Project description:Subcellular genomics shows pervasive within-mitochondrion single nucleotide variant heteroplasmy as revealed by single mitochondrion sequencing
Project description:The endobiotic flagellate Monocercomonoides exilis is the only eukaryote known to lose any form of mitochondria and all its associated proteins in its evolutionary past. This final stage of the mitochondrial evolutionary pathway may serve as a model to explain events at their very beginning such as the initiation of protein import. We have assessed the preadaptation of proteins in this mitochondrion-free cell to enter emerging mitochondria using a specifically designed in vitro assay. In this experiment, hydrogenosomes (reduced mitochondria) of Trichomonas vaginalis were incubated with a soluble protein pool derived from a cytosolic fraction of M. exilis, and proteins entering hydrogenosomes were subsequently detected by mass spectrometry. The assay detected 19 specifically and reproducibly imported proteins, and in 14 cases, import was confirmed by the overexpression of their tagged version in the T. vaginalis heterologous system and visualization by immunofluorescence and western blot. In most cases, only a small portion of the signal reached the hydrogenosomes, suggesting specific but inefficient transport. Most of these proteins represent enzymes of carbon metabolism, and none exhibited conspicuous signatures of proteins targeted to hydrogenosomes or mitochondria, which is consistent with their inefficient import. The observed phenomenon may resemble a primaeval type of protein import which might play a role in the establishing of the organelle and shaping of its proteome in the initial stages of endosymbiosis.
