Mutation of an unusual mitochondrial targeting sequence of SODB2 produces multiple targeting fates inToxoplasma gondii

Abstract

Proteins destined for the mitochondria travel an intricate pathway through two membranes, each with its own receptors and channels. These proteins interact with receptors via N-terminal presequences that form amphipathic helices. Generally, these helices contain abundant positive charges on one face and hydrophobic residues on the other, but share little primary sequence homology. While extensive research on mitochondrial import has been done in yeast and mammalian cells, little is known about import or contents of the single mitochondrion of Toxoplasma gondii, a parasite in the phylum Apicomplexa. We describe here the characterization of TgSODB2, a novel, mitochondrial superoxide dismutase in T. gondii with an unusual targeting sequence consisting of a hydrophobic segment resembling a signal peptide, followed by a presequence. We show that although the hydrophobic segment is competent to target a reporter protein to the secretory system, it is prevented from directing ER translocation when coupled with the presequence. When we mutated the only charged residue in the hydrophobic sequence, ER translocation is restored and the reporter targeted to the apicoplast, a chloroplast-like organelle found in most apicomplexans. The presequence that follows is predicted to form an amphipathic helix, but targeted the cytoplasm when the hydrophobic peptide is removed. In addition to having an unusual targeting sequence, TgSODB2 is only the second mitochondrially imported, iron-containing SOD to be described.

Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting

“…The decreased processing of ACP-GFP-HDEL as compared to ACP-GFP suggests a delay in reaching the apicoplast lumen, possibly resulting from interactions with Erd2. More compellingly, a mutant of mitochondrial T. gondii SODB2 mistargets to the apicoplast, yet can be retained in the ER by an ER retention sequence (Brydges and Carruthers, 2003). Finally, in the diatom Phaeodactylum tricornutum (red lineage) GFP fusions to a truncated, but functional, plastid targeting sequence can be retained in the ER by an ER retrieval sequence, whereas fusions with longer targeting sequences cannot (Apt et al, 2002).…”

Proliferation of Toxoplasma gondii in inflammatory macrophages in vivo is associated with diminished oxygen radical production in the host cell

“…On the other hand, although reactive oxygen species (ROS) can mediate parasite killing in cell culture models (Aline et al, 2002;Murray et al, 1985), this mechanism appears to be inessential in vivo (Alexander et al, 1997), and ROS production is not triggered by parasite invasion of cultured macrophages (Wilson et al, 1980). The effect of ROS in vivo might be obscured by the presence of additional antiparasitic functions; alternatively the parasite may possess effective defenses against ROS, as indicated by its expression of antioxidant enzymes (Brydges and Carruthers, 2003;Kwok et al, 2004). Thus the involvement of this arm of host defense, a critical element of the response to many pathogens, remains undefined for T. gondii.…”

Protein Trafficking to the Apicoplast: Deciphering the Apicomplexan Solution to Secondary Endosymbiosis

“…Interestingly, when an ER retrieval sequence is appended to reporters bearing wild-type bipartite targeting sequences in T. gondii or in P. falciparum, the reporters are not retained in the ER but rather show solely an apicoplast localization (15,81). However, when a mutated targeting sequence was used, the addition of the retrieval sequence allowed retention in the ER (9). These data suggest that a retrieval mechanism may be present, but wildtype apicoplast targeting is dominant over that retrieval.…”