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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.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.

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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Class Ib-mdm1 and Class IIIb-mdm1 cells display complete transfer of nuclear DNA to daughter buds. MYY709 (mdm1-204)  (A) and MYY715 (mdm1-227) (B and C) cells were grown at 23°C, incubated for 4 h at 37°C, and fixed and processed as described for  Fig. 1. (A and B) Cells were stained with anti-OM14 followed by fluorescein-conjugated goat anti–mouse IgG, DAPI, and anti-MDM1p  followed by rhodamine-conjugated donkey anti–rabbit IgG. (C) Cells were stained with anti-OM14 followed by fluorescein-conjugated  goat anti–mouse IgG, DAPI and calcofluor (Calc.; to visualize bud scars), and anti-tubulin followed by rhodamine-conjugated donkey  anti–rat IgG. Two representative cells are shown for each strain. Bars, 2 μm.
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Figure 3: Class Ib-mdm1 and Class IIIb-mdm1 cells display complete transfer of nuclear DNA to daughter buds. MYY709 (mdm1-204) (A) and MYY715 (mdm1-227) (B and C) cells were grown at 23°C, incubated for 4 h at 37°C, and fixed and processed as described for Fig. 1. (A and B) Cells were stained with anti-OM14 followed by fluorescein-conjugated goat anti–mouse IgG, DAPI, and anti-MDM1p followed by rhodamine-conjugated donkey anti–rabbit IgG. (C) Cells were stained with anti-OM14 followed by fluorescein-conjugated goat anti–mouse IgG, DAPI and calcofluor (Calc.; to visualize bud scars), and anti-tubulin followed by rhodamine-conjugated donkey anti–rat IgG. Two representative cells are shown for each strain. Bars, 2 μm.

Mentions: A distinct, novel nuclear phenotype was displayed by several other alleles (Class Ib and Class IIIb). In these strains, a significant fraction of the cells incubated at the nonpermissive temperature exhibited all of the nuclear DNA localized to the bud (Fig. 3, A and B). Double-label experiments in which cells were stained with both DAPI (to reveal DNA) and Calcofluor (to label bud scars on the mother portion of the cells) confirmed that daughter buds were, in fact, receiving all of the nuclear DNA in these cells (Fig. 3 C). Visualization of microtubules in Class Ib and Class IIIb cells by indirect immunofluorescence microscopy revealed that many of the nuclei in the buds possessed unelongated mitotic spindles (Fig. 3 C), indicating aberrant positioning of the premitotic spindle apparatus.


Mutational analysis of Mdm1p function in nuclear and mitochondrial inheritance.

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

Class Ib-mdm1 and Class IIIb-mdm1 cells display complete transfer of nuclear DNA to daughter buds. MYY709 (mdm1-204)  (A) and MYY715 (mdm1-227) (B and C) cells were grown at 23°C, incubated for 4 h at 37°C, and fixed and processed as described for  Fig. 1. (A and B) Cells were stained with anti-OM14 followed by fluorescein-conjugated goat anti–mouse IgG, DAPI, and anti-MDM1p  followed by rhodamine-conjugated donkey anti–rabbit IgG. (C) Cells were stained with anti-OM14 followed by fluorescein-conjugated  goat anti–mouse IgG, DAPI and calcofluor (Calc.; to visualize bud scars), and anti-tubulin followed by rhodamine-conjugated donkey  anti–rat IgG. Two representative cells are shown for each strain. Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141631&req=5

Figure 3: Class Ib-mdm1 and Class IIIb-mdm1 cells display complete transfer of nuclear DNA to daughter buds. MYY709 (mdm1-204) (A) and MYY715 (mdm1-227) (B and C) cells were grown at 23°C, incubated for 4 h at 37°C, and fixed and processed as described for Fig. 1. (A and B) Cells were stained with anti-OM14 followed by fluorescein-conjugated goat anti–mouse IgG, DAPI, and anti-MDM1p followed by rhodamine-conjugated donkey anti–rabbit IgG. (C) Cells were stained with anti-OM14 followed by fluorescein-conjugated goat anti–mouse IgG, DAPI and calcofluor (Calc.; to visualize bud scars), and anti-tubulin followed by rhodamine-conjugated donkey anti–rat IgG. Two representative cells are shown for each strain. Bars, 2 μm.
Mentions: A distinct, novel nuclear phenotype was displayed by several other alleles (Class Ib and Class IIIb). In these strains, a significant fraction of the cells incubated at the nonpermissive temperature exhibited all of the nuclear DNA localized to the bud (Fig. 3, A and B). Double-label experiments in which cells were stained with both DAPI (to reveal DNA) and Calcofluor (to label bud scars on the mother portion of the cells) confirmed that daughter buds were, in fact, receiving all of the nuclear DNA in these cells (Fig. 3 C). Visualization of microtubules in Class Ib and Class IIIb cells by indirect immunofluorescence microscopy revealed that many of the nuclei in the buds possessed unelongated mitotic spindles (Fig. 3 C), indicating aberrant positioning of the premitotic spindle apparatus.

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.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.

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