TbTim20 facilitates protein import at a low membrane potential in trypanosomes lacking the mitochondrial genome

Abstract

Protein import across the mitochondrial inner membrane typically depends on two protein translocases of the inner membrane (TIM) complexes and the membrane potential. The protozoan parasite Trypanosoma brucei, however, has a single, divergent TIM complex. Unlike other trypanosomal TIM subunits, TbTim20 is not essential for the normal growth of the insect or bloodstream forms of T. brucei, leaving its role uncertain. Specific mutations in the γ-subunit of the F₁F_O-ATPase, such as γL262P, permit bloodstream form trypanosomes to grow without mitochondrial DNA (kinetoplast or kDNA). Here, we show that RNAi-mediated depletion of TbTim20 inhibits growth of this cell line, but only if it lacks the kDNA. Titration of mitochondrial uncouplers and direct membrane potential measurements reveal that TbTim20 becomes more critical as the membrane potential decreases across all tested cell lines. Proteomic analysis of the uninduced and induced γL262P TbTim20-RNAi cell line, which lacks kDNA and exhibits the lowest membrane potential, shows depletion of a subset of imported proteins. This subset includes ATPase subunits, suggesting a mechanism by which TbTim20-silenced cell lines become more sensitive to uncouplers. Thus, we propose that TbTim20 supports the import of a subset of proteins whose import is hypersensitive to a low membrane potential.

Keywords: Trypanosoma brucei; TIM complex; kinetoplast DNA; mitochondrial membrane potential; mitochondrial protein import.