Fig. 4.

Simultaneous deletion of CQD1 and UPS1 impairs mitochondrial protein import and dynamics. (A) Analysis of steady-state levels of mitochondrial proteins in wild-type (WT) cells and cells lacking Ups1, Cqd1, or both. Cells were grown in synthetic complete medium containing galactose (SCGal), and whole-cell extracts were analyzed by immunoblotting. (B) Formation of mitochondrial protein complexes. Strains were grown in SCG. Isolated mitochondria were lysed in digitonin-containing buffer (3% w/v) and cleared lysates were subjected to BN-PAGE. The assembly of the TOM complex and respiratory chain super complexes were analyzed by immunoblotting using antibodies against Tom40 or Cyt1. (C) Deletion of CQD1 in cells lacking Ups1 exacerbates accumulation of the precursor of Mdj1. Cells were grown in SCD, whole-cell lysates were prepared and analyzed by immunoblotting with specific antibodies. Pgk1 served as a loading control. p, precursor of Mdj1; m, mature form of Mdj1. The quantification was obtained from three independent experiments and shows mean±s.d. of the ratio of the Mdj1 precursor to the total amount of Mdj1. Quantification was undertaken with Image Studio software. *P≤0.05 (unpaired two-tailed Student's t-test). (D) Simultaneous deletion of CQD1 and UPS1 leads to strongly reduced processing of Mgm1. Whole-cell lysates from cells grown in SCD were analyzed by immunoblotting. l, long isoform of Mgm1; s, short isoform of Mgm1. The quantification was obtained from three independent experiments and shows mean±s.d. of the ratio of the short form to the long form of Mgm1. Quantification was undertaken with Image Studio software. **P≤0.01 (unpaired two-tailed Student's t-test). (E) Mitochondria in cells lacking Ups1 and Cqd1 are highly fragmented. Mitochondria were labeled by expression of mKate targeted to mitochondria. Cells were grown in YPD and shifted to SCD, harvested in their logarithmic growth phase and immobilized on slides covered with concanavalin A. PC, phase contrast. For quantification 100 cells were counted for each strain. Scale bars: 4 µm. (F) The synthetic growth defect of the double deletion mutant Δcqd1 Δups1 is not caused by loss of mitochondrial DNA. Cells of the indicated strains were grown to logarithmic phase on YPD, shifted to SCD and growth was analyzed by drop dilution assay on SCD and YPG plates at 30°C. Images in A, B and F are representative of at least three repeats.