Characterization of the targeting and membrane insertion of mitochondrial tombusvirus replication proteins and tail-anchored proteins

There is lack of information on the biogenesis of the outer mitochondrial membrane proteins, including how they are targeted and inserted into mitochondria in plant cells. Here, we characterize the molecular mechanisms responsible for the sorting of the Tombusvirus replication proteins and a number of the C-terminal tail-anchored (TA) proteins to the outer mitochondrial membrane. Specifically, we examined the targeting information responsible for mitochondrial localization of the 36-kD RNA-binding membrane-bound replicase protein (p36) of Carnation Italian Ringspot Virus (CIRV). CIRV is a member of the Tombusvirus family of positive-strand RNA viruses and the predominant cytopathological features in the infected host cell include massive structural alterations of the mitochondria into the so-called mitochondrial-multivesicular bodies (mMVBs). We show that the targeting information responsible for the sorting of p36 to mitochondria/mMVBs consists of its two transmembrane domains (TMDs) and an intervening loop sequence. Using bimolecular fluorescent complementation (BiFC) assays, we show that p36 interacts with certain components of translocase complex in the mitochondrial membrane (TOM), but not with the sorting and assembly machinery (SAM).In terms of the targeting of TA proteins, we show using in vitro assays that the C-terminal tails (CTSs) of tung (Vernicia fordii) cytochrome b5 (Cb5) isoforms A and B acquire their ER-targeting specificity by preventing spontaneous insertion into incorrect membranes. In contrast, the CTS of the Cb5 isoform D acquires its mitochondrial-targeting specificity by promoting targeting to the correct membranes. We also show that several other putative and known mitochondrial TA proteins, many of which were identified in a recent bioinformatics screen, possess similar or distinct C-terminal targeting signals compared to those found in tung Cb5D, These results as well as the results of BiFC experiments reveal that different insertion pathways may be utilized for the targeting and insertion of different mitochondrial TA proteins in plant cells. Overall, these results provide insight to the cellular processes underlying plant mitochondrial outer membrane protein sorting in general.