Fig. 5.

Neither deletion of zinc finger like region nor alteration of residues in substrate-binding cleft affects maintenance of mtDNA. A) (Top) Scheme of Mdj1_ΔZ with dotted line indicating the deletion of the zinc finger like region (for details see Fig. 3A). (Bottom) Structural model of C module with ZFLR, CTD1 and CTD2 indicated and with residues of the substrate binding cleft red — Ile²⁰², blue — Leu²²², yellow — Phe²²⁴, green — Ile³⁰¹, orange — Ile³⁴³, magenta — Val³⁴⁵. Arrows indicate residues, which were altered in this study. B) mdj1-Δ [TETr-MDJ1] expressing Mdj1 (wt), Mdj1_ΔZ, Mdj1_LFI/AAA under the control of the MDJ1 promoter was grown in the presence of doxycycline. At the indicated number of generations after doxycycline addition, aliquots were collected and plated on glucose-based media. The percentage of respiring cells was determined as the ratio of the number of red colored versus the total number of colonies on plates. C) Distribution of Mdj1 variants after centrifugation of mitochondrial lysates. Mitochondrial lysates isolated from MDJ1/mdj1-Δ diploid cells expressing Mdj1 (wt) or the Mdj1 variants, Mdj1_ΔZ, or Mdj1_LFI/AAA were subjected to ultracentrifugation through a sucrose gradient. Each fraction was analyzed for protein content using immunoblot analysis with antibodies specific for Mdj1 or, as a control, Abf2. Mdj1_LFI/AAA GFP fusions were used to allow separation from wt Mdj1 during electrophoresis. D) Cellular localization of Mdj1 variants. Cells expressing GFP fusions of Mdj1_ΔZ (∆Z-GFP) or Mdj1_LFI/AAA (LFI/AAA-GFP) were analyzed by fluorescence microscopy. Cellular DNA was stained with DAPI. Overlay of the two images (MERGE). Size bars (2 μm) are shown. E) mdj1-Δ cells harboring plasmid-borne copies of wt MDJ1, ΔZ or LFI/AAA variants, as indicated, were plated as 10-fold dilutions on glucose-rich medium (top) or glycerol-rich medium (bottom) and incubated at indicated temperatures for 3 days (glucose) or 4 days (glycerol).