A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae

Abstract

The smm1 mutation suppresses defects in mitochondrial distribution and morphology caused by the mdm1-252 mutation in the yeast Saccharomyces cerevisiae. Cells harboring only the smm1 mutation themselves display temperature-sensitive growth and aberrant mitochondrial inheritance and morphology at the nonpermissive temperature. smm1 maps to RSP5, a gene encoding an essential ubiquitin-protein ligase. The smm1 defects are suppressed by overexpression of wild-type ubiquitin but not by overexpression of mutant ubiquitin in which lysine-63 is replaced by arginine. Furthermore, overexpression of this mutant ubiquitin perturbs mitochondrial distribution and morphology in wild-type cells. Site-directed mutagenesis revealed that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. A second mutation, smm2, which also suppressed mdm1-252 defects, but did not cause aberrant mitochondrial distribution and morphology, mapped to BUL1, encoding a protein interacting with Rsp5p. These results indicate that protein ubiquitination mediated by Rsp5p plays an essential role in mitochondrial inheritance, and reveal a novel function for protein ubiquitination.

Figure 1

Suppression of mdm1-252 defects by smm1 and smm2. (A) Cells harboring the mdm1-252 mutation (MYY721), or mdm1-252 together with the smm1 (MYY803) or smm2 (MYY804) mutation were cultured on YPD medium for 48 h at 37°C. (B) Strains MYY721, MYY803, and MYY804 were grown on YPD at 23°C, incubated for 4 h at 37°C, and mitochondria were visualized by staining with DASPMI and fluorescence microscopy. Shown are two representative cells for each strain. Bar, 2 μm.

Figure 2

smm1 and smm2 confer temperature-sensitive growth. Strains MYY803 and MYY804 were backcrossed to MYY291 to separate mdm1-252 from smm1 and smm2, respectively. Spores of the indicated genotypes were plated on YPD medium and incubated 48 h at 37°C. (A) Growth at 37°C of spores from a cross of MYY803 and MYY291; (B) growth at 37°C of spores from a cross of MYY804 and MYY291.

Figure 3

The smm1 mutation causes mitochondrial inheritance defects. Strain MYY809 (smm1) was cultured on YPD medium at 23°C and then incubated for 4 h at 23°C or 37°C. (A) Cells were stained with DASPMI to visualize mitochondria. Bar, 2 μm. (B) Cells which had been shifted to 37°C for 4 h were fixed with formaldehyde and processed for indirect immunofluorescence. Mitochondria were detected with anti-OM14 followed by fluorescein-conjugated donkey anti–mouse IgG (top). Mitochondrial and nuclear DNAs were stained with DAPI (middle). Mdm1p was detected with affinity-purified anti-Mdm1p followed by rhodamine-conjugated donkey anti– rabbit IgG (bottom). Shown are two representative cells. Bar, 2 μm.

Figure 4

Ubiquitin overexpression affects mitochondrial distribution and morphology. smm1 mutant cells (MYY823) and wild-type cells (MYY290) which harbored no plasmid (−), plasmid pUb (Ub), or plasmid pTER23 (K63R) were cultured at 23°C on minimal medium lacking uracil. CuSO₄ was added to 100 μM, and cells were incubated for 2 h at 23°C or 37°C. (A) Cells were fixed, stained with DAPI, and examined by fluorescence microscopy. Budded cells were scored as possessing empty buds if no mitochondrial DNA nucleoids were observed in the bud. Percentages of empty buds were determined for duplicate samples with at least 300 budded cells counted per sample. (B) Wild-type cells (MYY290) transformed with pUb (Ub) or pTER23 (Ub K63R) were incubated at 37°C for 2 h in the presence of 100 μm CuSO₄ and stained with DASPMI. Cells were examined by fluorescence (DASPMI) or phase-contrast (Phase) microscopy. Bar, 2 μm.

Figure 4

Ubiquitin overexpression affects mitochondrial distribution and morphology. smm1 mutant cells (MYY823) and wild-type cells (MYY290) which harbored no plasmid (−), plasmid pUb (Ub), or plasmid pTER23 (K63R) were cultured at 23°C on minimal medium lacking uracil. CuSO₄ was added to 100 μM, and cells were incubated for 2 h at 23°C or 37°C. (A) Cells were fixed, stained with DAPI, and examined by fluorescence microscopy. Budded cells were scored as possessing empty buds if no mitochondrial DNA nucleoids were observed in the bud. Percentages of empty buds were determined for duplicate samples with at least 300 budded cells counted per sample. (B) Wild-type cells (MYY290) transformed with pUb (Ub) or pTER23 (Ub K63R) were incubated at 37°C for 2 h in the presence of 100 μm CuSO₄ and stained with DASPMI. Cells were examined by fluorescence (DASPMI) or phase-contrast (Phase) microscopy. Bar, 2 μm.

Figure 5

Differential effects of rsp5 mutations on mitochondrial inheritance. Haploid rsp5-null strains harboring plasmids containing either wild-type RSP5 (MYY826) or mutant forms of rsp5 with the smm1 (MYY829), mdp1-1 (MYY832), mdp1-13 (MYY833), or mdp1-14 (MYY834) mutation were cultured on minimal medium lacking uracil at 23°C. Cells were incubated at 37°C for 2 h, stained with DASPMI, and examined by fluorescence microscopy. Percentages of empty buds were determined for budded cells in duplicate samples with at least 200 cells counted per sample.

Figure 6

Ubiquitin ligase activity of Rsp5p is required for mitochondrial inheritance. Cells harboring the smm1 mutation (MYY823) were transformed with centromere-based plasmids encoding wild-type RSP5 (pRS316-RSP5) or rsp5 bearing the C777A mutation (pRS316-CA). (A) Transformants were incubated on YPD medium at 37°C for 48 h. (B) Cells were cultured in minimal medium lacking uracil, shifted to 37°C for 4 h, stained with DASPMI, and examined by fluorescence microscopy. Bar, 2 μm.

Figure 7

Mutations in ubiquitin-conjugating enzymes cause defects in mitochondrial morphology and inheritance. Cells harboring null mutations in both ubc4 and ubc5 (LHY21) were cultured at 23°C on YPD medium, incubated at 37°C for 4 h, stained with DASPMI, and examined by fluorescence microscopy. Arrows indicate daughter buds without mitochondria. Bar, 2 μm.