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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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Lipid profile of mitochondria lacking GEP genes. CL and PE levels were determined by mass spectrometry in mitochondria isolated from wild type (WT) and Δphb1 cells, and cells lacking various GEP genes grown on galactose-containing media. Mean values of two mitochondrial lipid extracts are shown. Effects of Yme1 and Oxa1 on mitochondrial PE levels have been previously observed in cells grown on glucose-containing media (Nebauer et al., 2007).
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fig7: Lipid profile of mitochondria lacking GEP genes. CL and PE levels were determined by mass spectrometry in mitochondria isolated from wild type (WT) and Δphb1 cells, and cells lacking various GEP genes grown on galactose-containing media. Mean values of two mitochondrial lipid extracts are shown. Effects of Yme1 and Oxa1 on mitochondrial PE levels have been previously observed in cells grown on glucose-containing media (Nebauer et al., 2007).

Mentions: The regulation of the phospholipid composition of the inner membrane by Gep1 and Ups1 proteins together with their synthetic lethal interaction with prohibitins suggests that reduced levels of PE and CL are deleterious for inner membrane integrity in prohibitin-deficient cells. Accordingly, other GEP genes may affect the PE and CL levels in mitochondrial membranes as well. We therefore isolated mitochondria from various yeast strains lacking Phb1 or GEP genes, extracted membrane lipids, and determined PE and CL levels by mass spectrometry (Fig. 7). Strikingly, mitochondrial PE and/or CL were affected in the majority of 23 examined strains (Fig. 7). Only some strains showed normal PE and almost unaltered CL levels in mitochondria (Fig. 7). The latter group included cells lacking assembly factors of the FO particle of the F1FO ATP-synthase, like Atp10 and Atp23, that have been previously found to interact genetically with prohibitins (Osman et al., 2007), or Oxa1, for which a function during FO assembly was recently described (Jia et al., 2007). Deletion of PSD1 or CRD1 resulted in the expected drastic reduction of the PE or CL content of mitochondrial membranes, respectively (Fig. 7). Notably, we observed increased PE levels in cells showing a severely reduced CL content. This is in agreement with previous findings describing an increase in mitochondrial PE in Δcrd1 cells, and suggested a coordinated regulation of both phospholipids (Zhong et al., 2004). Moreover, membranes isolated from Phb1-deficient mitochondria contained reduced amounts of CL and slightly increased PE levels. Strikingly, the loss of a large number of GEP genes genetically interacting with prohibitins led to strongly reduced levels of PE and/or CL (Fig. 7). These included genes with functions for mitochondrial morphology and the assembly of β-barrel proteins (MDM10, MMM1, MDM31, MDM32, MDM34, and MDM35; Merz et al., 2007; Bolender et al., 2008), genes associated with the assembly of respiratory chain complexes (COX6, YTA10, and YTA12), and several uncharacterized open reading frames (GEP3-6). Our findings link the function of these genes to the mitochondrial lipid metabolism and point to a critical role of the PE and CL content of the inner membrane for the survival of prohibitin-deficient cells.


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)

Lipid profile of mitochondria lacking GEP genes. CL and PE levels were determined by mass spectrometry in mitochondria isolated from wild type (WT) and Δphb1 cells, and cells lacking various GEP genes grown on galactose-containing media. Mean values of two mitochondrial lipid extracts are shown. Effects of Yme1 and Oxa1 on mitochondrial PE levels have been previously observed in cells grown on glucose-containing media (Nebauer et al., 2007).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Lipid profile of mitochondria lacking GEP genes. CL and PE levels were determined by mass spectrometry in mitochondria isolated from wild type (WT) and Δphb1 cells, and cells lacking various GEP genes grown on galactose-containing media. Mean values of two mitochondrial lipid extracts are shown. Effects of Yme1 and Oxa1 on mitochondrial PE levels have been previously observed in cells grown on glucose-containing media (Nebauer et al., 2007).
Mentions: The regulation of the phospholipid composition of the inner membrane by Gep1 and Ups1 proteins together with their synthetic lethal interaction with prohibitins suggests that reduced levels of PE and CL are deleterious for inner membrane integrity in prohibitin-deficient cells. Accordingly, other GEP genes may affect the PE and CL levels in mitochondrial membranes as well. We therefore isolated mitochondria from various yeast strains lacking Phb1 or GEP genes, extracted membrane lipids, and determined PE and CL levels by mass spectrometry (Fig. 7). Strikingly, mitochondrial PE and/or CL were affected in the majority of 23 examined strains (Fig. 7). Only some strains showed normal PE and almost unaltered CL levels in mitochondria (Fig. 7). The latter group included cells lacking assembly factors of the FO particle of the F1FO ATP-synthase, like Atp10 and Atp23, that have been previously found to interact genetically with prohibitins (Osman et al., 2007), or Oxa1, for which a function during FO assembly was recently described (Jia et al., 2007). Deletion of PSD1 or CRD1 resulted in the expected drastic reduction of the PE or CL content of mitochondrial membranes, respectively (Fig. 7). Notably, we observed increased PE levels in cells showing a severely reduced CL content. This is in agreement with previous findings describing an increase in mitochondrial PE in Δcrd1 cells, and suggested a coordinated regulation of both phospholipids (Zhong et al., 2004). Moreover, membranes isolated from Phb1-deficient mitochondria contained reduced amounts of CL and slightly increased PE levels. Strikingly, the loss of a large number of GEP genes genetically interacting with prohibitins led to strongly reduced levels of PE and/or CL (Fig. 7). These included genes with functions for mitochondrial morphology and the assembly of β-barrel proteins (MDM10, MMM1, MDM31, MDM32, MDM34, and MDM35; Merz et al., 2007; Bolender et al., 2008), genes associated with the assembly of respiratory chain complexes (COX6, YTA10, and YTA12), and several uncharacterized open reading frames (GEP3-6). Our findings link the function of these genes to the mitochondrial lipid metabolism and point to a critical role of the PE and CL content of the inner membrane for the survival of prohibitin-deficient cells.

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