An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles inToxoplasma gondii

Abstract

The T. gondii protein superoxide dismutase 2 86 (TgSOD2) is dually targeted to the apicoplast and mitochondrion (Pino et al., 2007) and a 87 single point mutation in the N-terminus of a TgSOD2 GFP fusion protein shifts its 88 localization from the mitochondrion to the apicoplast (Brydges, 2003). Clearly, open 89 questions remain; does the dual targeting of proteins to the apicoplast and mitochondrion 90 occur due to superimposed signals at the N-terminus and if so, which signals? 91To understand mechanisms underlying dual targeting, we studied the antioxidant protein, 92 Thioredoxin peroxidase 1/2 (TgTPx1/2), an alternatively spliced form of T. gondii 93 thioredoxin peroxidase, TgTPx. TgTPx1/2 localizes to both the apicoplast and mitochondrion 94 in T. gondii parasites (Pino et al., 2007), reflecting the generation of oxidative radicals in 95 these sub-cellular compartments and their need to handle oxidative stress. Through a 96 combination of bioinformatics and mutational analyses we show that an N-terminal 97 ambiguous sequence directs the dual targeting of TgTPx1/2 to the apicoplast and 98 mitochondrion. This sequence shows an overlap between the signal peptide of the apicoplast 99 targeting signal and the predicted amphipathic helix of the mitochondrial targeting signal. 100 Interestingly, proteins with mutants in the N-terminus of TgTPx1/2 are found in either 101 organelle while a truncated N-terminus results in localization of a reporter protein to punctate 102 structures close to, but not overlapping with the apicoplast. These data suggest that 103 organellar receptors compete for the ambiguous N-terminus of TgTPx1/2 and this N-terminus 104 consists of an unusual sequence where the signals for localization to each organelle are 105 overlapping yet separable. 106 107 Materials and Methods 108 109 T. gondii culture, transfection and selection 110 T. gondii parasites (wild type RH strain) were grown in primary Human Foreskin fibroblasts 111 (HFF-1) cells. HFF-1 cells were obtained from ATCC and grown in Dulbecco's Modified 112 Eagle's Medium (Gibco TM , Mfr. No. 12100-046) containing 3.7 g/L sodium bicarbonate and 113 2.38 g/L HEPES, supplemented with 10% Cosmic calf serum (HyClone TM ) and 20 mg/L 114 Gentamicin. Transient transfections of extracellular T. gondii parasites were carried out at 115 1.5 kV, 50 Ω and 25 μF using Bio-Rad GenePulser Xcell system and subjected to 116 5 immunostaining 24 hours post transfection. Stable lines were generated by Restriction 117 Enzyme Mediated Integration (REMI) using NotI followed by selection of the parasites for 118 growth in 20 µM chloramphenicol.

Dually localised proteins found in both the apicoplast and mitochondrion utilize the Golgi‐dependent pathway for apicoplast targeting in Toxoplasma gondii

“…The single positively charged amino acid in the N‐terminus of Tg TPx1/2, an arginine at position 24, was replaced with alanine to generate the construct Tg TPx1/2(R24A)‐HA. Consistent with published data [Mastud & Patankar, 2019], this construct co‐localised with the apicoplast marker ACP and showed no mitochondrial localisation (Figure 3A). In some cases, in addition to apicoplast localisation, the protein also showed localisation to the ER [Mastud & Patankar, 2019].…”

“…Consistent with published data [Mastud & Patankar, 2019], this construct co‐localised with the apicoplast marker ACP and showed no mitochondrial localisation (Figure 3A). In some cases, in addition to apicoplast localisation, the protein also showed localisation to the ER [Mastud & Patankar, 2019]. An arginine at position 12 in the N‐terminus of Tg SOD2 was replaced with an alanine to generate Tg SOD2(R12A)‐HA.…”

“…Tg TPx1/2‐HA also localised to the apicoplast and the mitochondrion (Figure 2B), consistent with previously published data [Pino et al., 2007; Mastud & Patankar, 2019]. Interestingly, with the presence of a C‐terminal HDEL motif, the protein localised to the apicoplast and was also perinuclear and spread across the cell (Figure 2B).…”

“…Proteins targeted to both the mitochondrion and the apicoplast have developed overlapping signals of the apicoplast bipartite signalling sequence and the mitochondrial signalling sequence. The superimposition of these signals, rather than distinct signals to each of the organelles, may have led to the dual localised proteins developing an ambiguous N‐terminal signal sequence that has components of both the organellar signal sequences [Chew et al., 2003; Mastud & Patankar, 2019]. These N‐termini have evolved to dictate the localisation and consequently the pathway by which the protein reaches the apicoplast.…”

“…This was done by mutating the single positively charged amino acid in the N‐terminus of the protein. Previous experiments on dually localised proteins Tg TPx1/2 and Tg SOD2 have demonstrated that mutating the single positively charged amino acid of the ambiguous N‐terminal sequence completely abolished mitochondrial targeting [Brydges, 2003; Mastud & Patankar, 2019].…”