In Trichomonas vaginalis, the TvCyP1-catalyzed conformational switches of two glycinyl-prolyl imide bonds in Myb3 were previously shown to regulate the trafficking of Myb3 from cytoplasmic membrane compartments towards the nucleus. In this study, TvCyP2 was identified as a second cyclophilin that binds to Myb3 at the same dipeptide motifs. The enzymatic proficiency of TvCyP2, but not its binding to Myb3, was aborted by a mutation of Arg75 in the catalytic domain. TvCyP2 was localized to the endoplasmic reticulum with a weak signal that extensively extends into the cytoplasm as well as to the plasma membrane according to an immunofluorescence assay. Moreover, TvCyP2 was co-enriched with TvCyP1 and Myb3 in various membrane fractions purified by differential and gradient centrifugation. TvCyP2 was found to proficiently enzymatically regulate the distribution of TvCyP1 and Myb3 among purified membrane fractions, and to localize TvCyP1 in hydrogenosomes and on plasma membranes. Protein complexes immunoprecipitated from lysates of cells overexpressing TvCyP1 and TvCyP2 were found to share some common components, like TvCyP1, TvCyP2, TvBip, Myb3, TvHSP72, and the hydrogenosomal heat shock protein 70 (HSP70). Direct interaction between TvCyP1 and TvCyP2 was confirmed by a GST pull-down assay. Fusion of vesicles with hydrogenosomes was observed by transmission electron microscopy, whereas TvCyP1, TvCyP2, and Myb3 were each detected at the fusion junction by immunoelectron microscopy. These observations suggest that T. vaginalis may have evolved a novel protein trafficking pathway to deliver proteins among the endomembrane compartments, hydrogenosomes and plasma membranes.
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