Elp3 and RlmN: A tale of two mitochondrial tail-anchored radical SAM enzymes in Toxoplasma gondii.

Leah R Padgett,Michael J Holmes,William J Sullivan,Dragony Fu,Jenna M Lentini,Krista L Stilger,Gordon Langsley

doi:10.1371/journal.pone.0189688

Leah R Padgett, Michael J Holmes + Show 5 more

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https://doi.org/10.1371/journal.pone.0189688

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Abstract

Radical S-adenosylmethionine (rSAM) enzymes use a 5’-deoxyadensyl 5’-radical to methylate a wide array of diverse substrates including proteins, lipids and nucleic acids. One such enzyme, Elongator protein-3 (TgElp3), is an essential protein in Toxoplasma gondii, a protozoan parasite that can cause life-threatening opportunistic disease. Unlike Elp3 homologues which are present in all domains of life, TgElp3 localizes to the outer mitochondrial membrane (OMM) via a tail-anchored trafficking mechanism in Toxoplasma. Intriguingly, we identified a second tail-anchored rSAM domain containing protein (TgRlmN) that also localizes to the OMM. The transmembrane domain (TMD) on Toxoplasma Elp3 and RlmN homologues is required for OMM localization and has not been seen beyond the chromalveolates. Both TgElp3 and TgRlmN contain the canonical rSAM amino acid sequence motif (CxxxCxxC) necessary to form the 4Fe-4S cluster required for tRNA modifications. In E. coli, RlmN is responsible for the 2-methlyadenosine (m2A) synthesis at purine 37 in tRNA while in S. cerevisiae, Elp3 is necessary for the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at the wobble tRNA position. To investigate why these two rSAM enzymes localize to the mitochondrion in Toxoplasma, and whether or not TgRlmN and TgElp3 possess tRNA methyltransferase activity, a series of mutational and biochemical studies were performed. Overexpression of either TgElp3 or TgRlmN resulted in a significant parasite replication defect, but overexpression was tolerated if either the TMD or rSAM domain was mutated. Furthermore, we show the first evidence that Toxoplasma tRNAGlu contains the mcm5s2U modification, which is the putative downstream product generated by TgElp3 activity.

Highlights

Radical S-adenosylmethionine enzymes utilize a [4Fe-4S] cluster and SAM to generate a 5’-deoxyadenosyl radical intermediate required for methylation reactions
Normal growth rate is restored if the transmembrane domain (TMD) is removed or if the Radical S-adenosylmethionine (rSAM) domain is mutated, but not if the KAT domain is mutated. These results suggest that the rSAM domain of TgElp3 is critical for protein function, and confirms that TgElp3 activity is dependent on its localization to the outer mitochondrial membrane (OMM)
We determined that overexpression of TgElp3 or TgRlmN at the parasite mitochondrion results in a significant replication defect, but overexpression of these proteins bearing a mutant rSAM domain or lacking their TMD is well-tolerated and does not impair fitness

Summary

Introduction

Radical S-adenosylmethionine (rSAM) enzymes utilize a [4Fe-4S] cluster and SAM to generate a 5’-deoxyadenosyl radical intermediate required for methylation reactions. These enzymes are defined by the presence of a conserved cysteine motif that coordinates the formation of an [4Fe-4S] cluster. These motifs vary in the number of cysteines and amino acid length, the most common motif is CxxxCxxC. Studies have shown that rSAM enzymes play a pivotal role in the modification of RNA [1]. The study of rSAM enzymes is required to better understand the biological implications of RNA modifications

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