Minimal peptide length required to span the mitochondrial protein translocases in Trypanosoma brucei

•Spanning of ATOM-TIM supercomplexes requires at least 110 aa.•Formation of ATOM-TIM supercomplexes inhibits growth. Mitochondrial protein import depends on heterooligomeric translocases in the outer and inner membranes. Using import substrates consisting of various lengths of the N-terminal part of mitochondrial dihydrolipoamide dehydrogenase (LDH) fused to dihydrofolate reductase we present an in vivo analysis showing that in Trypanosoma brucei at least 96 aa of mature LDH are required to efficiently produce an import intermediate that spans both translocases. This is different to yeast, where around 50 aa are sufficient to achieve the same task and likely reflects the different arrangement and architecture of the trypanosomal mitochondrial translocases. Furthermore, we show that formation of the stuck import intermediate leads to a strong growth inhibition suggesting that, depending on the length of the LDH, the import channels in the translocases are quantitatively blocked.