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Cryptosporidium parvum Mitochondrial-Type HSP70 Targets Homologous and Heterologous Mitochondria (Slapeta and Keithly, 2004)

In this article they describe a HSP70 gene that they call mitochondrial, but all they really can say is that it has an homology with a mitochondrial gene. Since they assume that this gene has been derived from an endosymbiont, this biases the entire article, but still they have some interesting results. They show that the gene has a mitochondrion-like targeting presequence (NH2-side) that targets proteins (eg GFP) to the mirochondrion and still works to target this protein in yeast and toxoplasmosis. They interpret as a secondarily-reduced mitochondrion, although the data is also is perfect agreement with a common import pathway thta slowly evolved. They also confirm that Cryptosporidium has a mitochondrion (albeit with no genome).

 

Abstract: A mitochondrial HSP70 gene (Cp-mtHSP70) is described for the apicomplexan Cryptosporidium parvum, an agent of diarrhea in humans and animals. Mitochondrial HSP70 is known to have been acquired from the proto-mitochondrial endosymbiont. The amino acid sequence of Cp-mtHSP70 shares common domains with mitochondrial and proteobacterial homologues, including 34 amino acids of an NH2-terminal mitochondrion-like targeting presequence. Phylogenetic reconstruction places Cp-mtHSP70 within the mitochondrial clade of HSP70 homologues. Using reverse transcription-PCR, Cp-mtHSP70 mRNA was observed in C. parvum intracellular stages cultured in HCT-8 cells. Polyclonal antibodies to Cp-mtHSP70 recognize a 70-kDa protein in Western blot analysis of sporozoite extracts. Both fluorescein- and immunogold-labeled anti-Cp-mtHSP70 localize to a single mitochondrial compartment in close apposition to the nucleus. Furthermore, the NH2-terminal presequence of Cp-mtHSP70 can correctly target green fluorescent protein to the single mitochondrion of the apicomplexan Toxoplasma gondii and the mitochondrial network of the yeast Saccharomyces cerevisiae. When this presequence was truncated, the predicted amphiphilic -helix was shown to be essential for import into the yeast mitochondrion. These data further support the presence of a secondarily reduced relict mitochondrion in C. parvum.

Here we describe a C. parvum mtHSP70 (Cp-mtHSP70) that (i) is a nuclear gene with clear proto-mitochondrial origins, (ii) possesses a mitochondrial targeting sequence, and (iii) is part of the mitochondrial protein import machinery. A reporter gene (green fluorescent protein [GFP]) is used to show that the C. parvum presequence targets mitochondria in the apicomplexan T. gondii and the yeast Saccharomyces cerevisiae. Analyses of truncated C. parvum targeting presequences revealed the amino acids within the predicted helix of the presequence to be essential for import. Fluorescence and immunogold microscopy showed the localization of labeled polyclonal antibodies within the relict mitochondrion of C. parvum.

Nevertheless, evidence for the presence of a relict mitochondrion was obtained using FITC-labeled anti-Cp-mtHSP70. Here, for the first time, immunofluorescence of the mitochondrion was observed both in C. parvum sporozoites and in intracellular stages. These immunofluorescence data indicate not only that a relict mitochondrion is present in sporozoites but that this organelle occurs in developmental stages that are multiplying within host cells. Previously, only immunogold labeling of transmission electron micrograph sections by anti-CpCpn60 was able to clearly delineate the relict mitochondrion (40). Here we also show immunogold localization of anti-Cp-mtHSP70 to a compartment between the nucleus and crystalloid body identical with that for anti-CpCpn60 (40), further confirming that this organelle is a mitochondrion.

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