Skip to main content
. 2016 Nov 15;13(1):114–132. doi: 10.1080/15548627.2016.1252889

Figure 9.

Figure 9.

Mutation of the PY motif of MDM34 affects mitophagy. (A) The WT, MDM34-GFP, mdm34[3PA]-GFP, MDM34-MYC and mdm34[3PA]-MYC strains were grown to mid-exponential growth phase in YPD, washed twice in water and subjected to serial 5-fold dilution. Cells were then spotted onto solid complete medium containing glycerol as a carbon source (Gly) and supplemented with the indicated concentrations of rapamycin (Rapa). The plates were then incubated at 30°C. (B and C) The MDM34-MYC and mdm34[3PA]-MYC strains transformed with a plasmid encoding mitochondrion-targeted Rosella (mtRosella) were grown to very early exponential growth phase in YNB with glycerol as the carbon source and supplemented with the appropriate amino acids. After 24 h in glycerol-containing medium, the cultures were split in 2: one-half of the culture was treated with 200 nM rapamycin (Rapa) to induce mitophagy and the second-half of the culture was allowed to grow for 48 to 72 h. The cells of the first half of the culture were observed by fluorescence microscopy at time 0 (just before the addition of rapamycin) and after 24 h in the presence of rapamycin (A). Arrows indicate cells with mtRosella in the vacuole. (C) Total protein extracts were obtained from these cells after 0, 4 and 24 h of rapamycin treatment (C, left panel) and from the cells of the second culture after 48 and 72 h of culture in glycerol-containing medium (C, right panel). Protein extracts were analyzed by western immunobloting with antibodies against MYC, GFP (to detect full-length mtRosella and free GFP) and Pgk1, as a loading control. The ratio of free GFP to mtRosella was determined by densitometry with Image Lab 3.0.1 software (Bio-Rad). Fluorescence images and protein gel blots shown were representative images from 3 independent experiments.