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Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway.

Frederick RL, McCaffery JM, Cunningham KW, Okamoto K, Shaw JM - J. Cell Biol. (2004)

Bottom Line: Biol.Chem. 278:6495-6502), Gem1p is not required for pheromone-induced yeast cell death.Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.

ABSTRACT
Cell signaling events elicit changes in mitochondrial shape and activity. However, few mitochondrial proteins that interact with signaling pathways have been identified. Candidates include the conserved mitochondrial Rho (Miro) family of proteins, which contain two GTPase domains flanking a pair of calcium-binding EF-hand motifs. We show that Gem1p (yeast Miro; encoded by YAL048C) is a tail-anchored outer mitochondrial membrane protein. Cells lacking Gem1p contain collapsed, globular, or grape-like mitochondria. We demonstrate that Gem1p is not an essential component of characterized pathways that regulate mitochondrial dynamics. Genetic studies indicate both GTPase domains and EF-hand motifs, which are exposed to the cytoplasm, are required for Gem1p function. Although overexpression of a mutant human Miro protein caused increased apoptotic activity in cultured cells (Fransson et al., 2003. J. Biol. Chem. 278:6495-6502), Gem1p is not required for pheromone-induced yeast cell death. Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics.

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Mitochondrial morphology is abnormal in cells lacking Gem1p. Mitochondrial morphologies are shown for GEM1 cells (A), gem1Δ cells (B–D), and fusion-deficient fzo1Δ cells (E) expressing mito-GFP and grown in SDextrose medium at 30°C to mid-log phase (OD600 0.5–1.5). GFP fluorescent and DIC images are superimposed for each cell. The percentage of cells in a GEM1 or gem1Δ culture with the morphologies depicted in A–E is shown (bottom). n = 900. Bar, 5 μm.
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fig3: Mitochondrial morphology is abnormal in cells lacking Gem1p. Mitochondrial morphologies are shown for GEM1 cells (A), gem1Δ cells (B–D), and fusion-deficient fzo1Δ cells (E) expressing mito-GFP and grown in SDextrose medium at 30°C to mid-log phase (OD600 0.5–1.5). GFP fluorescent and DIC images are superimposed for each cell. The percentage of cells in a GEM1 or gem1Δ culture with the morphologies depicted in A–E is shown (bottom). n = 900. Bar, 5 μm.

Mentions: In wild-type yeast, mitochondria form a branched tubular network located at the cell cortex (Stevens, 1981; Koning et al., 1993). When visualized with a mitochondrial-targeted GFP (mito-GFP), 86.6% of cells in a wild-type population displayed a tubular mitochondrial network (Fig. 3 A). In contrast, cells lacking Gem1p had pronounced defects in mitochondrial distribution and morphology. As shown in Fig. 3 C, 53.7% of gem1Δ cells contained mitochondria that were globular with an irregular perimeter. Aberrant mitochondria of this type were never observed in wild-type strains (n > 1000). These large mitochondria did not appear to be collapsed tubules, as labeling with an outer membrane targeted form of GFP resulted in rim staining that enclosed matrix-targeted RFP (unpublished data). Other mitochondrial morphologies, including grape-like clusters (24%; Fig. 3 D) and collapsed mitochondria (16%; Fig. 3 B) were also observed in gem1Δ cells. The grape-like mitochondrial clusters were larger and fewer in number than mitochondrial fragments observed in a mitochondrial fusion mutant (fzo1Δ; Fig. 3 E). Collapsed mitochondria (Fig. 3 B) were frequently thicker than wild-type mitochondrial tubules and had variations in diameter along the length of the tubule. Because these collapsed mitochondria retain their tubular structure, they were not included in the “mutant” category during phenotypic quantification. Globular (Fig. 3 C), grape-like (Fig. 3 D), and fragmented (Fig. 3 E) mitochondria were scored in a single category, designated “Percent mutant” (Table I), unless otherwise indicated. A plasmid containing GEM1 expressed from its own promoter restored tubular networks in 69.3% of the gem1Δ population (Table I). In the FY strain background, deletion of gem1 had a less severe affect on mitochondrial morphology (unpublished data). Overexpression of full-length Gem1p or Gem1p(1-632) lacking the putative transmembrane domain from a galactose-inducible promoter did not cause defects in mitochondrial morphology in wild-type cells (unpublished data).


Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway.

Frederick RL, McCaffery JM, Cunningham KW, Okamoto K, Shaw JM - J. Cell Biol. (2004)

Mitochondrial morphology is abnormal in cells lacking Gem1p. Mitochondrial morphologies are shown for GEM1 cells (A), gem1Δ cells (B–D), and fusion-deficient fzo1Δ cells (E) expressing mito-GFP and grown in SDextrose medium at 30°C to mid-log phase (OD600 0.5–1.5). GFP fluorescent and DIC images are superimposed for each cell. The percentage of cells in a GEM1 or gem1Δ culture with the morphologies depicted in A–E is shown (bottom). n = 900. Bar, 5 μm.
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Related In: Results  -  Collection

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fig3: Mitochondrial morphology is abnormal in cells lacking Gem1p. Mitochondrial morphologies are shown for GEM1 cells (A), gem1Δ cells (B–D), and fusion-deficient fzo1Δ cells (E) expressing mito-GFP and grown in SDextrose medium at 30°C to mid-log phase (OD600 0.5–1.5). GFP fluorescent and DIC images are superimposed for each cell. The percentage of cells in a GEM1 or gem1Δ culture with the morphologies depicted in A–E is shown (bottom). n = 900. Bar, 5 μm.
Mentions: In wild-type yeast, mitochondria form a branched tubular network located at the cell cortex (Stevens, 1981; Koning et al., 1993). When visualized with a mitochondrial-targeted GFP (mito-GFP), 86.6% of cells in a wild-type population displayed a tubular mitochondrial network (Fig. 3 A). In contrast, cells lacking Gem1p had pronounced defects in mitochondrial distribution and morphology. As shown in Fig. 3 C, 53.7% of gem1Δ cells contained mitochondria that were globular with an irregular perimeter. Aberrant mitochondria of this type were never observed in wild-type strains (n > 1000). These large mitochondria did not appear to be collapsed tubules, as labeling with an outer membrane targeted form of GFP resulted in rim staining that enclosed matrix-targeted RFP (unpublished data). Other mitochondrial morphologies, including grape-like clusters (24%; Fig. 3 D) and collapsed mitochondria (16%; Fig. 3 B) were also observed in gem1Δ cells. The grape-like mitochondrial clusters were larger and fewer in number than mitochondrial fragments observed in a mitochondrial fusion mutant (fzo1Δ; Fig. 3 E). Collapsed mitochondria (Fig. 3 B) were frequently thicker than wild-type mitochondrial tubules and had variations in diameter along the length of the tubule. Because these collapsed mitochondria retain their tubular structure, they were not included in the “mutant” category during phenotypic quantification. Globular (Fig. 3 C), grape-like (Fig. 3 D), and fragmented (Fig. 3 E) mitochondria were scored in a single category, designated “Percent mutant” (Table I), unless otherwise indicated. A plasmid containing GEM1 expressed from its own promoter restored tubular networks in 69.3% of the gem1Δ population (Table I). In the FY strain background, deletion of gem1 had a less severe affect on mitochondrial morphology (unpublished data). Overexpression of full-length Gem1p or Gem1p(1-632) lacking the putative transmembrane domain from a galactose-inducible promoter did not cause defects in mitochondrial morphology in wild-type cells (unpublished data).

Bottom Line: Biol.Chem. 278:6495-6502), Gem1p is not required for pheromone-induced yeast cell death.Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.

ABSTRACT
Cell signaling events elicit changes in mitochondrial shape and activity. However, few mitochondrial proteins that interact with signaling pathways have been identified. Candidates include the conserved mitochondrial Rho (Miro) family of proteins, which contain two GTPase domains flanking a pair of calcium-binding EF-hand motifs. We show that Gem1p (yeast Miro; encoded by YAL048C) is a tail-anchored outer mitochondrial membrane protein. Cells lacking Gem1p contain collapsed, globular, or grape-like mitochondria. We demonstrate that Gem1p is not an essential component of characterized pathways that regulate mitochondrial dynamics. Genetic studies indicate both GTPase domains and EF-hand motifs, which are exposed to the cytoplasm, are required for Gem1p function. Although overexpression of a mutant human Miro protein caused increased apoptotic activity in cultured cells (Fransson et al., 2003. J. Biol. Chem. 278:6495-6502), Gem1p is not required for pheromone-induced yeast cell death. Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics.

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