Asymmetric Inheritance of Oxidatively Damaged Proteins During Cytokinesis

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Science  14 Mar 2003:
Vol. 299, Issue 5613, pp. 1751-1753
DOI: 10.1126/science.1080418


Carbonylated proteins were visualized in single cells of the budding yeast Saccharomyces cerevisiae, revealing that they accumulate with replicative age. Furthermore, carbonylated proteins were not inherited by daughter cells during cytokinesis. Mother cells of a yeast strain lacking the sir2 gene, a life-span determinant, failed to retain oxidatively damaged proteins during cytokinesis. These findings suggest that a genetically determined, Sir2p-dependent asymmetric inheritance of oxidatively damaged proteins may contribute to free-radical defense and the fitness of newborn cells.

In the yeastSaccharomyces cerevisiae, cell division is asymmetrical and some genetic material is unequally distributed (1). For example, the daughter cell does not inherit extrachromosomal ribosomal DNA circles (ERCs), which accumulate in mother cells during growth and have been suggested to cause replicative senescence (2). Reports have shown that cells lacking the silent information regulator, Sir2p, an NAD (nicotinamide adenine dinucleotide)–dependent histone deacetylase (3), contain more ERCs than their wild-type counterparts and have a reduced replicative potential (4). It has also been suggested that the age asymmetry may depend on partition of undamaged cellular components to the progeny (5) and that old mother cells show markers of oxidative stress (6). However, no information is available on whether oxidative damage, like ERCs, accumulates as a function of replicative age.

To determine whether protein carbonylation, an irreversible oxidative damage (7), increases with the yeast replicative age, we isolated cells of different age from a population growing exponentially in YPD medium by elutriation. Newly born cells are small and pass through the G1 phase until they reach the critical size required to enter S phase. Thus, elutriation fractions 1 and 2 contain cells of similar replicative age, but the cells of fraction 2 are chronologically older (Fig. 1) and a small portion of cells is budding in this fraction. Fraction 2 cells exhibited a fourfold higher level of oxidative damage than fraction 1 cells (Fig. 1), implying that oxidation of proteins occurs during the first G1 phase of newborn cells. Protein oxidation increased further as mother cells produce 10 or more daughters (Fig. 1, fraction 5). The results were confirmed with the use of biotin-streptavidin magnetic sorting (8) to isolate cells of different replicative age (9). To address how and when daughter cells rid themselves of damaged proteins, we visualized carbonylated proteins in situ using immunocytochemistry (9, 10). An uneven distribution of oxidized proteins between mother cells and buds was observed during cytokinesis (Fig. 2, A and B). Paraquat exposure increased the oxidative damage (per unit volume) in the bud (Fig. 2C), demonstrating that target carbonyl groups can be effectively derivatized in the bud compartment and reached by the antibodies. Analysis of carbonylation per total protein by dot-blot analysis confirmed a lower (3.6-fold) damage density in newborn daughters. In addition, planes separated by 0.1 μm in depth (Z-series) were scanned to construct three-dimensional images of carbonylated proteins, which confirmed an unequal density of oxidatively damaged proteins in the bud and mother cells (Fig. 2D). Buds contained active mitochondria (Fig. 2E), and in situ detection of superoxide (Fig. 2F) (11) and hydrogen peroxide (9) demonstrated that the production of reactive oxygen species (ROS) was not different in mothers and buds. The carbonyl signals partly colocalized with mitochondria, but carbonyls were also detected in areas free of mitochondria (Fig. 2G). In accordance with this, isolated mitochondria contained about 40% of total protein carbonyls in cell extracts (fig. S1).

Figure 1

Levels of oxidative protein damage as a function of replicative age. Average number of birth/bud scars (open bars) in five fractions obtained from elutriation experiments (9). Protein carbonyl levels (filled bars) in the five elutriated fractions were obtained by quantifying dot-blots as described (15). Relative values are shown, and the highest signal was arbitrarily assigned a value of 100. Lines on top of bars are standard deviations.

Figure 2

Distribution of oxidatively damaged proteins, mitochondria, and ROS during yeast cytokinesis. (A) Bright-field image and protein carbonyl image, detected by in situ immunofluorescence (ImFl Carbonyls), of budding yeast (9). (B) The carbonyl signal was quantified along the line drawn on the bright-field image of (A). The data were obtained by averaging 70 line intensity plots distanced by 0.1 μm from each other within the plane orthogonal to the plane of the picture along the line depicted in (A). (C) Protein carbonyls in budding wild-type cells treated with paraquat (400 μg/ml) 20 min before fixation. (D) A three-dimensional image was reconstructed with the LaserVox software (Bio-Rad) after confocal scanning of carbonylation signals in planes separated by 0.1 μm in depth. Four frames at different rotation angles are shown. Blue denotes the lowest and red the highest intensity. The bud is indicated with an arrow in the first panel. (E) Mitochondrial distribution analyzed with the membrane potential–dependent dye DiOC6. (F) Superoxide-ion detection in budding cells with dihydroethidium (DHE). (G) Colocalization of mitochondria and carbonylated proteins. The first panel shows the bright-field image; the second and third panels show carbonyls and mitochondria (detected with an antibody to Por1p), respectively. The last panel shows the merge between panels 2 and 3. Colocalized signals appear yellow.

To determine whether oxidized proteins were inherited asymmetrically during cytokinesis, we treated cells with paraquat to increase the level of carbonylated proteins twofold, and then the mother cells were allowed to produce offspring. After elutriation, carbonylation damage per total protein in the first-generation daughters was sixfold lower than in the mother cell (Table 1). Thus, the extra load of oxidative damage appeared to be retained in the mother cell and was not inherited by the daughter cell. Consistent with this notion, the rate of degradation of carbonylated proteins was similar in mother cell and bud compartments (fig. S2).

Table 1

Density of oxidative damage (carbonyls per total protein) in newborn daughters as compared with the mother determined by dot-blot analysis. The “daughter fraction ” is fraction 1 and the “mother fraction” is fractions 2, 3, 4, and 5 combined (Fig. 1). The analysis was performed in growing untreated cultures (−) and immediately after exposure to paraquat (400 μg/ml) for 20 min (+). After paraquat treatment, the cells were washed and allowed to divide once in YPD. Mothers and newborn daughters were then isolated by elutriation and scored for carbonyl levels (column “−→+”). The oxidation density in the daughters is related to that of the mother cell, which was assigned a value of 100. Numbers in parentheses are standard deviations.

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The ability of mother cells to retain oxidatively damaged proteins during cell division diminished with replicative age (Fig. 3A). In mutant cells lacking the oxidative stress defense genes sod1, sod2, orcta1 (12, 13), the overall protein oxidation was higher than in wild-type cells, but the low oxidation density in buds as compared with mothers was not affected (Fig. 3B). When mutants (sir2, sir3, andsir4) displaying a reduced life-span (4) were analyzed, only cells lacking sir2differed from the wild type (Fig. 3B). The sir2 mutants tend to simultaneously express the a and α mating-types genes (14). However, this phenotype was not linked to segregation defects because Δsir3 and Δsir4 mutants share this mating-type defect with the Δsir2 mutant (4). Deletinghml in a Δsir2 mutant did not correct for its failure to segregate carbonylated proteins (Fig. 3B). Young Δsir2 cells exhibited higher levels of oxidatively damaged proteins than young wild-type cells, and protein carbonyls were distributed evenly between mother and daughter cells during cell division regardless of the age of the Δsir2 mother cells (Fig. 3C). No difference in paraquat sensitivity, ROS production, or the state of respiration (15) was observed between the wild type and the Δsir2 mutant (fig. S3). This is consistent with a recent report demonstrating that the Δsir2 mutation does not affect the cells' ability to withstand external oxidative stress (16). In contrast to wild-type daughters, the first-generation daughters ofsir2 cells inherited the extra load of oxidatively damaged proteins after paraquat treatment (Table 1). This indicates that Δsir2 cells are defective in the segregation of oxidized proteins during cytokinesis.

Figure 3

Protein carbonylation in wild-type and Δsir2 mutant cells of different replicative age. (A) Oxidation density (carbonyls per unit volume) in mother cells (open bars) and buds (filled bars) in elutriated fractions. Fraction 1 is not displayed because cells isolated from this fraction are not budding. The damage density of mother cells in fraction 5 was assigned a value of 100. (B) Average oxidation density value (protein carbonyls per total protein) in daughter cells as compared with their mothers (assigned a value of 100) of wild-type, Δsod1, Δsod2, Δcta1, Δsir3, Δsir4, Δsir2, and Δsir2, hml strains. Numbers in parentheses indicate the level of protein carbonylation in mother cells relative to that in the wild type (wild-type cells were arbitrarily assigned a value of 100). (Inset) Protein carbonyls in budding Δsir2 cells. The upper panels show bright-field images, and the lower panels show protein carbonyls signals. (C) Oxidative damage density (carbonyls per unit volume) in mother cells (open bars) and daughters (filled bars) in elutriated fractions of the Δsir2 strain. The damage density of mother cells in fraction 5 was assigned a value of 100. Lines on top of bars are standard deviations.

The spatial distribution of F-actin during cytokinesis was atypical in the Δsir2 mutant but not in Δsir3and Δsir4 (fig. S4). Specifically, at the end of cytokinesis, actin was predominantly found in the daughter cells of Δsir2 mutants, whereas in wild-type, Δsir3, and Δsir4 cells, the actin is redistributed to both the mother and daughter cell (fig. S4). Inhibition of actin assembly by latrunculin A (Lat-A) abolished the ability of wild-type mother cells to retain oxidized proteins (fig. S4), suggesting that the actin skeleton is required for proper segregation of oxidized proteins.

In summary, oxidatively damaged proteins are inherited asymmetrically during yeast cytokinesis, and this process is Sir2p dependent. Although the data provided do not establish the capacity to segregate oxidative damage as a life-span determinant, they indicate a new aspect of cell division asymmetry and a mechanism for dealing with oxidative damage, which is likely important for the fitness of newly born cells.

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