Elongator protein 3 (Elp3) lysine acetyltransferase is a tail-anchored mitochondrial protein in Toxoplasma gondii.

Lysine acetylation has recently emerged as an important, widespread post-translational modification occurring on proteins that reside in multiple cellular compartments, including the mitochondria. However, no lysine acetyltransferase (KAT) has been definitively localized to this organelle to date. Here we describe the identification of an unusual homologue of Elp3 in early-branching protozoa in the phylum Apicomplexa. Elp3 is the catalytic subunit of the well-conserved transcription Elongator complex; however, Apicomplexa lack all other Elongator subunits, suggesting that the Elp3 in these organisms plays a role independent of transcription. Surprisingly, Elp3 in the parasites of this phylum, including Toxoplasma gondii (TgElp3), possesses a unique C-terminal transmembrane domain (TMD) that localizes the protein to the mitochondrion. As TgElp3 is devoid of known mitochondrial targeting signals, we used selective permeabilization studies to reveal that this KAT is oriented with its catalytic components facing the cytosol and its C-terminal TMD inserted into the outer mitochondrial membrane, consistent with a tail-anchored membrane protein. Elp3 trafficking to mitochondria is not exclusive to Toxoplasma as we also present evidence that a form of Elp3 localizes to these organelles in mammalian cells, supporting the idea that Elp3 performs novel functions across eukaryotes that are independent of transcriptional elongation. Importantly, we also present genetic studies that suggest TgElp3 is essential in Toxoplasma and must be positioned at the mitochondrial surface for parasite viability.

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

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.

Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii.

Mitochondria distribution in cells controls cellular physiology in health and disease. Here we describe the mitochondrial morphology and positioning found in the different stages of the lytic cycle of the eukaryotic single-cell parasite Toxoplasma gondii. The lytic cycle, driven by the tachyzoite life stage, is responsible for acute toxoplasmosis. It is known that whilst inside a host cell the tachyzoite maintains its single mitochondrion at its periphery. We found that upon parasite transition from the host cell to the extracellular matrix, mitochondrion morphology radically changes, resulting in a reduction in peripheral proximity. This change is reversible upon return to the host, indicating that an active mechanism maintains the peripheral positioning found in the intracellular stages. Comparison between the two states by electron microscopy identified regions of coupling between the mitochondrion outer membrane and the parasite pellicle, whose features suggest the presence of membrane contact sites, and whose abundance changes during the transition between intra- and extra-cellular states. These novel observations pave the way for future research to identify molecular mechanisms involved in mitochondrial distribution in Toxoplasma and the consequences of these mitochondrion changes on parasite physiology.

Resolution of Specific Nucleotide Mismatches by Wild-Type and AZT-Resistant Reverse Transcriptases during HIV-1 Replication.

A key contributor to HIV-1 genetic variation is reverse transcriptase errors. Some mutations result because reverse transcriptase (RT) lacks 3' to 5' proofreading exonuclease and can extend mismatches. However, RT also excises terminal nucleotides to a limited extent, and this activity contributes to AZT resistance. Because HIV-1 mismatch resolution has been studied in vitro but only indirectly during replication, we developed a novel system to study mismatched base pair resolution during HIV-1 replication in cultured cells using vectors that force template switching at defined locations. These vectors generated mismatched reverse transcription intermediates, with proviral products diagnostic of mismatch resolution mechanisms. Outcomes for wild-type (WT) RT and an AZT-resistant (AZT(R)) RT containing a thymidine analog mutation set-D67N, K70R, D215F, and K219Q-were compared. AZT(R) RT did not excise terminal nucleotides more frequently than WT, and for the majority of tested mismatches, both WT and AZT(R) RTs extended mismatches in more than 90% of proviruses. However, striking enzyme-specific differences were observed for one mispair, with WT RT preferentially resolving dC-rC pairs either by excising the mismatched base or switching templates prematurely, while AZT(R) RT primarily misaligned the primer strand, causing deletions via dislocation mutagenesis. Overall, the results confirmed HIV-1 RT's high capacity for mismatch extension during virus replication and revealed dramatic differences in aberrant intermediate resolution repertoires between WT and AZT(R) RTs on one mismatched replication intermediate. Correlating mismatch extension frequencies observed here with reported viral mutation rates suggests a complex interplay of nucleotide discrimination and mismatch extension drives HIV-1 mutagenesis.

A decade of epigenetic research in Toxoplasma gondii.

In the past 10 years, the field of parasitology has witnessed an explosion of studies investigating gene regulation. In this review, we will describe recent advances largely stemming from the study of Toxoplasma gondii, a significant opportunistic pathogen and useful model for other apicomplexan protozoa. Surprising findings have emerged, including the discovery of a wealth of epigenetic machinery in these primitive eukaryotes, unusual histone variants, and a battery of plant-like transcription factors. We will elaborate on how these unusual features impact parasite physiology and potential therapeutics as we summarize some of the key discoveries from the last decade. We will close by proposing a few questions to address in the next 10 years.