Characterization of human TIM22 translocase of inner mitochondrial membrane
Katarzyna Chojnacka and Agnieszka Chacinska
Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland
The vast majority of mitochondrial proteins (~99%) are nuclear encoded and require import from the cytosol via specialized mitochondrial translocases. Among them Translocase of the Inner Membrane (TIM22) mediates the import and insertion of multi-pass transmembrane proteins to the mitochondrial inner membrane (IM). TIM22 complex has been studied extensively in yeast. Although TIMM22 protein (the core subunit of the human TIM22 translocase) is conserved across species, its partner proteins in humans are yet unknown. The aim of this study is to functionally characterize hTIM22 complex. To identify the interactome of TIMM22 we applied a proteomic approach. We used His-TIMM22 as a bait and together with the bait, there was a specific enrichment of a previously not characterized protein, C19orf52. The specific TIMM22-C19orf52 interaction was further confirmed by another pull-down and western blotting using specific antibodies. In accordance with its predicted molecular weight of 29 kDa C19orf52 was termed TIMM29. We demonstrated that TIMM29 is an integral component of the IM. Next, to define the molecular function of TIMM29, we used RNA interference to reduce gene expression in HEK293T cells. TIMM29 was efficiently downregulated which caused a reduction of the TIMM22 protein level. Interestingly TIMM22 knockdown also caused a significant reduction in TIMM29 protein levels demonstrating an interdependence of these two proteins. Mature TIMM22 is oxidized in mitochondria thus we aimed to assess whether TIMM22 oxidation status affects the interaction with TIMM29. We overexpressed TIMM22 mutants lacking cysteine residues involved in disulfide bond formation; HisTIMM22‐C69S and HisTIMM22‐C160S. As a control we expressed HisTIMM22‐C138S, a mutant lacking the cysteine residue that does not participate in disulfide bond formation. The interaction between TIMM29 and both mutants HisTIMM22‐C69S and HisTIMM22‐C160S was abolished. In contrast TIMM29 was recovered in the eluate of the HisTIMM22‐C138S purification. Accordingly, TIMM29 can only be stably assembled with the oxidized TIMM22. These findings establish TIMM29 as a stable partner of TIMM22 protein in the human TIM22 complex.
The project is supported by the National Science Centre Grant 2016/20/S/NZ1/00423.
Credits: None available.
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