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 (LtErv) was shown to be incompatible with Saccharomyces cerevisiae Mia40 (ScMia40). Here we addressed structure-function relationships of ScErv1 and LtErv 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 ScErv1 and a variety of truncated LtErv constructs were able to rescue yeast cells that lack ScErv1. Yeast cells were also viable when only a single cysteine residue was replaced in LtErvC17S. Thus, the presence and position of the C-terminal arm and the kinetoplastida-specific second (KISS) domain of LtErv did not interfere with its functionality in the yeast system, whereas a relatively conserved cysteine residue before the flavodomain rendered LtErv incompatible with ScMia40. 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 LtErv constructs were able to rescue yeast cells that lack ScMia40. 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.

Testing the CRISPR-Cas9 and glmS ribozyme systems in Leishmania tarentolae

“…The results confirmed their expected relevance for parasite motility and drug susceptibility, respectively 12,13,33 . Furthermore, using the CRISPR-Cas9 system we were unable to delete the L. tarentolae gene that encodes the mitochondrial flavoenzyme Erv in accordance with previous results that were obtained for L. infantum using traditional genetics 35 . Hence, two unrelated genetic studies in Leishmania, as well as RNAi experiments in T. brucei 38,41 , show that Erv is crucial for parasite survival.…”

“…We previously showed that the gene encoding the mitochondrial flavoenzyme Erv cannot be deleted in the related human pathogen L. infantum using standard genetics 35 . This enzyme is thought to play an important role in mitochondrial protein import in kinetoplastid parasites 6,[36][37][38] .…”

Testing the CRISPR-Cas9 andglmSribozyme systems inLeishmania tarentolae

“…The results confirmed their expected relevance for parasite motility and drug susceptibility, respectively 12,13,33 . Furthermore, using the CRISPR-Cas9 system we were unable to delete the L. tarentolae gene that encodes the mitochondrial flavoenzyme Erv in accordance with previous results that were obtained for L. infantum using traditional genetics 35 . Hence, two unrelated genetic studies in Leishmania, as well as RNAi experiments in T. brucei 38,41 , show that Erv is crucial for parasite survival.…”

Protein Import into the Endosymbiotic Organelles of Apicomplexan Parasites

“…It also suggests that evolutionarily earlier versions of Erv1 may have fulfilled the role of both MIA components or that a non-Mia40 homologue existed in those putative early versions that performed its function [ 59 ]. However, a first set of cross-species complementation studies between yeast and the excavate organism Leishmania tarentolae place doubt on this model [ 60 ]. It will be interesting to examine similar cross-species work between yeast and apicomplexans.…”