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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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Related in: MedlinePlus

Impaired mitochondrial cristae morphogenesis in cells lacking Gep1 and Phb1. (A, left) Wild-type (WT), Δphb1, Δgep1, Δphb1[PHB1], and Δgep1Δphb1[PHB1] cells expressing mitochondria-targeted GFP or DsRed were grown to log phase in YPD medium in the presence or absence of doxycycline, and analyzed by differential interference contrast (left) and fluorescence (right) microscopy. Bar, 5 µm. (right) The bar graph indicates the percentage of wild type–like (light gray), fragmented (dark gray), and ball-shaped (black) mitochondria. n ≥ 100; data represent mean values ± SD of three independent experiments. (B, left) Wild type, Δphb1, and Δgep1 or Δphb1[PHB1] and Δgep1Δphb1[PHB1] cells depleted of prohibitin were grown to log phase in YPD medium containing doxycycline and analyzed by transmission electron microscopy. Bar, 500 nm. (right) The bar graph indicates the percentage of wild type–like (light gray) or clustered mitochondria (black), and other mitochondrial phenotypes (dark gray). n = 100. Two representative micrographs of Δgep1Δphb1[PHB1] cells depleted of prohibitin are shown. (C) The cristae/contour ratio was determined as described in Materials and methods. The quantification is illustrated in the bar graph. n ≥ 100.
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fig2: Impaired mitochondrial cristae morphogenesis in cells lacking Gep1 and Phb1. (A, left) Wild-type (WT), Δphb1, Δgep1, Δphb1[PHB1], and Δgep1Δphb1[PHB1] cells expressing mitochondria-targeted GFP or DsRed were grown to log phase in YPD medium in the presence or absence of doxycycline, and analyzed by differential interference contrast (left) and fluorescence (right) microscopy. Bar, 5 µm. (right) The bar graph indicates the percentage of wild type–like (light gray), fragmented (dark gray), and ball-shaped (black) mitochondria. n ≥ 100; data represent mean values ± SD of three independent experiments. (B, left) Wild type, Δphb1, and Δgep1 or Δphb1[PHB1] and Δgep1Δphb1[PHB1] cells depleted of prohibitin were grown to log phase in YPD medium containing doxycycline and analyzed by transmission electron microscopy. Bar, 500 nm. (right) The bar graph indicates the percentage of wild type–like (light gray) or clustered mitochondria (black), and other mitochondrial phenotypes (dark gray). n = 100. Two representative micrographs of Δgep1Δphb1[PHB1] cells depleted of prohibitin are shown. (C) The cristae/contour ratio was determined as described in Materials and methods. The quantification is illustrated in the bar graph. n ≥ 100.

Mentions: To assess mitochondrial morphology in cells lacking Gep1 and prohibitins, we expressed mitochondrially targeted GFP variants in wild-type, Δgep1, or Δphb1 cells and Δgep1 or Δgep1Δphb1 cells harboring [PHB1]. An inspection of these cells by fluorescence microscopy revealed the accumulation of ball-shaped and clustered mitochondria in Δgep1Δphb1 cells upon down-regulation of Phb1 (Fig. 2 A). In the absence of doxycycline, mitochondrial morphology was slightly impaired in these cells, most likely reflecting a deleterious effect of Phb1 overexpression. Notably, the aberrant morphology of mitochondria was not a consequence of cell death, as mitochondria were inspected while at least 85% of the cells were still viable as determined by FUN-1 staining (unpublished data). Deletion of PHB1 alone did not interfere with the formation of tubular mitochondria (Fig. 2 A).


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)

Impaired mitochondrial cristae morphogenesis in cells lacking Gep1 and Phb1. (A, left) Wild-type (WT), Δphb1, Δgep1, Δphb1[PHB1], and Δgep1Δphb1[PHB1] cells expressing mitochondria-targeted GFP or DsRed were grown to log phase in YPD medium in the presence or absence of doxycycline, and analyzed by differential interference contrast (left) and fluorescence (right) microscopy. Bar, 5 µm. (right) The bar graph indicates the percentage of wild type–like (light gray), fragmented (dark gray), and ball-shaped (black) mitochondria. n ≥ 100; data represent mean values ± SD of three independent experiments. (B, left) Wild type, Δphb1, and Δgep1 or Δphb1[PHB1] and Δgep1Δphb1[PHB1] cells depleted of prohibitin were grown to log phase in YPD medium containing doxycycline and analyzed by transmission electron microscopy. Bar, 500 nm. (right) The bar graph indicates the percentage of wild type–like (light gray) or clustered mitochondria (black), and other mitochondrial phenotypes (dark gray). n = 100. Two representative micrographs of Δgep1Δphb1[PHB1] cells depleted of prohibitin are shown. (C) The cristae/contour ratio was determined as described in Materials and methods. The quantification is illustrated in the bar graph. n ≥ 100.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Impaired mitochondrial cristae morphogenesis in cells lacking Gep1 and Phb1. (A, left) Wild-type (WT), Δphb1, Δgep1, Δphb1[PHB1], and Δgep1Δphb1[PHB1] cells expressing mitochondria-targeted GFP or DsRed were grown to log phase in YPD medium in the presence or absence of doxycycline, and analyzed by differential interference contrast (left) and fluorescence (right) microscopy. Bar, 5 µm. (right) The bar graph indicates the percentage of wild type–like (light gray), fragmented (dark gray), and ball-shaped (black) mitochondria. n ≥ 100; data represent mean values ± SD of three independent experiments. (B, left) Wild type, Δphb1, and Δgep1 or Δphb1[PHB1] and Δgep1Δphb1[PHB1] cells depleted of prohibitin were grown to log phase in YPD medium containing doxycycline and analyzed by transmission electron microscopy. Bar, 500 nm. (right) The bar graph indicates the percentage of wild type–like (light gray) or clustered mitochondria (black), and other mitochondrial phenotypes (dark gray). n = 100. Two representative micrographs of Δgep1Δphb1[PHB1] cells depleted of prohibitin are shown. (C) The cristae/contour ratio was determined as described in Materials and methods. The quantification is illustrated in the bar graph. n ≥ 100.
Mentions: To assess mitochondrial morphology in cells lacking Gep1 and prohibitins, we expressed mitochondrially targeted GFP variants in wild-type, Δgep1, or Δphb1 cells and Δgep1 or Δgep1Δphb1 cells harboring [PHB1]. An inspection of these cells by fluorescence microscopy revealed the accumulation of ball-shaped and clustered mitochondria in Δgep1Δphb1 cells upon down-regulation of Phb1 (Fig. 2 A). In the absence of doxycycline, mitochondrial morphology was slightly impaired in these cells, most likely reflecting a deleterious effect of Phb1 overexpression. Notably, the aberrant morphology of mitochondria was not a consequence of cell death, as mitochondria were inspected while at least 85% of the cells were still viable as determined by FUN-1 staining (unpublished data). Deletion of PHB1 alone did not interfere with the formation of tubular mitochondria (Fig. 2 A).

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