Loss of function of KRE5 suppresses temperature sensitivity of mutants lacking mitochondrial anionic lipids

Abstract

Disruption of PGS1, which encodes the enzyme that catalyzes the committed step of cardiolipin (CL) synthesis, results in loss of the mitochondrial anionic phospholipids phosphatidylglycerol (PG) and CL. The pgs1Delta mutant exhibits severe growth defects at 37 degrees C. To understand the essential functions of mitochondrial anionic lipids at elevated temperatures, we isolated suppressors of pgs1Delta that grew at 37 degrees C. One of the suppressors has a loss of function mutation in KRE5, which is involved in cell wall biogenesis. The cell wall of pgs1Delta contained markedly reduced beta-1,3-glucan, which was restored in the suppressor. Stabilization of the cell wall with osmotic support alleviated the cell wall defects of pgs1Delta and suppressed the temperature sensitivity of all CL-deficient mutants. Evidence is presented suggesting that the previously reported inability of pgs1Delta to grow in the presence of ethidium bromide was due to defective cell wall integrity, not from "petite lethality." These findings demonstrated that mitochondrial anionic lipids are required for cellular functions that are essential in cell wall biogenesis, the maintenance of cell integrity, and survival at elevated temperature.

Figure 1.

Disruption of PGS1 results in loss of mtDNA. (A) Isogenic wild-type (FGY3), ρ⁰, and pgs1Δ (QZY24B) cells were grown in YPD to early stationary phase. DNA was visualized by staining with DAPI as described in Materials and Methods. (B) Haploid pgs1Δ and ρ^- tester strains (100-107) were crossed on a YPD plate. Diploid cells were selected on synthetic minimal medium. Mitochondrial function was determined by assessing growth on nonfermentable medium (YPGE).

Figure 2.

KRE5 complements the suppressor phenotype. (A) Cells from wild-type (FGY3), ρ⁰, pgs1Δ (QZY24B), the suppressor (QZY11A), and the suppressor transformed with empty vector YCp50 (+vec), or with YCp50 containing KRE5 (+KRE5) were serially diluted, spotted on YPD plates, and incubated at the indicated temperatures. (B) Wild-type (FGY3), ρ⁰, pgs1Δ (QZY24B), and suppressor (QZY11A) cells were grown to mid-log phase in YPD and examined microscopically.

Figure 3.

The suppressor mutant carries a loss of function allele of KRE5. (A) DNA of the KRE5 locus from pgs1Δ (QZY24B) and the suppressor (QZY11A) was sequenced as described in Materials and Methods. The single nonsense mutation in the coding sequence of KRE5 causes deletion of 201 amino acids from the C terminus of the protein. (B) K1 killer toxin producing cells (T158c/S14a) were spotted on plates preseeded with wild-type (FGY3), ρ⁰, pgs1Δ (QZY24B), and suppressor (QZY11A) cells and incubated at 30°C for 2 d. Sensitivity to K1 killer toxin is indicated by the presence of a killing zone surrounding cells. (C) The suppressor mutant (QZY11A) was crossed to the wild-type strain (FGY3). Diploid cells were sporulated, and meiotic tetrad analysis was performed. Genotypes of the haploid spores from six tetrads are shown.

Figure 4.

Cell wall properties of pgs1Δ and the suppressor pgs1Δ kre5^W1166X. (A) Cells from wild-type (FGY3), ρ⁰, pgs1Δ (QZY24B), and the suppressor (QZY11A) mutant were serially diluted and spotted on YPD or YPDS plates supplemented with 5 μg/ml CFW or 5 mM caffeine. Cells were incubated at 30°C. (B) Transmission electron microscopy of the cell wall in wild-type (FGY3), ρ⁰, pgs1Δ (QZY24B), and the suppressor (QZY11A) was performed as described in Materials and Methods. The horizontal bar represents 500 nm.

Figure 5.

Increased chitin deposition in pgs1Δ and the suppressor mutant. (A) Cells from wild-type (FGY3), ρ⁰, pgs1Δ (QZY24B), and the suppressor (QZY11A) were grown in YPD to early stationary phase. Chitin was visualized by staining with Oregon Green 488 as described in Materials and Methods. Chitin distribution was visualized by focusing on two planes. (B) Cells from pgs1Δ (QZY24B) and the suppressor mutant (QZY11A) were grown in YPD in the presence or absence of 10 mM glucosamine at the indicated temperatures. Viable cells were determined by serial dilution and plating.

Figure 6.

Supplementation with sorbitol supports growth of CL-deficient mutants at 37°C. (A) Isogenic wild-type and pgs1Δ mutant cells from two genetic backgrounds (FGY3 ρ⁰ and GA74D ρ⁺) were serially diluted and spotted on YPD or YPDS plates and incubated at the indicated temperature. (B) Wild-type (FGY3) and crd1Δ (FGY2) cells were grown overnight in YPD. Single cells were seeded on YPD or YPDS plates and incubated at the indicated temperature.

Figure 7.

Pgs1Δ survives ethidium bromide mutagenesis in the presence of osmotic stabilizer. Wild-type GA74D3A and isogenic pgs1Δ mutant GA74D3c cells were streaked on synthetic minimal plates with ethidium bromide or sorbitol as indicated and incubated at 30°C.