Abstract
Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier translocase shares eight subunits with the presequence translocase. This indicates that T. brucei may have a single IM translocase that with compositional variations mediates import of presequence-containing and carrier proteins.
Highlights
Mitochondrial protein import is essential for all eukaryotes
It has previously been shown that in T. brucei, as might be expected, inhibition of import leads to a decline in the abundance of mitochondrial proteins[28]
Its ablation confirms that the protein is essential for mitochondrial protein import
Summary
Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. The proteins are first imported into mitochondria by the translocase of the outer mitochondrial membrane (TOM), which is the general entry gate for essentially all mitochondrial proteins. The presequence translocase-associated motor (PAM) in cooperation with the mitochondrial inner membrane (IM) potential drives translocation of substrate proteins or domains thereof into the matrix using mitochondrial heat shock protein 70 (mtHsp70)-mediated ATP hydrolysis[9,10]. Import of proteins across the IM requires the cooperation of the TOM and the TIM23 complexes This allows purification of a TOM-TIM-preprotein complex when tightly folded domains in the C-terminus of the import substrate prevent complete translocation across the outer mitochondrial membrane[12,13]. The archaic translocase of the outer membrane (ATOM), the trypanosomal analogue of the yeast TOM complex, consists of six subunits. The other four subunits are unique to trypanosomatids and evolved independently of any TOM complex subunits of other eukaryotic lineages[28,29]
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