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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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Gem1p is a single-pass outer mitochondrial membrane protein with its NH2 terminus exposed to the cytoplasm. (A) WCE from JSY7000 (GEM1) expressing GFP-Gem1p was fractionated by differential centrifugation to yield PMS and MITO pellet, and analyzed by SDS-PAGE and Western blotting with antibodies against GFP, the multi-pass outer mitochondrial membrane protein Porin, and the cytoplasmic protein 3-PGK. WCE and PMS, 1× cell equivalents; MITO, 10× cell equivalents. (B) Untreated, osmotically shocked (OS), or Triton X-100 solubilized (TX) mitochondria were treated with (+) or without (−) PK and analyzed by SDS-PAGE and Western blotting with antibodies specific for GFP, the integral outer membrane protein Fzo1p, the intermembrane space protein Cyb2p, and the matrix protein mtHsp70. (C) Mitochondria (200 μg) were treated with sodium carbonate (pH 11.5) and separated into supernatant (S) and pellet (P) fractions. One-sixth of the reaction was loaded in each lane (L, load, untreated mitochondria). Tim50p is a single-pass inner membrane protein. Tim44p is a peripheral membrane protein in the matrix. Mge1p is a soluble matrix protein.
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fig7: Gem1p is a single-pass outer mitochondrial membrane protein with its NH2 terminus exposed to the cytoplasm. (A) WCE from JSY7000 (GEM1) expressing GFP-Gem1p was fractionated by differential centrifugation to yield PMS and MITO pellet, and analyzed by SDS-PAGE and Western blotting with antibodies against GFP, the multi-pass outer mitochondrial membrane protein Porin, and the cytoplasmic protein 3-PGK. WCE and PMS, 1× cell equivalents; MITO, 10× cell equivalents. (B) Untreated, osmotically shocked (OS), or Triton X-100 solubilized (TX) mitochondria were treated with (+) or without (−) PK and analyzed by SDS-PAGE and Western blotting with antibodies specific for GFP, the integral outer membrane protein Fzo1p, the intermembrane space protein Cyb2p, and the matrix protein mtHsp70. (C) Mitochondria (200 μg) were treated with sodium carbonate (pH 11.5) and separated into supernatant (S) and pellet (P) fractions. One-sixth of the reaction was loaded in each lane (L, load, untreated mitochondria). Tim50p is a single-pass inner membrane protein. Tim44p is a peripheral membrane protein in the matrix. Mge1p is a soluble matrix protein.

Mentions: Subcellular fractionation studies confirmed that Gem1p is a mitochondrial protein (Fig. 7 A). When whole cell extracts (WCEs) from wild-type cells expressing GFP-Gem1p were separated into post-mitochondrial supernatant (PMS) and mitochondrial pellet (MITO) fractions, GFP-Gem1p was found in the MITO, along with the integral outer mitochondrial membrane protein porin. In contrast, GFP-Gem1p was not detected in the PMS, which contained the cytoplasmic protein 3-phosphoglycerate kinase (3-PGK).


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)

Gem1p is a single-pass outer mitochondrial membrane protein with its NH2 terminus exposed to the cytoplasm. (A) WCE from JSY7000 (GEM1) expressing GFP-Gem1p was fractionated by differential centrifugation to yield PMS and MITO pellet, and analyzed by SDS-PAGE and Western blotting with antibodies against GFP, the multi-pass outer mitochondrial membrane protein Porin, and the cytoplasmic protein 3-PGK. WCE and PMS, 1× cell equivalents; MITO, 10× cell equivalents. (B) Untreated, osmotically shocked (OS), or Triton X-100 solubilized (TX) mitochondria were treated with (+) or without (−) PK and analyzed by SDS-PAGE and Western blotting with antibodies specific for GFP, the integral outer membrane protein Fzo1p, the intermembrane space protein Cyb2p, and the matrix protein mtHsp70. (C) Mitochondria (200 μg) were treated with sodium carbonate (pH 11.5) and separated into supernatant (S) and pellet (P) fractions. One-sixth of the reaction was loaded in each lane (L, load, untreated mitochondria). Tim50p is a single-pass inner membrane protein. Tim44p is a peripheral membrane protein in the matrix. Mge1p is a soluble matrix protein.
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Related In: Results  -  Collection

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fig7: Gem1p is a single-pass outer mitochondrial membrane protein with its NH2 terminus exposed to the cytoplasm. (A) WCE from JSY7000 (GEM1) expressing GFP-Gem1p was fractionated by differential centrifugation to yield PMS and MITO pellet, and analyzed by SDS-PAGE and Western blotting with antibodies against GFP, the multi-pass outer mitochondrial membrane protein Porin, and the cytoplasmic protein 3-PGK. WCE and PMS, 1× cell equivalents; MITO, 10× cell equivalents. (B) Untreated, osmotically shocked (OS), or Triton X-100 solubilized (TX) mitochondria were treated with (+) or without (−) PK and analyzed by SDS-PAGE and Western blotting with antibodies specific for GFP, the integral outer membrane protein Fzo1p, the intermembrane space protein Cyb2p, and the matrix protein mtHsp70. (C) Mitochondria (200 μg) were treated with sodium carbonate (pH 11.5) and separated into supernatant (S) and pellet (P) fractions. One-sixth of the reaction was loaded in each lane (L, load, untreated mitochondria). Tim50p is a single-pass inner membrane protein. Tim44p is a peripheral membrane protein in the matrix. Mge1p is a soluble matrix protein.
Mentions: Subcellular fractionation studies confirmed that Gem1p is a mitochondrial protein (Fig. 7 A). When whole cell extracts (WCEs) from wild-type cells expressing GFP-Gem1p were separated into post-mitochondrial supernatant (PMS) and mitochondrial pellet (MITO) fractions, GFP-Gem1p was found in the MITO, along with the integral outer mitochondrial membrane protein porin. In contrast, GFP-Gem1p was not detected in the PMS, which contained the cytoplasmic protein 3-phosphoglycerate kinase (3-PGK).

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.

Show MeSH