Loss of mitochondrial DNA in the yeast cardiolipin synthase crd1 mutant leads to up-regulation of the protein kinase Swe1p that regulates the G2/M transition

Abstract

The anionic phospholipid cardiolipin and its precursor phosphatidylglycerol are synthesized and localized in the mitochondrial inner membrane of eukaryotes. They are required for structural integrity and optimal activities of a large number of mitochondrial proteins and complexes. Previous studies showed that loss of anionic phospholipids leads to cell inviability in the absence of mitochondrial DNA. However, the mechanism linking loss of anionic phospholipids to petite lethality was unclear. To elucidate the mechanism, we constructed a crd1Deltarho degrees mutant, which is viable and mimics phenotypes of pgs1Delta in the petite background. We found that loss of cardiolipin in rho degrees cells leads to elevated expression of Swe1p, a morphogenesis checkpoint protein. Moreover, the retrograde pathway is activated in crd1Deltarho degrees cells, most likely due to the exacerbation of mitochondrial dysfunction. Interestingly, the expression of SWE1 is dependent on retrograde regulation as elevated expression of SWE1 is suppressed by deletion of RTG2 or RTG3. Taken together, these findings indicate that activation of the retrograde pathway leads to up-regulation of SWE1 in crd1Deltarho degrees cells. These results suggest that anionic phospholipids are required for processes that are essential for normal cell division in rho degrees cells.

FIGURE 1.

Loss of mtDNA in crd1Δ results in slow growth and decreased PG. A, yeast cells (except for pgs1Δ) were grown in YPD medium to the early stationary phase, and cells were seeded on solid YPD. The pgs1Δ cells were precultured in YPDS containing 1 m sorbitol and then switched to YPD medium for 18 h and plated on solid YPD. B, cells were grown in YPD at 30 °C. Cell growth was monitored by A₅₅₀ at the indicated times. C, WT, WT rho°, crd1Δ, and crd1Δrho° cells were grown in YPD, and pgs1Δ was grown in YPDS, at 30 °C to the early stationary phase. Cells were harvested by centrifugation, and lipids were extracted and separated as described under “Experimental Procedures.” Phospholipids were separated by one-dimensional TLC and visualized by phosphorimaging. CL and PG were quantified using the ImageQuant software. The relative amount of ³²P in CL and PG is presented as a percentage of the ³²P incorporated into total phospholipids. Error bars represent the range of the three independent experiments. PA, phosphatidic acid; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine.

FIGURE 2.

Cell division defects in crd1Δrho° cells. A, WT, rho°, crd1Δ, and crd1Δrho° cells were cultured in YPD medium. The crd1Δrho° cells transformed with pYPGK18 or pYPGK18CRD1 were cultured in synthetic drop-out medium (Leu⁻). Cell morphology was examined and photographed microscopically after growth to the mid-log phase at 30 °C. The scale bar represents 3 μm. B, cells were grown to the mid-log phase in YPD at 30 °C. The nucleus was visualized by staining with DAPI as described under “Experimental Procedures.” The scale bar represents 2 μm. C, yeast cells were grown to mid-log phase in YPD medium at 30 °C and processed for flow cytometry as described under “Experimental Procedures” (1C, G₁ DNA content; 2C, G₂/M DNA content; 4C, greater than G₂/M DNA content). D, cells were grown to the early stationary phase in YPD at 30 °C. Chitin deposition was visualized by staining with WGA-Oregon Green 488 as described under “Experimental Procedures.” The scale bar represents 2 μm. E, cells transformed with the GFP-tagged Cdc3p were grown to the mid-log phase in synthetic drop-out medium at 30 °C, and living cells were examined directly under the fluorescence microscope. The scale bar represents 2 μm. All images in the same pattern were taken at the same magnification (×1000).

FIGURE 3.

Swe1p is up-regulated in crd1Δrho° cells. A, total RNA was extracted from cells grown to the mid-log phase in YPD at 30 °C. SWE1 mRNA levels were determined by real-time PCR as described under “Experimental Procedures.” Expression was normalized to the RNA levels of the internal control ACT1. Error bars represent the range of the three independent experiments. B, total protein was extracted from cells grown to the mid-log phase in YPD at 30 °C. Swe1p was identified by Western blot. Wild type cells transformed with pYPGK18SWE1 were used as a positive control, swe1Δ was used as a negative control, and α-tubulin was used as a loading control.

FIGURE 4.

Deletion of SWE1 in crd1Δrho° rescued cell morphology, chitin deposition, septin ring localization, and nuclear DNA content. A, the nucleus was visualized by staining with DAPI as described in the legend for Fig. 2. The scale bar represents 3 μm. B, chitin deposition was visualized by staining with WGA-Oregon Green 488 as described in the legend for Fig. 2. The scale bar represents 2 μm. C, Cdc3p-GFP was visualized as described in the legend for Fig. 2. The scale bar represents 2 μm. All images in the same pattern were taken at the same magnification (×1000). D, yeast cells were grown to mid-log phase in YPD medium at 30 °C and processed for flow cytometry as described under “Experimental Procedures” (1C, G₁ DNA content; 2C, G₂/M DNA content; 4C, greater than G₂/M DNA content).

FIGURE 5.

The crd1Δrho° mutant exhibited severe defects in mitochondrial integrity. Cells grown to the mid-log phase were incubated with DiOC₆(3) or MitoTracker Green FM and examined under the fluorescence microscope. In living cells containing COX4-DsRed, mitochondria were visualized directly under the fluorescence microscope after cells were grown to the mid-log phase. All images were taken at the same magnification (×1000). The scale bar represents 2 μm.

FIGURE 6.

Deletion of RTG2 or RTG3 suppressed elevated expression of SWE1 in crd1Δrho° cells. Total RNA was extracted from cells grown to the mid-log phase in YPD at 30 °C, and mRNA levels of CIT2, DLD3, and SWE1 were determined by real-time PCR as described under “Experimental Procedures.” Expression was normalized to the mRNA levels of the internal control ACT1. Error bars represent the range of the three independent experiments.

FIGURE 7.

Model for retrograde pathway-mediated cell division delay in crd1Δrho° cells. The loss of mtDNA in crd1Δ cells leads to exacerbation of mitochondrial dysfunction, which is sensed by Rtg2p. The Rtg2p transmits this signal to the heterodimer Rtg1p/Rtg3p, resulting in translocation of Rtg1p/Rtg3p from the cytoplasm to the nucleus where it activates transcription of SWE1. The up-regulation of Swe1p serves as a compensatory mechanism to delay the cell division, preventing the generation of abnormal daughter cells.