Intramolecular interactions control Vms1 translocation to damaged mitochondria

Abstract

Mitochondrial dysfunction is associated with the development of many age-related human diseases. Therefore recognizing and correcting the early signs of malfunctioning mitochondria is of critical importance for cellular welfare and survival. We previously demonstrated that VCP/Cdc48-associated mitochondrial stress responsive 1 (Vms1) is a component of a mitochondrial surveillance system that mediates the stress-responsive degradation of mitochondrial proteins by the proteasome. Here we propose novel mechanisms through which Vms1 monitors the status of mitochondria and is recruited to damaged or stressed mitochondria. We find that Vms1 contains a highly conserved region that is necessary and sufficient for mitochondrial targeting (the mitochondrial targeting domain [MTD]). Of interest, MTD-mediated mitochondrial targeting of Vms1 is negatively regulated by a direct interaction with the Vms1 N-terminus. Using laser-induced generation of mitochondrial reactive oxygen species, we also show that Vms1 is preferentially recruited to mitochondria subjected to oxidative stress. These studies define cellular and biochemical mechanisms by which Vms1 locali-zation to mitochondria is controlled to enable an efficient protein quality control system.

FIGURE 1:

Mitochondrial localization of Vms1 requires the MTD and is inhibited by the N-terminus. (A) Schematic representation of the domain structure of full-length Vms1 and deletion mutants. Full-length Vms1 contains a ZnF, an MTD, an ankyrin repeat, a predicted coiled-coil region (CC), and a VIM. The percentage identity between S. cerevisiae and human Vms1 is indicated for each region. (B) The vms1Δ strain containing both a plasmid expressing mito-RFP and a plasmid expressing the indicated GFP-tagged Vms1 deletion mutant was grown in SD-Ura-Leu. On reaching mid–log phase, the culture was either treated with vehicle (left) or 200 ng/ml rapamycin (right) for 3 h. Note that the deletion mutants were expressed as C-terminal GFP fusions under control of the native VMS1 promoter. Representative images are shown for each. (C) The vms1Δ mutant containing a plasmid expressing a C-terminus HA-epitope tagged full-length wild-type (cen) or MTD-only mutant (Vms1^MTD, 2μ) was grown in SGE-Ura medium. On reaching late log phase, cells were harvested and subjected to a mitochondrial isolation procedure as described. WCE, PMS, crude mitochondria (crude mito), and sucrose gradient–purified mitochondria (pure mito) were further analyzed for the presence of Vms1 by immunoblotting. Antibodies against actin (cytoplasm) or porin (mitochondria) were used to determine the purity of each fraction.

FIGURE 2:

Vms1^1-182 and Vms1^MTD physically interact in vivo. (A) The AH109 strain was transformed with plasmids expressing AD and DBD fusions with nothing (ev), the Vms1^1-182, or the Vms1^MTD. AD-ALIX and DBD-TSG101 were used as a positive control. Each strain was streaked on an SD-Trp-Leu-His plate and grown at 30°C for 3 d. (B) For coimmunoprecipitation studies, the vms1Δ strain was transformed with plasmids encoding the Vms1^1-182 and Vms1^MTD tagged with GFP, myc, or HA as indicated, all under control of the native VMS1 promoter. Each strain was grown to log phase in SD-Leu-Trp and harvested. The crude lysates from each strain were immunoprecipitated with anti-myc antibody, and Western blots were performed with anti-HA or anti-myc antibodies as indicated. Ten percent of crude lysate was loaded.

FIGURE 3:

Overexpression of Vms1^1-182 stabilizes Vms1^MTD and sequesters it in the cytoplasm. (A) The JRY1734 strain (pep4::HIS3 prb2::LEU2 bar1::HISG lys2::GAL1/10-GAL4) was transformed with plasmids expressing C-terminally HA-tagged Vms1^MTD (2μ) and empty vector (ev), N-terminally His₁₂-tagged Vms1^1-182, or Vms1^{1-182, MutD}. Each strain was grown in SGE-Ura-Trp media at 30°C. On reaching mid–log phase, galactose was added to a final concentration of 0.4%. After additional growth for 4 h, cells were harvested and subjected to mitochondrial isolation as described. Representative images of three independent experiments are shown. (B) Same set of experiments as in A, performed using C-terminally GFP-fused Vms1^MTD expressed under the VMS1 promoter rather than the HA-tagged Vms1^MTD (2μ).

FIGURE 4:

Vms1^11-55 is necessary and sufficient for interaction with Vms1^MTD. (A) The AH109 strain was transformed with plasmids expressing AD and DBD fusions with nothing (ev), Vms1^11-55, Vms1^MutB, Vms1^MutC, Vms1^MutD, or Vms1^MTD. AD-Vms1^1-182 and DBD-Vms1^MTD were used as a positive control. Each strain was streaked on both SD-Trp-Leu (left, control) and SD-Trp-Leu-His (right) plates and grown at 30°C for 3 d. (B) Sequence alignment of Vms1^11-55 among VMS1 orthologues from other species. (C) For coimmunoprecipitation studies, the vms1Δ strain was transformed with plasmids encoding Vms1^1-182, Vms1^{1-182, MutC}, Vms1^{1-182, MutD}, and Vms1^MTD tagged with myc or HA as indicated, all under control of the native VMS1 promoter. 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. Ten percent of crude lysate was loaded.

FIGURE 5:

Disruption of the Vms1 intramolecular interaction facilitates mitochondrial translocation of Vms1. (A) The vms1Δ strain containing a plasmid expressing GFP-tagged wild-type Vms1 or Vms1^MutD was grown in SD-Ura medium. On reaching log phase, cells were subjected to fluorescence microscopy to detect Vms1 localization. Representative images are shown. (B) The vms1Δ strain containing a plasmid expressing HA-tagged wild-type Vms1 or Vms1^MutD was grown in SD-Ura and harvested at log phase. Cells were then subjected to mitochondrial isolation and subsequent sucrose gradient purification. The same fraction of each sample was subjected to SDS–PAGE and subsequent Western blot. Antibodies against actin (cytoplasm) or porin (mitochondria) were used to determine the purity of each fraction. Representative images of three independent experiments are shown.

FIGURE 6:

Vms1 exhibits preferential localization to ROS-exposed mitochondria. (A) Yeast cells expressing Vms1-GFP were transformed with a mitochondrially targeted KillerRed, which generates superoxide and singlet oxygen upon irradiation with green light. Cells were irradiated for 15 min with 572-nm light. After irradiation, Vms1-GFP localization was monitored by wide-field fluorescence microscopy every 10 min for 60 min. (B) The cells described were irradiated with a 543-nm HeNe laser at 100% intensity for 1 min and only in a small region of the cell (indicated by the yellow dot), which contained mitochondria. Vms1-GFP localization was monitored by confocal fluorescence microscopy for 60 min after irradiation. (C) Quantification of Vms1-GFP localization to irradiated mitochondria, nonirradiated mitochondria, and the cytosol is shown as described in Materials and Methods.

FIGURE 7:

Intramolecular interactions regulate Vms1 localization to mitochondria. In resting and unstressed conditions, Vms1 is maintained in the cytosol through a stable intramolecular interaction between residues 1–182 and the MTD. Under stress conditions, specifically those conditions that necessitate the recruitment of the ubiquitin/proteasome system to mitochondria, a mitochondrial damage signal accumulates. Vms1 binds to mitochondria possessing this signal, which further promotes the disruption of the intramolecular interaction between residues 1–182 and the MTD, enabling a stable MTD/mitochondrial interaction.