Figure 6.

Stress-induced Mdv1p mitochondrial localization requires cyclin C. (A) A wild-type strain transformed with Mdv1-GFP and mt-DsRed expressing plasmids were analyzed by confocal microscopy before and following H₂O₂ treatment (1 mM for 2h). White arrows indicate sites of active fission, blue arrows identify Mdv1p foci not associated with the mitochondria. (B) Mdv1p subcellular localization patterns in WT and cnc1Δ strains before and following H₂O₂ stress (1 mM for 2 h) were determined by confocal microscopy. The categories for Mdv1-GFP localization patterns (Schauss et al., 2006) are given on the left. Values are mean ±s.e.m. **=p<0.01. (C) Wild-type strain expressing cyclin C-YFP, Mdv1p-DsRed and mt-CFP was subjected to H₂O₂ stress (1 mM for 2 h). Subcellular localization of the proteins and mitochondria was monitored by fluorescence microscopy. The percentage of cyclin C-YFP foci associating with Mdv1p-DsRed is indicated (n=3). Arrows indicate regions of co-localization between cyclin C-YFP and Mdv1p in the enlarged images. (D) and (E) A two-hybrid reporter strain (gal1-HIS3, PJ69-4A) transformed with Gal4p DNA binding domain (DBD) and activator fusion protein combinations as indicated was patched onto medium selecting for reporter gene activation (-His). The previously reported Mdv1-DBD and Fis1-AD interaction (Griffin et al., 2005) was used as a positive control. Vec = vector control. (F) Extracts prepared from unstressed and stressed wild-type strain expressing endogenous cyclin C-TAP and Mdv1p-HA were incubated with αTAP or αHA antibodies then probed for the presence of cyclin C-TAP (top panel). Control immunoprecipitation of Mdv1p-HA is shown in the bottom panel. The control extracts not expressing endogenous Mdv1p-HA (−) are indicated. (G) A wild-type strain expressing mt-CFP, cyclin C^HADΔ-YFP and Mdv1p-dsRed was grown to mid-log phase then examined by fluorescence microscopy. The arrows indicate areas of co-localization between the two proteins and the mitochondria. See also Figure S5.