Mpa1 prevents Ppr10 degradation by Lon1. (A) Deletion of mpa1 reduces Ppr10 protein levels. WT and Δmpa1 cells expressing chromosomally 13Myc-tagged Ppr10 were grown for the indicated time points (in hours), and the Ppr10 protein levels were monitored by western blotting with anti-c-Myc Ab. The results are representative of three independent experiments. (B) Reduction of Ppr10 protein levels by mpa1 deletion can be rescued by expression of WT Mpa1. Δmpa1 cells expressing chromosomally 13Myc-tagged Ppr10 were transformed with an integrated empty vector pJK148 or an integrated vector expressing 3HA-tagged Mpa1 under the control of its native promoter, and WT cells expressing chromosomally 13Myc-tagged Ppr10 were transformed with pJK148. Cells were grown for 18 h. Ppr10 protein levels were visualized by western blotting with anti-c-Myc Ab. (C) The Mpa1 protein levels are not affected in the absence of Ppr10. WT and Δppr10 cells expressing chromosomally 3HA-tagged Mpa1 were grown for the indicated time points, and the Mpa1 protein levels were monitored by western blotting with anti-HA Ab. (D) Deletion of lon1 restores the Ppr10 protein levels. WT, Δmpa1, Δlon1 and Δmpa1Δlon1 cells expressing chromosomally encoded 13Myc-tagged Ppr10 were grown for the indicated times, and Ppr10 protein levels were monitored by western blotting with anti-c-Myc Ab. For A–D, extracts were prepared by alkaline extraction. Sla1 serves as a loading control. (E) Deletion of lon1 cannot rescue the respiratory growth defect of Δmpa1 cells. A 10-fold dilution spot assay of cells were spotted on YES+Glu, YES+Gly or YES+Gal and incubated at 30°C. (F) Deletion of lon1 cannot restore the protein levels of mitochondrial-encoded proteins. Mitochondrial extracts were prepared from WT, Δlon1, Δmpa1 and Δlon1Δmpa1 cells by spheroplast lysis, and analyzed by western blotting using anti-Cob1, Cox1, Cox2 and Cox3 Abs. Hsp60 serves as a loading control.