Protein Import into the Endosymbiotic Organelles of Apicomplexan Parasites

Mallo, Natalia; Fellows, Justin D.; Johnson, Carla J.; Sheiner, Lilach

doi:10.3390/genes9080412

Cited by 22 publications

(18 citation statements)

References 135 publications

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“…To fully understand and explore mitochondrial functions it is necessary to create an inventory of experimentally validated mitochondrial proteins. To date only 88 Toxoplasma mitochondrial proteins have been experimentally validated (summarised in (Mallo et al , )). Establishing the proteome through organelle isolation has been a challenge to the apicomplexan field partially due to the association between the mitochondrion and the apicoplast (Kobayashi et al , ; Nishi et al , ; and our unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

Lacombe

Maclean

Ovciarikova

et al. 2019

Molecular Microbiology

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Summary Apicomplexan parasites cause diseases such as malaria and toxoplasmosis. The apicomplexan mitochondrion shows striking differences from common model organisms, including fundamental processes such as mitochondrial translation. Despite evidence that mitochondrial translation is essential for parasite survival, it is largely understudied. Progress has been restricted by the absence of functional assays to detect apicomplexan mitochondrial translation, a lack of knowledge of proteins involved in the process and the inability to identify and detect mitoribosomes. We report the localization of 12 new mitochondrial proteins, including 6 putative mitoribosomal proteins. We demonstrate the integration of three mitoribosomal proteins in macromolecular complexes, and provide evidence suggesting these are apicomplexan mitoribosomal subunits, detected here for the first time. Finally, a new analytical pipeline detected defects in mitochondrial translation upon depletion of the small subunit protein 35 (TgmS35), while other mitochondrial functions remain unaffected. Our work lays a foundation for the study of apicomplexan mitochondrial translation.

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Section: Discussionmentioning

confidence: 99%

Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

Lacombe

Maclean

Ovciarikova

et al. 2019

Molecular Microbiology

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“…In order to rule out the possibility of differential Manuscript to be reviewed 247 splicing being responsible for dual targeting, in this study, cDNA of TgTPx1/2 was amplified 248 using primers that are specific to the TgTPx1/2 spliced form of the mRNA. 249 Proteins that are trafficked to endosymbiotic organelles are generally cleaved on import (Waller 250 et al, 1998;Toursel et al, 2000;Mallo et al, 2018). To investigate the processing of TgTPx1/2, 251 Western blot analysis of lysates from parasites expressing TgTPx1/2-HA was carried out using 252 anti-HA antibodies.…”

Section: Resultsmentioning

confidence: 99%

Peer Review #1 of "An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii (v0.1)"

Besteiro¹

2019

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Toxoplasma gondii harbors two endosymbiotic organelles: a relict plastid, the apicoplast and a mitochondrion. The parasite expresses an antioxidant protein, thioredoxin peroxidase 1/2 (TgTPx1/2), that is dually targeted to these organelles. Nuclear-encoded proteins such as TgTPx1/2 are trafficked to the apicoplast via a secretory route through the endoplasmic reticulum (ER) and to the mitochondrion via a non-secretory pathway comprising of translocon uptake. Given the two distinct trafficking pathways for localization to the two organelles, the signals in TgTPx1/2 for this dual targeting are open areas of investigation. Here we show that the signals for apicoplast and mitochondrial trafficking lie in the N-terminal 50 amino acids of the protein and are overlapping.Interestingly, mutational analysis of the overlapping stretch shows that despite this overlap, the signals for individual organellar uptake can be easily separated. Further, deletions in the N-terminus also reveal a 10 amino acid stretch that is responsible for targeting the protein from punctate structures surrounding the apicoplast into the organelle itself. Collectively, results presented in this report suggest that an ambiguous signal sequence for organellar uptake combined with a hierarchy of recognition by the protein trafficking machinery drives the dual targeting of TgTPx1/2. PeerJ reviewing PDF | Abstract 24 Toxoplasma gondii harbors two endosymbiotic organelles: a relict plastid, the apicoplast and a 25 mitochondrion. The parasite expresses an antioxidant protein, thioredoxin peroxidase 1/2 26 (TgTPx1/2), that is dually targeted to these organelles. Nuclear-encoded proteins such as 27 TgTPx1/2 are trafficked to the apicoplast via a secretory route through the endoplasmic 28 reticulum (ER) and to the mitochondrion via a non-secretory pathway comprising of translocon 29 uptake. Given the two distinct trafficking pathways for localization to the two organelles, the 30 signals in TgTPx1/2 for this dual targeting are open areas of investigation. Here we show that the 31 signals for apicoplast and mitochondrial trafficking lie in the N-terminal 50 amino acids of the 32 protein and are overlapping. Interestingly, mutational analysis of the overlapping stretch shows 33 that despite this overlap, the signals for individual organellar uptake can be easily separated. 34 Further, deletions in the N-terminus also reveal a 10 amino acid stretch that is responsible for 35 targeting the protein from punctate structures surrounding the apicoplast into the organelle itself. 36 Collectively, results presented in this report suggest that an ambiguous signal sequence for 37 organellar uptake combined with a hierarchy of recognition by the protein trafficking machinery 38 drives the dual targeting of TgTPx1/2. 39 40 Introduction 41 Endosymbiotic organelles, plastids and mitochondria, carry out specialized and essential 42 functions in eukaryotes. The proteins required for these functions are predominantly 43 nuclear-encoded and are trafficked from their site of s...

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“…To fully understand and explore mitochondrial functions it is necessary to create an inventory of mitochondrial proteins. To date only 88 Toxoplasma mitochondrial proteins have been experimentally validated (summarised in (Mallo et al, 2018)). Establishing the proteome through organelle isolation has been a challenge to the apicomplexan field partially due to the association between the mitochondrion and the apicoplast (Kobayashi et al, 2007; Nishi et al, 2008; and our unpublished data).…”

Section: Discussionmentioning

confidence: 99%

In silico screen identifies a new Toxoplasma gondii mitochondrial ribosomal protein essential for mitochondrial translation

Lacombe

Maclean

Ovciarikova

et al. 2019

Preprint

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22 23 24 2 Summary 25 26Apicomplexan parasites cause diseases such as malaria and toxoplasmosis. The 27 apicomplexan mitochondrion shows striking differences from common model organisms, 28including in fundamental processes such as mitochondrial translation. Despite evidence 29 that mitochondrial translation is essential for parasites survival, it is largely understudied. 30Progress has been restricted by the absence of functional assays to detect 31 apicomplexan mitochondrial translation, a lack of knowledge of proteins involved in the 32 process and the inability to identify and detect mitoribosomes. 33 Using mRNA expression patterns, 279 candidate mitochondrial housekeeping 34 components were identified in Toxoplasma. 11 were validated, including the 35 mitoribosomal small subunit protein 35 (TgmS35). TgmS35 tagging enabled the 36 detection of a macromolecular complex corresponding to the mitoribosomal small 37 subunit for the first time in apicomplexans. A new analytical pipeline detected defects in 38 mitochondrial translation upon TgmS35 depletion, while other mitochondrial functions 39 remain unaffected. Our work lays a foundation for the study of apicomplexan 40 mitochondrial translation. 41 42 43 Abbreviated summary 44 45 46The apicomplexan mitochondrion is divergent and essential yet poorly studied. 47Mitochondrial translation is predicted to utilize ribosomes assembled from fragmented 48 rRNA but this was never shown. Knowing the mitochondrial protein content is critical for 49 these studies. We identified 11 new mitochondrial proteins via in-silico searches. 50 Tagging and depletion of a mitoribosomal small subunit protein enabled the first 51 detection of a macromolecular ribosomal complex, and provided proof of principle for our 52 new mitochondrial translation analytic pipeline. 53 54 55 3 Introduction 56 57 Mitochondria are organelles of central importance to eukaryotic cells, providing key 58 nutrients and metabolites. Mitochondria are present in virtually all eukaryotic cells, 59 excluding a rare case of secondary loss (Karnkowska et al., 2016; Karnkowska and 60 Hampl, 2016). The recent interest in evolutionary cell biology has resulted in an 61 increasing appreciation of the diverse features of mitochondria found in divergent 62 organisms, which includes differences in fundamental pathways such as protein 63 translation (e.g. Ramrath et al., 2018). However, most of our knowledge of mitochondrial 64 biology is still based on studies in organisms that represent a small proportion of the 65 range of eukaryotic diversity, thus limiting our understanding. One of the challenges to 66 the study of mitochondrial biology in diverse organisms is the lack of tools to identify and 67 functionally characterise genes encoding species or phylum specific mitochondrial 68 proteins. 70Apicomplexa is a phylum of parasitic protists with high impact on human health globally, 71 which includes the malaria causing Plasmodium spp and the causative agent of 72 toxoplasmosis, Toxoplasma gondii. Apicomplexans represent o...

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Protein Import into the Endosymbiotic Organelles of Apicomplexan Parasites

Cited by 22 publications

References 135 publications

Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

Peer Review #1 of "An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii (v0.1)"

In silico screen identifies a new Toxoplasma gondii mitochondrial ribosomal protein essential for mitochondrial translation

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