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Mitochondrial IMS can enhance the stability of the bacterial trimeric autotransporter YadA when the Telatinib Biological Activity latter is expressed in yeast cells and gets assembled into the mitochondrial OM18. We next wanted to test whether this influence can be observed also for other bacterial OMPs and how the chaperone's contribution is dependent on the presence of POTRA domains. To that aim we employed cells depleted for endogenous Tob55 and complemented with plasmid-encoded Tob55, POTRA-less Tob55, or the latter fused to bacterial POTRA5. OmpA or PhoE were co-expressed with either Skp or SurA in these cells. Of note, both bacterial chaperones are targeted to the mitochondrial IMS in these cells18. The levels of OmpA were not significantly altered by the presence of Skp or SurA (Fig. 5a,b). The apparent independency of OmpA levels of the presence of periplasmic chaperones or the various Tob55/BamA hybrids raises the theoretical possibility that the membrane assembly of OmpA can occur without the involvement of the mitochondrial import system. To test this possibility we monitored the levels of OmpA in a strain where the expression of Tob55 is controlled by the inducible GAL10 promoter. Similarly to other mitochondrial -barrel proteins like Porin and Tom40, the detected levels of OmpA were dramatically reduced upon depletion of Tob55 (Supplementary Fig. S6), suggesting that OmpA requires the TOB complex for its biogenesis. In contrast to the behavior of OmpA, the co-expression of Skp dramatically enhanced the levels of PhoE in all tested cells. Interestingly, the presence of SurA moderately increased the levels of PhoE in cells harboring native Tob55 but resulted in decreased PhoE amounts in cells containing the POTRA-less Tob55 variant (Fig. 5c,d). The elevated levels of PhoE upon co-expression with Skp raised the question whether the latter simply stabilizes more substrate molecules in the mitochondrial IMS or can also enhance their membrane integration. ToScientific RepoRts | 6:39053 | DOI: 10.1038/srepwww.nature.com/scientificreports/Figure 4. Cells harboring hybrid Tob55/BamA proteins contain lower levels of PhoE. (a) Crude mitochondria were isolated from Tob55-depleted cells co-expressing OmpA and either plasmid-encoded Tob55 or the indicated hybrid protein. Proteins were analyzed by SDS-PAGE and immunodetection with antibodies against OmpA, and the indicated mitochondrial proteins. (b) Crude mitochondria were isolated from Tob55-depleted cells co-expressing PhoE and the indicated hybrid protein. Further treatment and analysis were as described in part (a). (c) The bands of at least three experiments as those described in (a) and (b) were quantified and their means ?s.d. are presented. To allow comparison among the various samples, the levels of Tom20 were taken as internal reference. The intensities of the bands from cells transformed with native Tob55 (construct A) were set to 100 . **p value