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Mutational analysis of Mdm1p function in nuclear and mitochondrial inheritance.

Fisk HA, Yaffe MP - J. Cell Biol. (1997)

Bottom Line: Class I and II mutants also exhibited altered mitochondrial morphology, possessing primarily small, round mitochondria instead of the extended tubular structures found in wild-type cells.The mutations defining all three allelic classes mapped to two distinct domains within the Mdm1p protein.These results support a model of Mdm1p function in which a network comprised of multimeric assemblies of the protein mediates two distinct cellular processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Nuclear and mitochondrial transmission to daughter buds of Saccharomyces cerevisiae depends on Mdm1p, an intermediate filament-like protein localized to numerous punctate structures distributed throughout the yeast cell cytoplasm. These structures disappear and organelle inheritance is disrupted when mdm1 mutant cells are incubated at the restrictive temperature. To characterize further the function of Mdm1p, new mutant mdm1 alleles that confer temperature-sensitive growth and defects in organelle inheritance but produce stable Mdm1p structures were isolated. Microscopic analysis of the new mdm1 mutants revealed three phenotypic classes: Class I mutants showed defects in both mitochondrial and nuclear transmission; Class II alleles displayed defective mitochondrial inheritance but had no effect on nuclear movement; and Class III mutants showed aberrant nuclear inheritance but normal mitochondrial distribution. Class I and II mutants also exhibited altered mitochondrial morphology, possessing primarily small, round mitochondria instead of the extended tubular structures found in wild-type cells. Mutant mdm1 alleles affecting nuclear transmission were of two types: Class Ia and IIIa mutants were deficient for nuclear movement into daughter buds, while Class Ib and IIIb mutants displayed a complete transfer of all nuclear DNA into buds. The mutations defining all three allelic classes mapped to two distinct domains within the Mdm1p protein. Genetic crosses of yeast strains containing different mdm1 alleles revealed complex genetic interactions including intragenic suppression, synthetic phenotypes, and intragenic complementation. These results support a model of Mdm1p function in which a network comprised of multimeric assemblies of the protein mediates two distinct cellular processes.

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New mdm1 alleles define three phenotypic classes. MYY290 (MDM1) (A), MYY720 (mdm1-251) (B), MYY721 (mdm1-252)  (C), and MYY702 (mdm1-200) (D) cells were grown on YPD medium at 23°C, incubated at 37°C for 4 h, fixed with formaldehyde, and  processed for indirect immunofluorescence microscopy. Mitochondria were detected with a mouse monoclonal antibody against OM14  (a mitochondrial outer-membrane protein) followed by fluorescein-conjugated goat anti–mouse IgG. Nuclear and mitochondrial DNAs  were visualized by DAPI staining. Mdm1p structures were detected using affinity-purified, anti-Mdm1p antibodies followed by  rhodamine-conjugated donkey anti–rabbit IgG. For each strain, two representative cells are shown. Bars, 2 μm.
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Figure 1: New mdm1 alleles define three phenotypic classes. MYY290 (MDM1) (A), MYY720 (mdm1-251) (B), MYY721 (mdm1-252) (C), and MYY702 (mdm1-200) (D) cells were grown on YPD medium at 23°C, incubated at 37°C for 4 h, fixed with formaldehyde, and processed for indirect immunofluorescence microscopy. Mitochondria were detected with a mouse monoclonal antibody against OM14 (a mitochondrial outer-membrane protein) followed by fluorescein-conjugated goat anti–mouse IgG. Nuclear and mitochondrial DNAs were visualized by DAPI staining. Mdm1p structures were detected using affinity-purified, anti-Mdm1p antibodies followed by rhodamine-conjugated donkey anti–rabbit IgG. For each strain, two representative cells are shown. Bars, 2 μm.

Mentions: To characterize the effects of the new mutations on organelle inheritance, cells were analyzed by fluorescence and indirect immunofluorescence microscopy (Fig. 1). An initial examination of cells treated with the mitochondria-specific, vital dye, 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI), revealed that only a subset of the new alleles caused defects in mitochondrial distribution. Further analysis of both nuclei and mitochondria by indirect immunofluorescence microscopy permitted the assignment of mutant alleles to three phenotypic classes (Table II). Whereas wild-type cells incubated at 37°C displayed normal organelle distribution and stable Mdm1p structures (Fig. 1 A), Class I mutants displayed defects in both mitochondrial and nuclear transmission to buds (Fig. 1 B), similar to the original mdm1-1 allele. Class II alleles appeared to affect only mitochondrial inheritance, with normal nuclear behavior (Fig. 1 C). Finally, Class III mutants displayed aberrant nuclear transmission but normal mitochondrial distribution (Fig. 1 D). Both dominant and recessive Class I and Class III alleles were identified, but both Class II alleles obtained were dominant. All of the mdm1 mutant cells possessed stable Mdm1p-punctate structures (Fig. 1, B–D), in contrast to the Mdm1p structures observed in the original mdm1-1 strain, which disappeared after incubation at 37°C (McConnell and Yaffe, 1992). Additionally, immunoblot analysis of cellular extracts revealed no difference in Mdm1p levels between wild-type and mutant cells grown at the permissive or nonpermissive temperature (data not shown), indicating that the new mutant phenotypes were not caused by unstable forms of Mdm1p. In some cells there appeared to be a concentration of Mdm1p staining at or around the nucleus; this staining was observed in both wild-type and mutant cells and did not appear to correspond to a particular phase of the cell cycle. Like the original mdm1-1 mutant, none of the new mutant alleles appeared to alter actin distribution (to cortical patches and cables) at either permissive or nonpermissive temperature (data not shown).


Mutational analysis of Mdm1p function in nuclear and mitochondrial inheritance.

Fisk HA, Yaffe MP - J. Cell Biol. (1997)

New mdm1 alleles define three phenotypic classes. MYY290 (MDM1) (A), MYY720 (mdm1-251) (B), MYY721 (mdm1-252)  (C), and MYY702 (mdm1-200) (D) cells were grown on YPD medium at 23°C, incubated at 37°C for 4 h, fixed with formaldehyde, and  processed for indirect immunofluorescence microscopy. Mitochondria were detected with a mouse monoclonal antibody against OM14  (a mitochondrial outer-membrane protein) followed by fluorescein-conjugated goat anti–mouse IgG. Nuclear and mitochondrial DNAs  were visualized by DAPI staining. Mdm1p structures were detected using affinity-purified, anti-Mdm1p antibodies followed by  rhodamine-conjugated donkey anti–rabbit IgG. For each strain, two representative cells are shown. Bars, 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2141631&req=5

Figure 1: New mdm1 alleles define three phenotypic classes. MYY290 (MDM1) (A), MYY720 (mdm1-251) (B), MYY721 (mdm1-252) (C), and MYY702 (mdm1-200) (D) cells were grown on YPD medium at 23°C, incubated at 37°C for 4 h, fixed with formaldehyde, and processed for indirect immunofluorescence microscopy. Mitochondria were detected with a mouse monoclonal antibody against OM14 (a mitochondrial outer-membrane protein) followed by fluorescein-conjugated goat anti–mouse IgG. Nuclear and mitochondrial DNAs were visualized by DAPI staining. Mdm1p structures were detected using affinity-purified, anti-Mdm1p antibodies followed by rhodamine-conjugated donkey anti–rabbit IgG. For each strain, two representative cells are shown. Bars, 2 μm.
Mentions: To characterize the effects of the new mutations on organelle inheritance, cells were analyzed by fluorescence and indirect immunofluorescence microscopy (Fig. 1). An initial examination of cells treated with the mitochondria-specific, vital dye, 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI), revealed that only a subset of the new alleles caused defects in mitochondrial distribution. Further analysis of both nuclei and mitochondria by indirect immunofluorescence microscopy permitted the assignment of mutant alleles to three phenotypic classes (Table II). Whereas wild-type cells incubated at 37°C displayed normal organelle distribution and stable Mdm1p structures (Fig. 1 A), Class I mutants displayed defects in both mitochondrial and nuclear transmission to buds (Fig. 1 B), similar to the original mdm1-1 allele. Class II alleles appeared to affect only mitochondrial inheritance, with normal nuclear behavior (Fig. 1 C). Finally, Class III mutants displayed aberrant nuclear transmission but normal mitochondrial distribution (Fig. 1 D). Both dominant and recessive Class I and Class III alleles were identified, but both Class II alleles obtained were dominant. All of the mdm1 mutant cells possessed stable Mdm1p-punctate structures (Fig. 1, B–D), in contrast to the Mdm1p structures observed in the original mdm1-1 strain, which disappeared after incubation at 37°C (McConnell and Yaffe, 1992). Additionally, immunoblot analysis of cellular extracts revealed no difference in Mdm1p levels between wild-type and mutant cells grown at the permissive or nonpermissive temperature (data not shown), indicating that the new mutant phenotypes were not caused by unstable forms of Mdm1p. In some cells there appeared to be a concentration of Mdm1p staining at or around the nucleus; this staining was observed in both wild-type and mutant cells and did not appear to correspond to a particular phase of the cell cycle. Like the original mdm1-1 mutant, none of the new mutant alleles appeared to alter actin distribution (to cortical patches and cables) at either permissive or nonpermissive temperature (data not shown).

Bottom Line: Class I and II mutants also exhibited altered mitochondrial morphology, possessing primarily small, round mitochondria instead of the extended tubular structures found in wild-type cells.The mutations defining all three allelic classes mapped to two distinct domains within the Mdm1p protein.These results support a model of Mdm1p function in which a network comprised of multimeric assemblies of the protein mediates two distinct cellular processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
Nuclear and mitochondrial transmission to daughter buds of Saccharomyces cerevisiae depends on Mdm1p, an intermediate filament-like protein localized to numerous punctate structures distributed throughout the yeast cell cytoplasm. These structures disappear and organelle inheritance is disrupted when mdm1 mutant cells are incubated at the restrictive temperature. To characterize further the function of Mdm1p, new mutant mdm1 alleles that confer temperature-sensitive growth and defects in organelle inheritance but produce stable Mdm1p structures were isolated. Microscopic analysis of the new mdm1 mutants revealed three phenotypic classes: Class I mutants showed defects in both mitochondrial and nuclear transmission; Class II alleles displayed defective mitochondrial inheritance but had no effect on nuclear movement; and Class III mutants showed aberrant nuclear inheritance but normal mitochondrial distribution. Class I and II mutants also exhibited altered mitochondrial morphology, possessing primarily small, round mitochondria instead of the extended tubular structures found in wild-type cells. Mutant mdm1 alleles affecting nuclear transmission were of two types: Class Ia and IIIa mutants were deficient for nuclear movement into daughter buds, while Class Ib and IIIb mutants displayed a complete transfer of all nuclear DNA into buds. The mutations defining all three allelic classes mapped to two distinct domains within the Mdm1p protein. Genetic crosses of yeast strains containing different mdm1 alleles revealed complex genetic interactions including intragenic suppression, synthetic phenotypes, and intragenic complementation. These results support a model of Mdm1p function in which a network comprised of multimeric assemblies of the protein mediates two distinct cellular processes.

Show MeSH
Related in: MedlinePlus