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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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Gag3p is a peripheral protein of the mitochondrial outer membrane. A, Cells expressing HA-tagged Gag3p (MYY2016) or myc-tagged Dnm1p (MYY1202) were grown on semisynthetic lactate medium (Daum et al. 1982). Cells were homogenized and subcellular fractions were isolated by differential centrifugation. The homogenate (hom), low-speed pellet (LSP), mitochondrial (mito), intermediate-speed pellet (ISP), high-speed pellet (HSP), and the cytosolic fractions (cyto) were analyzed by SDS-PAGE and immunoblotting to detect Gag3p, Dnm1p, the mitochondrial outer membrane protein OM45, and the cytosolic protein glucose-6-phosphate dehydrogenase (G6PDH). Each lane contained 10 μg total protein. B, Trypsin treatment of mitochondria. Isolated mitochondria containing HA-tagged Gag3p were treated with trypsin in the presence (+) or absence (−) of deoxycholate detergent and analyzed by SDS-PAGE and immunoblotting. Samples were tested for the presence of Gag3p, the outer membrane protein Tom70p, the intermembrane space protein cytochrome b2, and the mitochondrial matrix protein Mas2p. C, Gag3p is peripherally associated with mitochondrial membranes. Isolated mitochondria containing HA-tagged Gag3p were treated with 1 M NaCl, 0.1 M Na2CO3, or 8 M urea. Mitochondria were centrifuged, and the resulting pellet (p) and supernatant (s) fractions were analyzed by SDS-PAGE and immunoblotting for Gag3p and OM45.
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Figure 4: Gag3p is a peripheral protein of the mitochondrial outer membrane. A, Cells expressing HA-tagged Gag3p (MYY2016) or myc-tagged Dnm1p (MYY1202) were grown on semisynthetic lactate medium (Daum et al. 1982). Cells were homogenized and subcellular fractions were isolated by differential centrifugation. The homogenate (hom), low-speed pellet (LSP), mitochondrial (mito), intermediate-speed pellet (ISP), high-speed pellet (HSP), and the cytosolic fractions (cyto) were analyzed by SDS-PAGE and immunoblotting to detect Gag3p, Dnm1p, the mitochondrial outer membrane protein OM45, and the cytosolic protein glucose-6-phosphate dehydrogenase (G6PDH). Each lane contained 10 μg total protein. B, Trypsin treatment of mitochondria. Isolated mitochondria containing HA-tagged Gag3p were treated with trypsin in the presence (+) or absence (−) of deoxycholate detergent and analyzed by SDS-PAGE and immunoblotting. Samples were tested for the presence of Gag3p, the outer membrane protein Tom70p, the intermembrane space protein cytochrome b2, and the mitochondrial matrix protein Mas2p. C, Gag3p is peripherally associated with mitochondrial membranes. Isolated mitochondria containing HA-tagged Gag3p were treated with 1 M NaCl, 0.1 M Na2CO3, or 8 M urea. Mitochondria were centrifuged, and the resulting pellet (p) and supernatant (s) fractions were analyzed by SDS-PAGE and immunoblotting for Gag3p and OM45.

Mentions: To determine the site of Gag3p activity, a yeast strain was created in which the chromosomal copy of GAG3 was replaced by a version of the gene encoding a form of Gag3p with an HA-epitope tag at the COOH terminus. This tagged protein was fully functional, as cells expressing only this variant gene displayed mitochondrial morphology indistinguishable from that of wild-type cells (data not shown). Cells expressing the tagged protein were homogenized and subcellular fractions were isolated by differential centrifugation. Immunoblot analysis revealed that Gag3p was enriched in the mitochondrial fraction (Fig. 4 A). This pattern was similar to that observed for the mitochondrial outer membrane protein, OM45, but contrasted with that found for glucose-6-phosphate dehydrogenase, a cytosolic protein. An identical pattern of Gag3p fractionation with mitochondria was observed in Δdnm1, gag2, and double mutant Δdnm1 gag2 cells, indicating that the mitochondrial association of Gag3p is independent of functional Dnm1p or Gag2p (data not shown). In contrast to Gag3p, Dnm1p was essentially absent from the mitochondrial fraction and largely recovered in the cytosolic fraction (Fig. 4 A). This distribution agrees with that previously reported for Dnm1p in subcellular fractions (Otsuga et al. 1998), although indirect immunofluorescence microscopy revealed a mitochondrial localization for Dnm1p (Otsuga et al. 1998; Sesaki and Jensen 1999). This discrepancy between microscopic localization and recovery in isolated subcellular fractions may reflect a weak association or transient interaction of Dnm1p with mitochondria. Although we have been unable to detect Gag3p by indirect immunofluorescence microscopy (data not shown), its association with mitochondria is strong enough to persist through purification of the organelle.


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

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

Gag3p is a peripheral protein of the mitochondrial outer membrane. A, Cells expressing HA-tagged Gag3p (MYY2016) or myc-tagged Dnm1p (MYY1202) were grown on semisynthetic lactate medium (Daum et al. 1982). Cells were homogenized and subcellular fractions were isolated by differential centrifugation. The homogenate (hom), low-speed pellet (LSP), mitochondrial (mito), intermediate-speed pellet (ISP), high-speed pellet (HSP), and the cytosolic fractions (cyto) were analyzed by SDS-PAGE and immunoblotting to detect Gag3p, Dnm1p, the mitochondrial outer membrane protein OM45, and the cytosolic protein glucose-6-phosphate dehydrogenase (G6PDH). Each lane contained 10 μg total protein. B, Trypsin treatment of mitochondria. Isolated mitochondria containing HA-tagged Gag3p were treated with trypsin in the presence (+) or absence (−) of deoxycholate detergent and analyzed by SDS-PAGE and immunoblotting. Samples were tested for the presence of Gag3p, the outer membrane protein Tom70p, the intermembrane space protein cytochrome b2, and the mitochondrial matrix protein Mas2p. C, Gag3p is peripherally associated with mitochondrial membranes. Isolated mitochondria containing HA-tagged Gag3p were treated with 1 M NaCl, 0.1 M Na2CO3, or 8 M urea. Mitochondria were centrifuged, and the resulting pellet (p) and supernatant (s) fractions were analyzed by SDS-PAGE and immunoblotting for Gag3p and OM45.
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Figure 4: Gag3p is a peripheral protein of the mitochondrial outer membrane. A, Cells expressing HA-tagged Gag3p (MYY2016) or myc-tagged Dnm1p (MYY1202) were grown on semisynthetic lactate medium (Daum et al. 1982). Cells were homogenized and subcellular fractions were isolated by differential centrifugation. The homogenate (hom), low-speed pellet (LSP), mitochondrial (mito), intermediate-speed pellet (ISP), high-speed pellet (HSP), and the cytosolic fractions (cyto) were analyzed by SDS-PAGE and immunoblotting to detect Gag3p, Dnm1p, the mitochondrial outer membrane protein OM45, and the cytosolic protein glucose-6-phosphate dehydrogenase (G6PDH). Each lane contained 10 μg total protein. B, Trypsin treatment of mitochondria. Isolated mitochondria containing HA-tagged Gag3p were treated with trypsin in the presence (+) or absence (−) of deoxycholate detergent and analyzed by SDS-PAGE and immunoblotting. Samples were tested for the presence of Gag3p, the outer membrane protein Tom70p, the intermembrane space protein cytochrome b2, and the mitochondrial matrix protein Mas2p. C, Gag3p is peripherally associated with mitochondrial membranes. Isolated mitochondria containing HA-tagged Gag3p were treated with 1 M NaCl, 0.1 M Na2CO3, or 8 M urea. Mitochondria were centrifuged, and the resulting pellet (p) and supernatant (s) fractions were analyzed by SDS-PAGE and immunoblotting for Gag3p and OM45.
Mentions: To determine the site of Gag3p activity, a yeast strain was created in which the chromosomal copy of GAG3 was replaced by a version of the gene encoding a form of Gag3p with an HA-epitope tag at the COOH terminus. This tagged protein was fully functional, as cells expressing only this variant gene displayed mitochondrial morphology indistinguishable from that of wild-type cells (data not shown). Cells expressing the tagged protein were homogenized and subcellular fractions were isolated by differential centrifugation. Immunoblot analysis revealed that Gag3p was enriched in the mitochondrial fraction (Fig. 4 A). This pattern was similar to that observed for the mitochondrial outer membrane protein, OM45, but contrasted with that found for glucose-6-phosphate dehydrogenase, a cytosolic protein. An identical pattern of Gag3p fractionation with mitochondria was observed in Δdnm1, gag2, and double mutant Δdnm1 gag2 cells, indicating that the mitochondrial association of Gag3p is independent of functional Dnm1p or Gag2p (data not shown). In contrast to Gag3p, Dnm1p was essentially absent from the mitochondrial fraction and largely recovered in the cytosolic fraction (Fig. 4 A). This distribution agrees with that previously reported for Dnm1p in subcellular fractions (Otsuga et al. 1998), although indirect immunofluorescence microscopy revealed a mitochondrial localization for Dnm1p (Otsuga et al. 1998; Sesaki and Jensen 1999). This discrepancy between microscopic localization and recovery in isolated subcellular fractions may reflect a weak association or transient interaction of Dnm1p with mitochondria. Although we have been unable to detect Gag3p by indirect immunofluorescence microscopy (data not shown), its association with mitochondria is strong enough to persist through purification of the organelle.

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