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Gag3p, an outer membrane protein required for fission of mitochondrial tubules.

Fekkes P, Shepard KA, Yaffe MP - J. Cell Biol. (2000)

Bottom Line: Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane.This association requires neither the DNM1 nor GAG2 gene products.These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules.

View Article: PubMed Central - PubMed

Affiliation: University of California, San Diego, Division of Biology, Section of Cell and Developmental Biology, La Jolla, California 92093, USA.

ABSTRACT
Mitochondrial morphology and function depend on MGM1, a Saccharomyces cerevisiae gene encoding a dynamin-like protein of the mitochondrial outer membrane. Here, we show that mitochondrial fragmentation and mitochondrial genome loss caused by lesions in MGM1 are suppressed by three novel mutations, gag1, gag2, and gag3 (for glycerol-adapted growth). Cells with any of the gag mutations displayed aberrant mitochondrial morphology characterized by elongated, unbranched tubes and highly fenestrated structures. Additionally, each of the gag mutations prevented mitochondrial fragmentation caused by loss of the mitochondrial fusion factor, Fzo1p, or by treatment of cells with sodium azide. The gag1 mutation mapped to DNM1 that encodes a dynamin-related protein required for mitochondrial fission. GAG3 encodes a novel WD40-repeat protein previously found to interact with Dnm1p in a two-hybrid assay. Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane. This association requires neither the DNM1 nor GAG2 gene products. However, the localization of Dnm1p to the mitochondrial outer membrane is substantially reduced by the gag2 mutation, but unaffected by loss of Gag3p. These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules.

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Localization of Dnm1p in gag mutant cells. Wild-type (MYY290), gag2 (MYY981), and gag3 (MYY2017) cells expressing GFP-tagged Dnm1p were cultured on minimal glucose medium, stained with MitoTracker Red CMXRos, and analyzed by fluorescence microscopy. Pseudocolor was added to the digitized image. GFP-labeling is shown in green, MitoTracker is shown in red, and the merged images are shown on the right. Bar, 2 μm.
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Figure 5: Localization of Dnm1p in gag mutant cells. Wild-type (MYY290), gag2 (MYY981), and gag3 (MYY2017) cells expressing GFP-tagged Dnm1p were cultured on minimal glucose medium, stained with MitoTracker Red CMXRos, and analyzed by fluorescence microscopy. Pseudocolor was added to the digitized image. GFP-labeling is shown in green, MitoTracker is shown in red, and the merged images are shown on the right. Bar, 2 μm.

Mentions: To examine the role of the GAG2 and GAG3 gene products in localization of Dnm1p to the mitochondrial surface, the distribution of GFP-tagged Dnm1p was determined in gag2 and gag3 mutant cells by fluorescence microscopy (Fig. 5). Mitochondria in these same cells were labeled with MitoTracker red dye. As described previously (Otsuga et al. 1998; Sesaki and Jensen 1999), in wild-type cells, Dnm1p was localized to punctate structures largely associated with mitochondrial tubules (Fig. 5). This pattern was also observed in gag3 cells, despite their abnormal mitochondrial morphology (Fig. 5). In gag2 cells, however, a substantial fraction of Dnm1p appeared to be displaced from the mitochondrial network and randomly distributed through the cytoplasm. Interestingly, Dnm1p appeared to be associated in punctate structures, even when not localized to the mitochondria (Fig. 5). Quantitative analysis of this distribution indicated that 94% (± 4%, n = 319) of fluorescent Dnm1p spots were associated with mitochondria in wild-type cells, and 89% (± 5%, n = 384) in gag3- cells. In contrast, only 46% (± 14%, n = 642) of Dnm1p structures were localized to mitochondria in gag2 cells. These results indicate that the GAG2 gene product plays a role in promoting the interaction of Dnm1p with the mitochondrial surface, but that Gag3p does not mediate this interaction.


Gag3p, an outer membrane protein required for fission of mitochondrial tubules.

Fekkes P, Shepard KA, Yaffe MP - J. Cell Biol. (2000)

Localization of Dnm1p in gag mutant cells. Wild-type (MYY290), gag2 (MYY981), and gag3 (MYY2017) cells expressing GFP-tagged Dnm1p were cultured on minimal glucose medium, stained with MitoTracker Red CMXRos, and analyzed by fluorescence microscopy. Pseudocolor was added to the digitized image. GFP-labeling is shown in green, MitoTracker is shown in red, and the merged images are shown on the right. Bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192644&req=5

Figure 5: Localization of Dnm1p in gag mutant cells. Wild-type (MYY290), gag2 (MYY981), and gag3 (MYY2017) cells expressing GFP-tagged Dnm1p were cultured on minimal glucose medium, stained with MitoTracker Red CMXRos, and analyzed by fluorescence microscopy. Pseudocolor was added to the digitized image. GFP-labeling is shown in green, MitoTracker is shown in red, and the merged images are shown on the right. Bar, 2 μm.
Mentions: To examine the role of the GAG2 and GAG3 gene products in localization of Dnm1p to the mitochondrial surface, the distribution of GFP-tagged Dnm1p was determined in gag2 and gag3 mutant cells by fluorescence microscopy (Fig. 5). Mitochondria in these same cells were labeled with MitoTracker red dye. As described previously (Otsuga et al. 1998; Sesaki and Jensen 1999), in wild-type cells, Dnm1p was localized to punctate structures largely associated with mitochondrial tubules (Fig. 5). This pattern was also observed in gag3 cells, despite their abnormal mitochondrial morphology (Fig. 5). In gag2 cells, however, a substantial fraction of Dnm1p appeared to be displaced from the mitochondrial network and randomly distributed through the cytoplasm. Interestingly, Dnm1p appeared to be associated in punctate structures, even when not localized to the mitochondria (Fig. 5). Quantitative analysis of this distribution indicated that 94% (± 4%, n = 319) of fluorescent Dnm1p spots were associated with mitochondria in wild-type cells, and 89% (± 5%, n = 384) in gag3- cells. In contrast, only 46% (± 14%, n = 642) of Dnm1p structures were localized to mitochondria in gag2 cells. These results indicate that the GAG2 gene product plays a role in promoting the interaction of Dnm1p with the mitochondrial surface, but that Gag3p does not mediate this interaction.

Bottom Line: Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane.This association requires neither the DNM1 nor GAG2 gene products.These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules.

View Article: PubMed Central - PubMed

Affiliation: University of California, San Diego, Division of Biology, Section of Cell and Developmental Biology, La Jolla, California 92093, USA.

ABSTRACT
Mitochondrial morphology and function depend on MGM1, a Saccharomyces cerevisiae gene encoding a dynamin-like protein of the mitochondrial outer membrane. Here, we show that mitochondrial fragmentation and mitochondrial genome loss caused by lesions in MGM1 are suppressed by three novel mutations, gag1, gag2, and gag3 (for glycerol-adapted growth). Cells with any of the gag mutations displayed aberrant mitochondrial morphology characterized by elongated, unbranched tubes and highly fenestrated structures. Additionally, each of the gag mutations prevented mitochondrial fragmentation caused by loss of the mitochondrial fusion factor, Fzo1p, or by treatment of cells with sodium azide. The gag1 mutation mapped to DNM1 that encodes a dynamin-related protein required for mitochondrial fission. GAG3 encodes a novel WD40-repeat protein previously found to interact with Dnm1p in a two-hybrid assay. Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane. This association requires neither the DNM1 nor GAG2 gene products. However, the localization of Dnm1p to the mitochondrial outer membrane is substantially reduced by the gag2 mutation, but unaffected by loss of Gag3p. These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules.

Show MeSH
Related in: MedlinePlus