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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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GAG genes participate in mitochondrial fission. A, Cells harboring GFP-labeled mitochondria and mutant for FZO1 alone (strain MYY2005) or together with either dnm1/gag1 (strain MYY2007; left), gag2 (strain MYY2009; center), or gag3 (strain MYY2011; right) mutations were visualized by fluorescence microscopy. B, Cells with the fzo1 mutation (MYY2005) or the double mutants dnm1 fzo1 (MYY2007), gag2 fzo1 (MYY2009), or gag3 fzo1 (MYY2011) were plated on glycerol-medium and cultured for 3 d at 30°C. C, Wild-type (MYY1200), dnm1/gag1 (MYY2013), gag2 (MYY2029), and gag3 (MYY2031) cells with GFP-labeled mitochondria were treated with 0.5 mM sodium azide and examined immediately (top) or after 30 min (bottom) by fluorescence microscopy. Bar, 2 μm.
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Figure 3: GAG genes participate in mitochondrial fission. A, Cells harboring GFP-labeled mitochondria and mutant for FZO1 alone (strain MYY2005) or together with either dnm1/gag1 (strain MYY2007; left), gag2 (strain MYY2009; center), or gag3 (strain MYY2011; right) mutations were visualized by fluorescence microscopy. B, Cells with the fzo1 mutation (MYY2005) or the double mutants dnm1 fzo1 (MYY2007), gag2 fzo1 (MYY2009), or gag3 fzo1 (MYY2011) were plated on glycerol-medium and cultured for 3 d at 30°C. C, Wild-type (MYY1200), dnm1/gag1 (MYY2013), gag2 (MYY2029), and gag3 (MYY2031) cells with GFP-labeled mitochondria were treated with 0.5 mM sodium azide and examined immediately (top) or after 30 min (bottom) by fluorescence microscopy. Bar, 2 μm.

Mentions: Recently, two groups have demonstrated that Dnm1p is an essential component of the mitochondrial fission machinery (Bleazard et al. 1999; Sesaki and Jensen 1999). The similarity of the gag2 and gag3 mutant phenotypes to that of dnm1 suggested that they also participate in mitochondrial fission. To test this possibility, the gag mutants were crossed to a yeast strain deleted for the mitochondrial fusion factor gene, FZO1. Cells lacking Fzo1p display fragmented mitochondria that readily lose mitochondrial DNA (Hermann et al. 1998; Rapaport et al. 1998), and the development of these phenotypes depends on Dnm1p (i.e., a dnm1 mutation prevents mitochondrial fragmentation and genome loss in an fzo1 mutant; Bleazard et al. 1999; Sesaki and Jensen 1999). Analysis of gag2 fzo1 and gag3 fzo1 double mutants revealed that these new gag lesions similarly prevented the fragmentation of mitochondrial tubules (Fig. 3 A) and allowed growth on glycerol-containing medium (Fig. 3 B), indicating that mitochondrial genome loss was prevented.


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

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

GAG genes participate in mitochondrial fission. A, Cells harboring GFP-labeled mitochondria and mutant for FZO1 alone (strain MYY2005) or together with either dnm1/gag1 (strain MYY2007; left), gag2 (strain MYY2009; center), or gag3 (strain MYY2011; right) mutations were visualized by fluorescence microscopy. B, Cells with the fzo1 mutation (MYY2005) or the double mutants dnm1 fzo1 (MYY2007), gag2 fzo1 (MYY2009), or gag3 fzo1 (MYY2011) were plated on glycerol-medium and cultured for 3 d at 30°C. C, Wild-type (MYY1200), dnm1/gag1 (MYY2013), gag2 (MYY2029), and gag3 (MYY2031) cells with GFP-labeled mitochondria were treated with 0.5 mM sodium azide and examined immediately (top) or after 30 min (bottom) by fluorescence microscopy. Bar, 2 μm.
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Related In: Results  -  Collection

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Figure 3: GAG genes participate in mitochondrial fission. A, Cells harboring GFP-labeled mitochondria and mutant for FZO1 alone (strain MYY2005) or together with either dnm1/gag1 (strain MYY2007; left), gag2 (strain MYY2009; center), or gag3 (strain MYY2011; right) mutations were visualized by fluorescence microscopy. B, Cells with the fzo1 mutation (MYY2005) or the double mutants dnm1 fzo1 (MYY2007), gag2 fzo1 (MYY2009), or gag3 fzo1 (MYY2011) were plated on glycerol-medium and cultured for 3 d at 30°C. C, Wild-type (MYY1200), dnm1/gag1 (MYY2013), gag2 (MYY2029), and gag3 (MYY2031) cells with GFP-labeled mitochondria were treated with 0.5 mM sodium azide and examined immediately (top) or after 30 min (bottom) by fluorescence microscopy. Bar, 2 μm.
Mentions: Recently, two groups have demonstrated that Dnm1p is an essential component of the mitochondrial fission machinery (Bleazard et al. 1999; Sesaki and Jensen 1999). The similarity of the gag2 and gag3 mutant phenotypes to that of dnm1 suggested that they also participate in mitochondrial fission. To test this possibility, the gag mutants were crossed to a yeast strain deleted for the mitochondrial fusion factor gene, FZO1. Cells lacking Fzo1p display fragmented mitochondria that readily lose mitochondrial DNA (Hermann et al. 1998; Rapaport et al. 1998), and the development of these phenotypes depends on Dnm1p (i.e., a dnm1 mutation prevents mitochondrial fragmentation and genome loss in an fzo1 mutant; Bleazard et al. 1999; Sesaki and Jensen 1999). Analysis of gag2 fzo1 and gag3 fzo1 double mutants revealed that these new gag lesions similarly prevented the fragmentation of mitochondrial tubules (Fig. 3 A) and allowed growth on glycerol-containing medium (Fig. 3 B), indicating that mitochondrial genome loss was prevented.

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