A single-cysteine mutant and chimeras of essential Leishmania Erv can complement the loss of Erv1 but not of Mia40 in yeast

Abstract Mia40/CHCHD4 and Erv1/ALR are essential for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals. In contrast, many protists, including important apicomplexan and kinetoplastid parasites, lack Mia40. Furthermore, the Erv homolog of the model parasite Leishmania tarentolae ( Lt Erv) was shown to be incompatible with Saccharomyces cerevisiae Mia40 ( Sc Mia40). Here we addressed structure-function relationships of Sc Erv1 and Lt Erv as well as their compatibility with the oxidative protein folding system in yeast using chimeric, truncated, and mutant Erv constructs. Chimeras between the N-terminal arm of Sc Erv1 and a variety of truncated Lt Erv constructs were able to rescue yeast cells that lack Sc Erv1. Yeast cells were also viable when only a single cysteine residue was replaced in Lt Erv C17S . Thus, the presence and position of the C-terminal arm and the kinetoplastida-specific second (KISS) domain of Lt Erv did not interfere with its functionality in the yeast system, whereas a relatively conserved cysteine residue before the flavodomain rendered Lt Erv incompatible with Sc Mia40. The question whether parasite Erv homologs might also exert the function of Mia40 was addressed in another set of complementation assays. However, neither the KISS domain nor other truncated or mutant Lt Erv constructs were able to rescue yeast cells that lack Sc Mia40. The general relevance of Erv and its candidate substrate small Tim1 was analyzed for the related parasite L. infantum . Repeated unsuccessful knockout attempts suggest that both genes are essential in this human pathogen and underline the potential of mitochondrial protein import pathways for future intervention strategies.