Intramolecular interactions control Vms1 translocation to damaged mitochondria

A highly conserved middle region of Vms1 is necessary and sufficient for mitochondrial targeting (MTD). MTD-mediated mitochondrial targeting of Vms1 is negatively regulated by a direct interaction with the Vms1 N-terminus. Under conditions in which selected mitochondria are damaged, Vms1 is selectively recruited to damaged mitochondria.

. The vms1Δ growth defects cannot be rescued by Vms1 deletion mutants.
(A) WT and the vms1Δ strains were transformed with empty vector (ev), a plasmid containing the VMS1 gene (pVMS1), or plasmids containing various deletion mutants of Vms1 as described in Figure 2 (pvms1-182-end to 182-417).
Each strain was grown to saturation in liquid medium and serial dilutions were spotted on both SD-Ura and SD-Ura + rapamycin (20ng/ml) and grown at 30°f or 3 and 7 days, respectively. (B) The vms1Δ strain was transformed with plasmids encoding Cdc48-myc and either Vms1 1-182 -HA, Vms1-HA, or empty vector as indicated. Each strain was grown to log phase in SD-Leu-Trp and harvested. The crude lysates from each strain were immunoprecipitated with anti-HA antibody and Western blots were performed with anti-HA or anti-myc antibodies as indicated.
44 Figure S3. Schematic representation of Vms1 deletion mutants generated and tested for interaction in the yeast two-hybrid assay A series of Vms1 mutants was generated as above. Mutants exhibiting interaction are shown in black while those that failed to interact are shown in red. Figure S4. Vms1 11-end exhibits similar localization pattern to the Vms1 wild type and Vms1 11-end, MutA is constitutively localized to mitochondria.
The vms1Δ strain was transformed with C-terminus GFP tagged full-length wild type Vms1, Vms1 11-end , or Vms1 11-end, MutA . Each transformant was grown to log phase in SD-Ura media and subjected to fluorescence microscopy.
Representative images are shown. Figure S5. Vms1 11-end, MutA is dysfunctional due to its instability.
(A) WT and the vms1Δ strains were transformed with either empty vector (ev), a plasmid containing C-terminus HA tagged full-length wild type Vms1, Vms1 11-end , or Vms1 11-end, MutA . Each strain was then grown to saturation in liquid medium and serial 5-fold dilutions were spotted on both SD-Ura and SD-Ura + rapamycin (20ng/ml) and grown at 30° for 3 and 7 days, respectively. (B) The same transformed cells grown to mid log phase in SD-Ura media were either left as they were or treated with rapamycin (200ng/ml) for 3 hours. The same amount of cells was then harvested to prepare a whole cell extract (WCE).
Identical amounts of WCE were subjected to SDS-PAGE followed by Western blot. The levels of each Vms1 variant were determined by using anti-HA antibody. An antibody against actin was utilized as a loading control. WT and the vms1Δ strains were transformed with empty vector (ev), a plasmid containing C-terminus HA-tagged wild type VMS1 (Vms1 WT -HA), VMS1 MutC and VMS1 MutD . Each strain was grown to saturation in SD-Ura media and a serial 5fold dilution of equal numbers of cells was spotted on both SD-Ura (control) and SD-Ura+rapamycin (20ng/ml) and grown at 30° for 3 and 7 days, respectively. Figure S7. Vms1 1-182, T48A, S127A, S132A, T137A, S138A, S139A mutant exhibits almost identical affinity to Vms1 MTD compared with that of Vms1 1-182, WT .
(A) A schematic representation of phosphorylation sites identified in purified Vms1. (B) The vms1Δ strain was transformed with either empty vector (ev), Vms1 MTD -myc, Vms1 1-182, WT -HA or Vms1 1-182, T48A, S127A, S132A, T137A, S138A, S139A -HA as indicated. Each strain was then grown to log phase and harvested. The crude lysates from each strain were immunoprecipitated with anti-HA antibody and 46 (C) The vms1Δ strain was transformed with both mtRFP and C-terminus GFPfused full-length wild type Vms1 or Vms1 S/T->A mutant. Each cell was then grown to log phase in SD-Ura-Leu medium and subjected to imaging. Representative images are shown.    Heo, Figure S1 WT+e.v.