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The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria.

Osman C, Haag M, Potting C, Rodenfels J, Dip PV, Wieland FT, Brügger B, Westermann B, Langer T - J. Cell Biol. (2009)

Bottom Line: We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner.Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells.We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, Centre for Molecular Medicine (CMMC), Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50674, Germany.

ABSTRACT
Prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.

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Gep1 affects mitochondrial morphology and Mgm1 processing under respiring conditions. (A, left) Wild-type (WT), Δphb1, and Δgep1 cells expressing mitochondria-targeted GFP were grown to log phase in YP medium containing glycerol (YPG) and analyzed as in Fig. 2 A. Bar, 5 µm. (right) The bar graph indicates the percentage of wild type–like (light gray), fragmented (dark gray), and short tubular, partially fragmented (black) mitochondria. Data represent mean values ± SD of three independent experiments. (B) Decreased PE levels impair Mgm1 processing. Extracts of the indicated cells grown in YPG were analyzed by SDS-PAGE and immunoblotted using Mgm1- and Tom40-specific antisera. A quantification of the immunoblots is shown in the bottom panel. The percentage of S-Mgm1 was calculated from the ratio S-Mgm1/(S-Mgm1 + L-Mgm1). Data represent mean values ± SD of four independent experiments. *, P < 0.05; **, P < 0.01. L, long isoform of Mgm1; S, short isoform of Mgm1.
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fig3: Gep1 affects mitochondrial morphology and Mgm1 processing under respiring conditions. (A, left) Wild-type (WT), Δphb1, and Δgep1 cells expressing mitochondria-targeted GFP were grown to log phase in YP medium containing glycerol (YPG) and analyzed as in Fig. 2 A. Bar, 5 µm. (right) The bar graph indicates the percentage of wild type–like (light gray), fragmented (dark gray), and short tubular, partially fragmented (black) mitochondria. Data represent mean values ± SD of three independent experiments. (B) Decreased PE levels impair Mgm1 processing. Extracts of the indicated cells grown in YPG were analyzed by SDS-PAGE and immunoblotted using Mgm1- and Tom40-specific antisera. A quantification of the immunoblots is shown in the bottom panel. The percentage of S-Mgm1 was calculated from the ratio S-Mgm1/(S-Mgm1 + L-Mgm1). Data represent mean values ± SD of four independent experiments. *, P < 0.05; **, P < 0.01. L, long isoform of Mgm1; S, short isoform of Mgm1.

Mentions: When cells were grown on glycerol-containing medium, ∼80% of Δgep1 and ∼60% of Δphb1 cells contained an at least partially fragmented mitochondrial network, which indicates that both proteins are crucial for mitochondrial morphology under conditions of increased mitochondrial demand (Fig. 3 A). Inner membrane fusion and the formation of cristae depend on the dynamin-like GTPase Mgm1 (Meeusen et al., 2006), which undergoes proteolytic processing by the rhomboid protease Pcp1 in the inner membrane. Cleavage results in the accumulation of two isoforms, L- and S-Mgm1, that functionally cooperate during inner membrane fusion (Sesaki et al., 2003). Interestingly, deletion of GEP1 significantly impaired the formation of S-Mgm1 in cells grown under respiring conditions (Fig. 3 B). These findings identify Gep1 as a novel modulator of Mgm1 processing in the inner membrane and suggest that the unbalanced accumulation of L- and S-Mgm1 causes deficiencies in cristae morphogenesis in Δgep1 mitochondria.


The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria.

Osman C, Haag M, Potting C, Rodenfels J, Dip PV, Wieland FT, Brügger B, Westermann B, Langer T - J. Cell Biol. (2009)

Gep1 affects mitochondrial morphology and Mgm1 processing under respiring conditions. (A, left) Wild-type (WT), Δphb1, and Δgep1 cells expressing mitochondria-targeted GFP were grown to log phase in YP medium containing glycerol (YPG) and analyzed as in Fig. 2 A. Bar, 5 µm. (right) The bar graph indicates the percentage of wild type–like (light gray), fragmented (dark gray), and short tubular, partially fragmented (black) mitochondria. Data represent mean values ± SD of three independent experiments. (B) Decreased PE levels impair Mgm1 processing. Extracts of the indicated cells grown in YPG were analyzed by SDS-PAGE and immunoblotted using Mgm1- and Tom40-specific antisera. A quantification of the immunoblots is shown in the bottom panel. The percentage of S-Mgm1 was calculated from the ratio S-Mgm1/(S-Mgm1 + L-Mgm1). Data represent mean values ± SD of four independent experiments. *, P < 0.05; **, P < 0.01. L, long isoform of Mgm1; S, short isoform of Mgm1.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654118&req=5

fig3: Gep1 affects mitochondrial morphology and Mgm1 processing under respiring conditions. (A, left) Wild-type (WT), Δphb1, and Δgep1 cells expressing mitochondria-targeted GFP were grown to log phase in YP medium containing glycerol (YPG) and analyzed as in Fig. 2 A. Bar, 5 µm. (right) The bar graph indicates the percentage of wild type–like (light gray), fragmented (dark gray), and short tubular, partially fragmented (black) mitochondria. Data represent mean values ± SD of three independent experiments. (B) Decreased PE levels impair Mgm1 processing. Extracts of the indicated cells grown in YPG were analyzed by SDS-PAGE and immunoblotted using Mgm1- and Tom40-specific antisera. A quantification of the immunoblots is shown in the bottom panel. The percentage of S-Mgm1 was calculated from the ratio S-Mgm1/(S-Mgm1 + L-Mgm1). Data represent mean values ± SD of four independent experiments. *, P < 0.05; **, P < 0.01. L, long isoform of Mgm1; S, short isoform of Mgm1.
Mentions: When cells were grown on glycerol-containing medium, ∼80% of Δgep1 and ∼60% of Δphb1 cells contained an at least partially fragmented mitochondrial network, which indicates that both proteins are crucial for mitochondrial morphology under conditions of increased mitochondrial demand (Fig. 3 A). Inner membrane fusion and the formation of cristae depend on the dynamin-like GTPase Mgm1 (Meeusen et al., 2006), which undergoes proteolytic processing by the rhomboid protease Pcp1 in the inner membrane. Cleavage results in the accumulation of two isoforms, L- and S-Mgm1, that functionally cooperate during inner membrane fusion (Sesaki et al., 2003). Interestingly, deletion of GEP1 significantly impaired the formation of S-Mgm1 in cells grown under respiring conditions (Fig. 3 B). These findings identify Gep1 as a novel modulator of Mgm1 processing in the inner membrane and suggest that the unbalanced accumulation of L- and S-Mgm1 causes deficiencies in cristae morphogenesis in Δgep1 mitochondria.

Bottom Line: We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner.Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells.We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, Centre for Molecular Medicine (CMMC), Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50674, Germany.

ABSTRACT
Prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.

Show MeSH
Related in: MedlinePlus