correct mitochondrial function, a plethora of quality control factors govern mitochondrial protein import at various steps, thereby ensuring proper protein import into mitochondria. However, our understanding of quality control mechanisms is still limited. In this research study, we aimed to identify quality control factors that monitor the TOM complex. <br /> Using affinity purifications, we identified a new quality control factor, Pth2, which operates at the TOM complex and promotes the removal of accumulated precursor proteins. Upon clogging of the TOM complex, Pth2 interacts with the ubiquitin-binding protein Dsk2 (sole yeast homolog of human, ubiquilins) which delivers ubiquitylated substrates to the proteasome for degradation. We observed two experimentally independent functions of Pth2 in quality control. <br /> One related to its role in the removal of non-imported precursor proteins during mitochondrial import. The other function of Pth2 was related to its peptidyl tRNA hydrolase activity linked to the quality control pathway in clearing translation-stalled polypeptides from the ribosome. In addition, our data led to the discovery of Fmp52, a novel regulatory protein at the TOM complex. We observed that Fmp52 is present both in mitochondria as well as in the cytosol. <br /> Furthermore, we observed that Fmp52 is stabilized upon proteasomal inhibition and clogging of the TOM complex. Thus, Fmp52 plays a regulatory role in mitochondrial quality control. <br /> In conclusion, we identified two new factors operating at the TOM complex. These factors play an essential role in maintaining the TOM complex in an import-competent state, allowing efficient protein import via the cellular gateway.