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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 and Ups1 regulate the phospholipid composition of mitochondrial membranes. (A and B) Phospholipid profile of mitochondria lacking Gep1-like proteins. Phospholipids were extracted from mitochondria isolated from the indicated strains (ΔΔΔ: Δgep1Δgep2Δups1) and analyzed by TLC (A) and mass spectrometry (B). Mean values ± SD obtained from at least two independent mitochondrial isolations; samples analyzed in duplicate are shown in B. Asterisks indicate unidentified lipid species. (C) Cell growth in the absence of Gep1-like proteins. Fivefold serial dilutions of the indicated cells were spotted on YPD plates. Strains were grown at 30°C. (D and E) Phospholipid profile of mitochondria in cells overexpressing Gep1-like proteins. Mitochondrial phospholipids were analyzed by TLC (D) and mass spectrometry (E). Mean values ± SD obtained from three mitochondrial isolations, each analyzed in duplicate, are shown in E. (F) Impaired cell growth upon Gep1 overexpression. Gep1-like proteins were expressed in wild-type cells from high-copy plasmids under the control of the GAL promoter. Fivefold serial dilutions of the cells were grown on synthetic media containing galactose as the carbon source (SCGal) at 30°C.
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fig6: Gep1 and Ups1 regulate the phospholipid composition of mitochondrial membranes. (A and B) Phospholipid profile of mitochondria lacking Gep1-like proteins. Phospholipids were extracted from mitochondria isolated from the indicated strains (ΔΔΔ: Δgep1Δgep2Δups1) and analyzed by TLC (A) and mass spectrometry (B). Mean values ± SD obtained from at least two independent mitochondrial isolations; samples analyzed in duplicate are shown in B. Asterisks indicate unidentified lipid species. (C) Cell growth in the absence of Gep1-like proteins. Fivefold serial dilutions of the indicated cells were spotted on YPD plates. Strains were grown at 30°C. (D and E) Phospholipid profile of mitochondria in cells overexpressing Gep1-like proteins. Mitochondrial phospholipids were analyzed by TLC (D) and mass spectrometry (E). Mean values ± SD obtained from three mitochondrial isolations, each analyzed in duplicate, are shown in E. (F) Impaired cell growth upon Gep1 overexpression. Gep1-like proteins were expressed in wild-type cells from high-copy plasmids under the control of the GAL promoter. Fivefold serial dilutions of the cells were grown on synthetic media containing galactose as the carbon source (SCGal) at 30°C.

Mentions: Several lines of evidence point to overlapping activities of Gep1 family members: first, both Δgep1 and Δups1 show a synthetic lethal interaction with prohibitin mutants (Table I); and second, overexpression of Gep2 promoted growth of Δgep1Δphb1 cells and restored PE accumulation in Gep1-deficient mitochondria (Fig. 4 A, B). We therefore determined the accumulation of PE in Δgep1, Δgep2, and Δups1 mitochondria both by TLC and by mass spectrometry (Fig. 6, A and B). In contrast to GEP1, deletion of GEP2 and UPS1 did not affect PE levels within mitochondria (Fig. 6, A and B). Our analysis revealed, however, a crucial role of Ups1 for CL levels. CL is a dimeric phosphoglycerolipid predominantly present in mitochondria, where it is synthesized by the CL synthase Crd1 in the inner membrane (Schlame, 2008). CL was decreased approximately sevenfold in Δups1 mitochondria but remained unaffected in the absence of Gep1 or Gep2 (Fig. 6, A and B). Growth of Δups1 cells was severely impaired on glucose-containing medium (Fig. 6 C), a phenotype reminiscent of yeast cells lacking Pgs1 that catalyzes the rate-limiting step of CL biosynthesis. Deletion of GEP1 and GEP2, however, did not affect cell growth under these conditions (Fig. 6 C). Similar to cells lacking Psd1, Δgep1 cells exhibited an increased tendency to lose mitochondrial DNA under these conditions (unpublished data). We conclude that the accumulation of CL in mitochondria depends on Ups1, whereas Gep1 controls mitochondrial PE.


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 and Ups1 regulate the phospholipid composition of mitochondrial membranes. (A and B) Phospholipid profile of mitochondria lacking Gep1-like proteins. Phospholipids were extracted from mitochondria isolated from the indicated strains (ΔΔΔ: Δgep1Δgep2Δups1) and analyzed by TLC (A) and mass spectrometry (B). Mean values ± SD obtained from at least two independent mitochondrial isolations; samples analyzed in duplicate are shown in B. Asterisks indicate unidentified lipid species. (C) Cell growth in the absence of Gep1-like proteins. Fivefold serial dilutions of the indicated cells were spotted on YPD plates. Strains were grown at 30°C. (D and E) Phospholipid profile of mitochondria in cells overexpressing Gep1-like proteins. Mitochondrial phospholipids were analyzed by TLC (D) and mass spectrometry (E). Mean values ± SD obtained from three mitochondrial isolations, each analyzed in duplicate, are shown in E. (F) Impaired cell growth upon Gep1 overexpression. Gep1-like proteins were expressed in wild-type cells from high-copy plasmids under the control of the GAL promoter. Fivefold serial dilutions of the cells were grown on synthetic media containing galactose as the carbon source (SCGal) at 30°C.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig6: Gep1 and Ups1 regulate the phospholipid composition of mitochondrial membranes. (A and B) Phospholipid profile of mitochondria lacking Gep1-like proteins. Phospholipids were extracted from mitochondria isolated from the indicated strains (ΔΔΔ: Δgep1Δgep2Δups1) and analyzed by TLC (A) and mass spectrometry (B). Mean values ± SD obtained from at least two independent mitochondrial isolations; samples analyzed in duplicate are shown in B. Asterisks indicate unidentified lipid species. (C) Cell growth in the absence of Gep1-like proteins. Fivefold serial dilutions of the indicated cells were spotted on YPD plates. Strains were grown at 30°C. (D and E) Phospholipid profile of mitochondria in cells overexpressing Gep1-like proteins. Mitochondrial phospholipids were analyzed by TLC (D) and mass spectrometry (E). Mean values ± SD obtained from three mitochondrial isolations, each analyzed in duplicate, are shown in E. (F) Impaired cell growth upon Gep1 overexpression. Gep1-like proteins were expressed in wild-type cells from high-copy plasmids under the control of the GAL promoter. Fivefold serial dilutions of the cells were grown on synthetic media containing galactose as the carbon source (SCGal) at 30°C.
Mentions: Several lines of evidence point to overlapping activities of Gep1 family members: first, both Δgep1 and Δups1 show a synthetic lethal interaction with prohibitin mutants (Table I); and second, overexpression of Gep2 promoted growth of Δgep1Δphb1 cells and restored PE accumulation in Gep1-deficient mitochondria (Fig. 4 A, B). We therefore determined the accumulation of PE in Δgep1, Δgep2, and Δups1 mitochondria both by TLC and by mass spectrometry (Fig. 6, A and B). In contrast to GEP1, deletion of GEP2 and UPS1 did not affect PE levels within mitochondria (Fig. 6, A and B). Our analysis revealed, however, a crucial role of Ups1 for CL levels. CL is a dimeric phosphoglycerolipid predominantly present in mitochondria, where it is synthesized by the CL synthase Crd1 in the inner membrane (Schlame, 2008). CL was decreased approximately sevenfold in Δups1 mitochondria but remained unaffected in the absence of Gep1 or Gep2 (Fig. 6, A and B). Growth of Δups1 cells was severely impaired on glucose-containing medium (Fig. 6 C), a phenotype reminiscent of yeast cells lacking Pgs1 that catalyzes the rate-limiting step of CL biosynthesis. Deletion of GEP1 and GEP2, however, did not affect cell growth under these conditions (Fig. 6 C). Similar to cells lacking Psd1, Δgep1 cells exhibited an increased tendency to lose mitochondrial DNA under these conditions (unpublished data). We conclude that the accumulation of CL in mitochondria depends on Ups1, whereas Gep1 controls mitochondrial PE.

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