T H E J O U R N A L O F C E L L B I O L O G Y
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 184 No. 4 583–596
M. Haag and C. Potting contributed equally to this paper.
Correspondence to Thomas Langer: Thomas.Langer@uni-koeln.de
Abbreviations used in this paper: CL, cardiolipin; PE, phosphatidylethanol-
amine; PS, phosphatidylserine; SGA, synthetic genetic array.
Biological membranes are complex structures made up of a multi-
plicity of membrane lipids and proteins controlling essential
cellular processes. This is exemplifi ed by mitochondria, double-
membrane bound organelles with essential roles in diverse meta-
bolic and cellular signaling pathways ( Chan, 2006 ; McBride
et al., 2006 ). The mitochondrial inner membrane is considered to
be the most protein-rich cellular membrane, whose functional
impairment is associated with aging, myopathies, and neurologi-
cal disorders in humans ( Chan, 2006 ). It harbors the multisubunit
complexes of the respiratory chain, key enzymes of many cata-
bolic and anabolic pathways, and various protein translocases
with crucial roles during mitochondrial biogenesis. Mitochondria
form an interconnected, tubular network, which undergoes dy-
namic changes by balanced fusion and fi ssion events ( Hoppins
et al., 2007 ). Mitochondrial biogenesis therefore requires adjust-
ing and coordinating the assembly of proteins, encoded by both
nuclear and mitochondrial genomes, and membrane lipids, some
of which are synthesized within mitochondria. However, although
protein transport to mitochondrial membranes has been exten-
sively studied, mechanisms of lipid import into and within mito-
chondria are only poorly understood.
Prohibitins form an evolutionary conserved family of mem-
brane proteins with a variety of suggested activities in different
cellular compartments ( Rajalingam et al., 2005 ; Kasashima et al.,
2006 ; Wang et al., 2008 ). Cell proliferation depends on prohibi-
tins targeted to mitochondria ( Merkwirth et al., 2008 ), where two
homologous subunits, Phb1 and Phb2, assemble into multimeric,
high – molecular weight complexes in the inner membrane ( Coates
et al., 1997 ; Berger and Yaffe, 1998 ; Tatsuta et al., 2005 ). Prohi-
bitins have been found to be part of mitochondrial nucleoids
prohibitins are essential in higher eukaryotes, prohibitin-
defi cient yeast cells are viable and exhibit a reduced rep-
licative life span. Here, we defi ne 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
rohibitin ring complexes in the mitochondrial inner
membrane regulate cell proliferation as well as the
dynamics and function of mitochondria. Although
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-
specifi c but coordinated manner. Lipid profi ling by mass
spectrometry of GEP-defi cient mitochondria reveals a
critical role of cardiolipin and phosphatidylethanolamine
for survival of prohibitin-defi cient cells. We propose that
prohibitins control inner membrane organization and in-
tegrity by acting as protein and lipid scaffolds.
The genetic interactome of prohibitins: coordinated
control of cardiolipin and phosphatidylethanolamine
by conserved regulators in mitochondria
Christof Osman , 1 Mathias Haag , 3 Christoph Potting , 1 Jonathan Rodenfels , 4 Phat Vinh Dip , 1 Felix T. Wieland , 3
Britta Br ü gger , 3 Benedikt Westermann , 4 and Thomas Langer 1,2
1 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
2 Max-Planck-Institute for Biology of Aging, Cologne, Germany
3 Heidelberg University Biochemistry Centre, Heidelberg 69120, Germany
4 Institute for Cell Biology and Electron Microscopy Laboratory, University of Bayreuth, Bayreuth 95440, Germany
© 2009 Osman et al. This article is distributed under the terms of an Attribution–
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Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
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JCB • VOLUME 184 • NUMBER 4 • 2009 584
which were previously found to genetically interact with each
other ( Dimmer et al., 2005 ). Diverse functions have been associ-
ated with another group of eight GEP genes, which include the
synthesis of CL (by the CL synthase Crd1) and the synthesis of
PE (by the phosphatidylserine [PS] decarboxylase Psd1). Finally,
growth of prohibitin-defi cient cells was found to depend on eight
GEP genes of unknown function ( Table I ).
Mitochondrial inner membrane integrity
depends on Gep1 and prohibitins
The open reading frame YLR168c , which was termed GEP1 , at-
tracted our attention, as it showed a strong genetic interaction with
prohibitin genes and belongs to a highly conserved gene family,
including previously described UPS1 / Preli1 genes ( Fig. 1 A ; Dee
and Moffat, 2005 ; Sesaki et al., 2006 ). Similar to Ups1 and
PRELI1, Gep1 has been identifi ed in the mitochondrial pro-
teome ( Kumar et al., 2002 ). To assess mitochondrial defects in
the absence of both Gep1 and Phb1, we generated ? gep1 ? phb1
cells expressing Phb1 from a tetracycline-regulatable promoter
([ PHB1 ]). These cells grew normally on both fermentable and
nonfermentable carbon sources under nonrepressing conditions,
whereas addition of the tetracycline analogue doxycycline shut
off PHB1 expression and inhibited cell growth ( Fig. 1 B ).
Immunoblot analysis of cell extracts revealed that outer mem-
brane proteins accumulated normally in these cells ( Fig. 1 C ).
However, the loss of Phb1 was accompanied by a decrease of
mitochondrial inner membrane and matrix proteins, which sug-
gests functional defi cits in the inner membrane ( Fig. 1 C ). We
therefore determined in Phb1-depleted cells the formation of
the mitochondrial membrane potential, which is required for
protein transport across and into the inner membrane and thereby
essential for cell survival. Depletion of Phb1 decreased the
membrane potential in ? phb1 [ PHB1 ] cells and led to its com-
plete dissipation in ? gep1 ? phb1 [ PHB1 ] cells ( Fig. 1 D ). These
fi ndings point to essential functions of both genes for the integ-
rity of the inner membrane and provide an explanation for the
synthetic lethal interaction of GEP1 and PHB1.
Requirement of Gep1 for mitochondrial
To assess mitochondrial morphology in cells lacking Gep1 and
prohibitins, we expressed mitochondrially targeted GFP variants
in wild-type, ? gep1 , or ? phb1 cells and ? gep1 or ? gep1 ? phb1
cells harboring [ PHB1 ]. An inspection of these cells by fl uores-
cence microscopy revealed the accumulation of ball-shaped
and clustered mitochondria in ? gep1 ? phb1 cells upon down-
regulation of Phb1 ( Fig. 2 A ). In the absence of doxycycline,
mitochondrial morphology was slightly impaired in these cells,
most likely refl ecting 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 in-
terfere with the formation of tubular mitochondria ( Fig. 2 A ).
The analysis of mitochondrial ultrastructure by trans-
mission electron microscopy revealed swollen and clustered
mitochondria in ? 40% of ? gep1 ? phb1 [ PHB1 ] but not ? phb1
( Bogenhagen et al., 2003 ), to affect the organization of mitochon-
drial DNA ( Kasashima et al., 2008 ), and to protect endothelial
cells against reactive oxygen – induced senescence ( Kasashima
et al., 2008 ; Schleicher et al., 2008 ). Fibroblasts lacking prohibi-
tins show aberrant mitochondrial cristae and exhibit a decreased
resistance toward apoptosis ( Merkwirth et al., 2008 ). These defi -
ciencies are caused by an impaired processing of the dynamin-like
GTPase OPA1 ( Merkwirth et al., 2008 ), a core component of the
mitochondrial fusion machinery ( Hoppins et al., 2007 ), which
is mutated in dominant optic atrophy ( Alexander et al., 2000 ;
Delettre et al., 2000 ).
Despite recent progress in understanding of cellular roles
of prohibitins, their molecular activity remained largely elu-
sive ( Mishra et al., 2006 ). Prohibitin complexes assemble with
m -AAA proteases in the mitochondrial inner membrane and
thereby modulate the turnover of nonnative membrane proteins
( Steglich et al., 1999 ). Therefore, they have been proposed to
exert chaperone-like activity ( Nijtmans et al., 2002 ). However,
the ring structure of purifi ed prohibitin complexes ( Tatsuta et al.,
2005 ) and the sequence similarity of prohibitins to lipid raft –
associated proteins of the SPFH family ( Browman et al., 2007 )
are also consistent with a scaffolding function of prohibitin com-
plexes in the inner membrane.
Notably, although they are required for embryonic develop-
ment in mice, Caenorhabditis elegans , and Drosophila melano-
gaster , deletion of prohibitin genes in yeast leads to premature
ageing but does not affect cell survival ( Coates et al., 1997 ; Artal-
Sanz et al., 2003 ; Merkwirth et al., 2008 ). We therefore searched
for yeast genes whose function is essential for cell growth in the
absence of prohibitins. Functional studies on the uncovered ge-
netic network identifi ed novel modulators of the phospholipid
composition of the inner membrane and link the function of pro-
hibitin complexes to cardiolipin (CL) and phosphatidylethanol-
amine (PE), nonbilayer phospholipids critical for mitochondrial
structure and integrity.
Defi ning the genetic interactome
To identify genes genetically interacting with prohibitins, a syn-
thetic genetic array (SGA) analysis was performed using ? phb1
cells and a collection of 4,850 yeast strains lacking nonessential
genes. Moreover, we tested candidate genes and reexamined pre-
viously described genetic interactors of prohibitins in the same
yeast strain background. In this way, we identifi ed 35 GEP genes
(for genetic interactors of prohibitins), whose deletion caused le-
thality or severely impaired growth of prohibitin-defi cient yeast
cells on glucose-containing media ( Table I ). Consistent with a
mitochondrial function of prohibitin complexes, 31 genes code
for mitochondrial proteins. Interestingly, 19 of the genetic inter-
actors fall into two functional classes: genes with functions dur-
ing the assembly of the respiratory chain, and genes required for
the maintenance of mitochondrial morphology ( Dimmer et al.,
2002 ) and the assembly of ? -barrel proteins in the outer mem-
brane ( Meisinger et al., 2007 ). The latter group includes the genes
MMM1 , MDM10 , MDM12 , MDM31 , MDM32 , and MDM34 ,
585GENETIC INTERACTOME OF PROHIBITINS • Osman et al.
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 ). Interest-
ingly, deletion of GEP1 signifi cantly impaired the formation of
S-Mgm1 in cells grown under respiring conditions ( Fig. 3 B ).
These fi ndings identify Gep1 as a novel modulator of Mgm1
processing in the inner membrane and suggest that the unbal-
anced accumulation of L- and S-Mgm1 causes defi ciencies in
cristae morphogenesis in ? gep1 mitochondria.
Gep1 regulates the accumulation of PE
To further defi ne the role of Phb1 and Gep1 for mitochondrial
morphogenesis and Mgm1 processing, we screened for genes
whose overexpression promotes growth of cells lacking both genes.
? gep1 ? phb1 cells expressing plasmid-borne PHB1 were trans-
formed with a multicopy yeast library. After plasmid shuffl ing,
[ PHB1 ] cells depleted of Phb1 ( Fig. 2 B ). We carefully quanti-
fi ed the surface of cristae membranes and related it to the sur-
face of the mitochondrial contours in ultrathin sections of
Gep1- and Phb1-defi cient cells ( Fig. 2 C ). The surface of cris-
tae membranes decreased only slightly in ? phb1 [ PHB1 ] cells
but was reduced dramatically by ? 80% in ? gep1 ? phb1 [ PHB1 ]
cells upon down-regulation of Phb1 ( Fig. 2 C ), which demon-
strates that Gep1 and Phb1 concomitantly affect mitochondrial
When cells were grown on glycerol-containing medium,
? 80% of ? gep1 and ? 60% of ? phb1 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
Table I. GEP genes
Functional groupGene ORFLocalization Genetic interaction
Respiratory chain assembly YTA10 a
MMM1 a , b
MDM10 a , b
MDM12 a , b
Mitochondrial morphology/ ? -barrel assembly
M, N, C
C, cytosol; GD, growth defect associated with double mutant; IM, inner membrane; IMS, intermembrane space; M, mitochondria; MA, matrix; N, nucleus; ND, not
determined; OM, outer membrane; SL, synthetically lethal.
a Previously described synthetic lethal interactions ( Berger and Yaffe, 1998 ; Steglich et al., 1999 ; Birner et al., 2003 ; Osman et al., 2007 ).
b Genetic interactors identifi ed by sporulation and tetrad dissection only.
JCB • VOLUME 184 • NUMBER 4 • 2009 586
Strikingly, overexpression of the PS synthase Cho1 was also
found to completely restore growth of ? gep1 ? phb1 cells, linking
functional defects in these cells to the cellular phospholipid
metabolism ( Fig. 4 A ). PS, which is synthesized by Cho1 in the
ER, is transported to mitochondria and decarboxylated to form
the previously uncharacterized GEP2 gene ( YDR185c ), which
codes for a homologue of Gep1, was identifi ed as a multicopy
suppressor of the synthetic lethal interaction of ? gep1 and
? phb1 ( Fig. 4 A ), indicating overlapping functions of members
of this conserved protein family.
Figure 1. Phb1 is required for mitochondrial inner membrane integrity in the absence of Gep1. (A) Multiple sequence alignment (score matrix: Blosum62)
of Gep1, Gep2, Ups1, and human homologues. The conserved MSF1 ? /PRELI domain is depicted. Numbers refer to amino acids. Black highlighting
indicates full conservation across all species; gray highlighting represents amino acids with similar properties conserved in at least three of the analyzed
sequences. (B) Synthetic lethal interaction of ? gep1 and ? phb1 . Fivefold serial dilutions of cell suspensions were spotted on glucose (Glc)- or galactose
(Gal)-containing YP plates, which were supplemented with 2 μ g/ml doxycycline (Dox) when indicated and incubated at 30 ° C. WT, wild type. (C) Steady-
state levels of mitochondrial proteins in ? gep1 ? phb1 [ PHB1 ] cells. Mitochondria were isolated from cells grown on glucose-containing media in the pres-
ence of doxycycline for different time periods. Mitochondrial proteins were analyzed by SDS-PAGE and immunoblotting. IM, inner membrane; OM, outer
membrane; M, matrix; L, long isoform of Mgm1; S, short isoform of Mgm1. (D) Dissipation of the membrane potential in mitochondria lacking Gep1 and
Phb1. Mitochondria were isolated from cells grown for 12 h on glucose-containing media in the presence or absence of doxycycline and stained with the
potential-sensitive dye 3,3 ? -dipropylthiadicarbocyanine iodide (DiDC 3 (5)).
587GENETIC INTERACTOME OF PROHIBITINS • Osman et al.
Figure 2. Impaired mitochondrial cristae morphogenesis in cells lacking Gep1 and Phb1. (A, left) Wild-type (WT), Δ phb1 , Δ gep1 , Δ phb1 [ PHB1 ], and
Δ gep1 Δ phb1 [ PHB1 ] cells expressing mitochondria-targeted GFP or DsRed were grown to log phase in YPD medium in the presence or absence of doxycy-
cline, and analyzed by differential interference contrast (left) and fl uorescence (right) microscopy. Bar, 5 μ m. (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 indepen-
dent experiments. (B, left) Wild type, ? phb1 , and ? gep1 or Δ phb1 [ PHB1 ] and Δ gep1 Δ phb1 [ 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 Δ gep1 Δ phb1 [ PHB1 ] cells depleted of prohibitin are shown. (C) The cristae/contour ratio was determined as described in Materials and methods. The
quantifi cation is illustrated in the bar graph. n ≥ 100.
JCB • VOLUME 184 • NUMBER 4 • 2009 588
Psd1 within mitochondria or by Psd2 in the Golgi apparatus
( Voelker, 2004 ). To examine which of these pathways is af-
fected by Gep1, we deleted GEP1 in ? psd1 and ? psd2 cells
and examined cell growth both on fermentable and nonfer-
mentable carbon sources ( Fig. 4 C ). The absence of Gep1 did
not impair growth of cells defi cient of Psd1 at any tempera-
ture, which is consistent with an epistatic relationship of these
genes ( Fig. 4 C ). However, growth of ? gep1 ? psd2 cells was
inhibited on nonfermentable carbon sources at 37 ° C ( Fig. 4 C ),
which suggests that Gep1 specifi cally affects mitochondrial
PE. The synthesis of PE within mitochondria was previously
found to be essential for the viability of yeast mutants lacking
the CL synthase Crd1, which is localized in the mitochondrial
inner membrane ( Gohil et al., 2005 ). Consistent with the sig-
nifi cantly reduced levels of PE in ? gep1 mitochondria, we did
not obtain viable double mutant offspring after tetrad dissec-
tion of diploid cells heterozygous for deletions in GEP1 and
CRD1 indicating synthetic lethality ( Fig. 4 D ). We conclude
from these experiments that Gep1 is required for the accumu-
lation of PE in mitochondria.
These fi ndings raise the possibility that an altered phos-
pholipid composition of the inner membrane impairs Mgm1
cleavage and causes an altered cristae morphogenesis in the
absence of Gep1. We therefore assessed processing of Mgm1
in mitochondria lacking Psd1 or Crd1 ( Fig. 3 B ). Deletion of
PSD1 , as well as that of GEP1 , impaired the formation of
S-Mgm1, whereas Mgm1 cleavage was not affected in the ab-
sence of CRD1 under these conditions ( Fig. 3 B ). We conclude
that the phospholipid composition, in particular the PE con-
tent, is crucial for effi cient Mgm1 processing by rhomboid in
the inner membrane.
Gep1 is dispensable for PE synthesis but
controls its stability in mitochondria
To unravel the molecular basis of impaired PE accumulation in
the absence of Gep1, we fi rst determined the localization of
Gep1 within mitochondria. A Gep1 variant carrying a C-terminal
hemagglutinin tag was expressed in ? gep1 cells and in ? gep1
? phb1 cells. Expression of this variant promoted growth of
? gep1 ? phb1 cells, demonstrating that the C-terminal extension
did not interfere with Gep1 function (unpublished data). Mito-
chondria were isolated from ? gep1 cells and subjected to pro-
tease treatment. Gep1 was degraded only under hypotonic
con ditions, which resulted in osmotic disruption of the outer
membrane, indicating that Gep1 is localized in the intermem-
brane space ( Fig. 5 A ). A hydropathy blot did not provide any
evidence for the presence of a membrane-spanning segment in
Gep1. However, Gep1 was found in the pellet fraction after car-
bonate extraction of mitochondrial membranes, which indicates
a tight membrane association ( Fig. 5 A ).
As Gep1 may directly regulate Psd1 in the inner mem-
brane, we assessed the enzymatic activity of Psd1 in wild-type
and ? gep1 mitochondria. After osmotic disruption of the outer
membrane, mitoplasts were incubated with fl uorescently la-
beled PS (NBD-PS), which is converted to NBD-PE ( Fig. 5 B ).
NBD-PE did not accumulate in ? psd1 mitochondria, which
demonstrates that NBD-PE is formed by Psd1. The synthesis
PE by Psd1 in the inner membrane ( Voelker, 2005 ). Psd1 is a
bifunctional protein, which is required for both PE synthesis
and the regulation of multidrug resistance ( Gulshan et al., 2008 ).
It has been observed to genetically interact with prohibitins
( Table I ; Birner et al., 2003 ), indicating a complex network of
genetic interactions between prohibitins, Gep1, and the cellular
We therefore determined the mitochondrial phospholipid
profi le in ? gep1 cells by TLC. Whereas the majority of mito-
chondrial phospholipids accumulated at normal levels in the
absence of Gep1, PE was present at signifi cantly reduced lev-
els in ? gep1 mitochondria ( Fig. 4 B ). Overexpression of Cho1
or Gep2, but not of Ups1, restored normal PE levels in Gep1-
defi cient mitochondria ( Fig. 4 B ). Further genetic experiments
corroborated the role of Gep1 for the formation of cellular PE.
Cell survival depends on PE, which is synthesized either by
Figure 3. Gep1 affects mitochondrial morphology and Mgm1 processing
under respiring conditions. (A, left) Wild-type (WT), Δ phb1 , and Δ gep1
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 μ m.
(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
immuno blotted using Mgm1- and Tom40-specifi c antisera. A quantifi ca-
tion 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.
589GENETIC INTERACTOME OF PROHIBITINS • Osman et al.
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 ? ups1 mitochondria but remained unaffected in
the absence of Gep1 or Gep2 ( Fig. 6, A and B ). Growth of ? ups1
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. Dele-
tion of GEP1 and GEP2 , however, did not affect cell growth
under these conditions ( Fig. 6 C ). Similar to cells lacking Psd1,
? gep1 cells exhibited an increased tendency to lose mito-
chondrial DNA under these conditions (unpublished data). We
conclude that the accumulation of CL in mitochondria de-
pends on Ups1, whereas Gep1 controls mitochondrial PE.
Strikingly, deletion of GEP1 restored normal CL levels in
membranes of ? ups1 mitochondria ( Fig. 6, A and B ) and sup-
pressed growth defects associated with the loss of Ups1 ( Fig. 6 C ).
In contrast, neither CL levels nor cell growth were affected
upon deletion of GEP2 in ? ups1 cells, although Gep1 and Gep2
share 55% identical amino acids ( Fig. 6, A – C ). PE, however,
remained reduced in ? gep1 ? ups1 mitochondria and accumulated
at similar levels as in ? gep1 mitochondria ( Fig. 6, A and B ),
which demonstrates that the absence of Ups1 does not alleviate
the requirement of Gep1 for the accumulation of PE. Thus, a
of NBD-PE was not affected in the absence of Gep1 ( Fig. 5 B ).
We observed a slightly but signifi cantly increased rate of NBD-PE
synthesis in ? gep1 mitochondria, which may indicate an allevi-
ated product inhibition of Psd1 in these mitochondria.
These results were substantiated by pulse-labeling experi-
ments in vivo, which were performed in a ? psd2 strain back-
ground to exclude masking effects of nonmitochondrial PE
synthesis ( Fig. 5 C ). We labeled ? psd2 and ? psd2 ? gep1 cells
with [ 3 H]serine and monitored the incorporation of 3 H in PE
within mitochondria. Deletion of GEP1 did not affect mito-
chondrial PE synthesis by mitochondrial Psd1 ( Fig. 5 C ). Pulse
chase experiments, however, revealed a signifi cantly decreased
stability of newly synthesized PE in the absence of Gep1 with-
out a signifi cant accumulation of other lipid species ( Fig. 5 D ).
We conclude that the decreased PE concentration in the absence
of Gep1 is not caused by an impaired Psd1 activity or uptake of
its substrate PS but instead refl ects a reduced stability of PE
within ? gep1 mitochondria.
Lipid-specifi c functions of Gep1-like
proteins for PE and CL
Several lines of evidence point to overlapping activities of
Gep1 family members: fi rst, both ? gep1 and ? ups1 show a
synthetic lethal interaction with prohibitin mutants ( Table I ); and
second, overexpression of Gep2 promoted growth of ? gep1 ? phb1
cells and restored PE accumulation in Gep1-defi cient mito-
chondria ( Fig. 4 A, B ). We therefore determined the accumula-
tion of PE in ? gep1 , ? gep2 , and ? ups1 mitochondria both by
Figure 4. The accumulation of PE in mitochon-
dria depends on Gep1. (A) Overexpression of
Gep2 or Cho1 allows growth of ? gep1 ? phb1
cells. ? gep1 ? phb1 [ PHB1 ] cells overexpress-
ing Phb1, Gep2, or Cho1 were incubated at
30 ° C on media with or without 5 ? fl uoroorotic
acid, which prevents growth of cells harboring
the [ PHB1 ] expression plasmid. (B) TLC analy-
sis of mitochondrial phospholipids isolated
from ? gep1 cells overexpressing Cho1, Ups1,
or Gep2. The asterisk indicates an unidentifi ed
lipid species. (C) Gep1 and Psd2 interact ge-
netically. Yeast cells were spotted on YP plates
containing glucose (YPD) or glycerol (YPG)
and incubated at 30 ° C or 37 ° C. (D) Synthetic
lethal interaction of ? gep1 and ? crd1 . A dip-
loid strain heterozygous for deletions of GEP1
and CRD1 was subjected to sporulation and
tetrad dissection. Arrowheads indicate inviable
double mutant progeny.
JCB • VOLUME 184 • NUMBER 4 • 2009 590
in ? ups1 cells upon deletion of GEP1 and the reduction of CL
levels upon Gep1 overexpression suggest a competition be-
tween Gep1 and Ups1.
Survival of prohibitin-defi cient cells
depends on the lipid composition of
The regulation of the phospholipid composition of the inner
membrane by Gep1 and Ups1 proteins together with their syn-
thetic lethal interaction with prohibitins suggests that reduced
levels of PE and CL are deleterious for inner membrane integ-
rity in prohibitin-defi cient 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 ma-
jority 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
F O particle of the F 1 F O ATP-synthase, like Atp10 and Atp23,
that have been previously found to interact genetically with
complex functional network of Gep1-like proteins controls mito-
chondrial PE and CL.
To further defi ne the functional interdependence of PE
and CL pathways, we generated yeast strains overexpressing
Gep1, Gep2, or Ups1 from galactose-inducible promoters.
Overexpression of Gep1 impaired cell growth on galactose-
containing medium, whereas increased cellular levels of Gep2
or Ups1 did not interfere with cell growth ( Fig. 6 F ). Mito-
chondria were isolated from these cells, and the phospholipid
profi le was determined by TLC and mass spectrometry ( Fig. 6,
D and E ). In agreement with the observed growth defect, we
noted a signifi cantly reduced CL content of mitochondria con-
taining overexpressed Gep1 ( Fig. 6 D, E ). Moreover, PE levels
were increased, whereas other phospholipids were present
at normal levels in these mitochondria ( Fig. 6, D and E ; and
unpublished data). PE and CL were not altered in a statisti-
cally signifi cant manner upon overexpression of Ups1 or Gep2
( Fig. 6, D and E ).
Collectively, these experiments defi ne lipid-specifi c activ-
ities of Gep1-like proteins for CL and PE and, at the same time,
point to common steps in the regulation of both phospholipids
within mitochondria. Both the observed restoration of CL levels
Figure 5. Gep1 is required for the stability
of mitochondrial PE. (A) Localization of Gep1
in the mitochondrial intermembrane space.
(left) Isolated mitochondria were treated with
Na 2 CO 3 , pH 11.5 (T), and split into a soluble
(S) and insoluble (P) fractions by ultracentri-
fugation. The asterisk indicates an unspecifi c
cross-reaction of the antiserum. (right) Mito-
chondria or mitoplasts, generated by osmotic
disruption of the outer membrane (SW), were
treated with 50 μ g/ml trypsin and analyzed
by SDS-PAGE and immunoblotting. Tom70, an
outer membrane protein; Yme1, an integral
inner membrane protein exposed to the IMS;
and Hsp60, a soluble matrix protein, served
as controls. (B) Psd1 activity in the absence of
Gep1. Mitoplasts of WT or ? gep1 cells were
incubated with NBD-PS for indicated time pe-
riods. Phospholipids were fractionated by TLC
and fl uorescent lipids were quantifi ed by fl uor
imaging. Equal loading was monitored by mo-
lybdenum blue staining. NBD-PE accumulating
in wild-type mitochondria after 20 min was set
to 100%. Data represent ± SD of four inde-
pendent experiments. **, P < 0.01. (C) Mito-
chondrial PE synthesis in Gep1-defi cient cells.
? psd2 and ? psd2 ? gep1 cells were incubated
with [ 3 H]serine for 0, 20, 40, or 60 min. Phos-
pholipids extracted from crude mitochondrial
isolations were subjected to TLC. PE was recov-
ered from the TLC plate and radioactivity was
determined by liquid scintillation counting.
Labeled PE accumulating in ? psd2 cells after
60 min was set to 100%. Data represent mean
values ± SD of fi ve independent experiments.
(D) PE stability is decreased in the absence of
Gep1. ? psd2 and ? psd2 ? gep1 cells were
incubated with [ 3 H]serine for 10 min, and,
after addition of excess serine and further in-
cubation for the time points indicated, PE was
quantifi ed as in C. Labeled PE accumulating in
? psd2 cells at time point 0 was set to 100%.
Data represent mean values ± SD of four in-
dependent experiments. *, P < 0.05; **, P <
0.01; ***, P < 0.001.
591GENETIC INTERACTOME OF PROHIBITINS • Osman et al.
morphology and the assembly of ? -barrel proteins ( MDM10 ,
MMM1 , MDM31 , MDM32 , MDM34 , and MDM35 ; Merz et al.,
2007 ; Bolender et al., 2008 ), genes associated with the assem-
bly of respiratory chain complexes ( COX6 , YTA10 , and YTA12 ),
and several uncharacterized open reading frames ( GEP3-6 ).
Our fi ndings link the function of these genes to the mitochon-
drial lipid metabolism and point to a critical role of the PE and
CL content of the inner membrane for the survival of prohibitin-
defi cient cells.
The network of prohibitin-interacting genes unraveled by our
SGA analysis demonstrates an intimate functional relationship
prohibitins ( Osman et al., 2007 ), or Oxa1, for which a function
during F O assembly was recently described ( Jia et al., 2007 ).
Deletion of PSD1 or CRD1 resulted in the expected drastic re-
duction 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 agree-
ment with previous fi ndings describing an increase in mitochon-
drial PE in ? crd1 cells, and suggested a coordinated regulation
of both phospholipids ( Zhong et al., 2004 ). Moreover, mem-
branes isolated from Phb1-defi cient mitochondria contained re-
duced 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
Figure 6. Gep1 and Ups1 regulate the
phospholipid composition of mitochondrial
membranes. (A and B) Phospholipid profi le
of mitochondria lacking Gep1-like proteins.
Phospholipids were extracted from mitochon-
dria isolated from the indicated strains ( ? ? ? :
? gep1 ? gep2 ? ups1 ) 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
unidentifi ed lipid species. (C) Cell growth in
the absence of Gep1-like proteins. Fivefold se-
rial dilutions of the indicated cells were spotted
on YPD plates. Strains were grown at 30 ° C.
(D and E) Phospholipid profi le of mitochondria in
cells overexpressing Gep1-like proteins. Mito-
chondrial phospholipids were analyzed by TLC
(D) and mass spectrometry (E). Mean values ±
SD obtained from three mitochondrial isola-
tions, each analyzed in duplicate, are shown
in E. (F) Impaired cell growth upon Gep1
overexpression. Gep1-like proteins were ex-
pressed in wild-type cells from high-copy plas-
mids 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.
JCB • VOLUME 184 • NUMBER 4 • 2009 592
proteins like fl otillins/reggies, which are distantly related to
prohibitins and were found to induce microdomains in the
plasma membrane and to modulate the assembly of signaling
complexes ( Frick et al., 2007 ; Langhorst et al., 2008 ).
Altered levels of CL or PE compromise mitochondrial
activities and are associated with many pathophysiological
states, but the mechanisms that determine the phospholipid
composition of mitochondrial membranes are poorly under-
stood. Our fi ndings identify the conserved family of Gep1-like
proteins as novel membrane-associated regulators of CL and
PE in mitochondrial membranes. Gep1 is essential for the ac-
cumulation of PE, whereas Ups1 is required for the accumula-
tion of CL in mitochondrial membranes. Strikingly, deletion
of GEP1 in ? ups1 cells restored CL levels in mitochondria,
which suggests competition between Gep1 and Ups1. Consis-
tently, overexpression of Gep1 reduces CL in mitochondria.
Thus, the phospholipid content of mitochondrial membranes
critically depends on the level of Gep1. The competitive ac-
tion of Gep1 and Ups1 allows the adjustment of relative PE
and CL levels simply by modulating the amounts or the avail-
ability of the regulatory proteins. These fi ndings demonstrate
that mitochondrial levels of PE and CL are regulated coordi-
nately by related proteins and are therefore in agreement with
previous notions that cells may require a critical amount of
these nonbilayer-forming phospholipids ( Zhong et al., 2004 ;
Gohil et al., 2005 ).
Gep1-like proteins likely exert a regulatory role during
membrane biogenesis, as the mitochondrial phospholipid profi le
is only modestly altered in cells lacking all Gep1-like proteins.
The decrease of the PE content of mitochondrial membranes in
the absence of Gep1 is not caused by an impaired PE synthesis.
Rather, PE does not accumulate stably in Gep1-defi cient mito-
chondria, suggesting that Gep1 inhibits either a PE-specifi c li-
pase or the export of PE from mitochondria. Accordingly, the
competition of Gep1 and Ups1 may determine the specifi city
of mitochondrial phospholipases or lipid transport processes.
of prohibitins to the lipid composition of mitochondrial mem-
branes ( Fig. 8 A ). While they are dispensable for yeast cell
growth under normal conditions, prohibitins are essential for
the integrity of the inner membrane and cell survival if mem-
branes are defi cient for CL or PE. Both CL and PE have similar
physical properties and cluster into nonbilayer, hexagonal phase
structures in lipid membranes ( de Kruijff, 1997 ). Disturbances
in both biosynthetic pathways are synthetic lethal in yeast
( Gohil et al., 2005 ) and bacteria, illustrating that the physical
similarities of CL and PE are of functional relevance in vivo.
Consistently, clusters of PE and CL have been detected in bacte-
rial membranes ( Matsumoto et al., 2006 ). It is therefore con-
ceivable that defi ned lipid clusters with specifi c functions exist
in the inner membrane of mitochondria. Contact sites between
inner and outer mitochondrial membrane, at which import of
nuclear-encoded mitochondrial proteins occurs ( Reichert and
Neupert, 2002 ) and which have been linked to phospholipid
transport processes ( Simbeni et al., 1990 ), were found to be
enriched in PE and CL, and may represent such specialized
membrane domains. Ringlike prohibitin complexes may serve
as membrane organizers and affect the distribution of CL and PE
in the membrane bilayer ( Fig. 8 B ). If CL and PE are present
only at low concentrations, this function may become essential
for inner membrane integrity and membrane-associated pro-
cesses. Such an activity of prohibitins is in perfect accordance
with the predicted function of prohibitins as protein scaffolds,
and may ensure the recruitment of membrane proteins to a spe-
cifi c lipid environment. This includes m -AAA proteases that as-
semble with prohibitins into large supercomplexes in the inner
membrane ( Steglich et al., 1999 ). It should be noted that a
fencelike activity of prohibitin ring complexes could also en-
sure the formation of protein-free domains in the mitochondrial
inner membrane, which is considered to be the most protein-
rich cellular membrane. These proposed functions of prohibi-
tins as membrane organizers are reminiscent of other SPFH
Figure 7. Lipid profi le of mitochondria lacking GEP genes. CL and PE levels were determined by mass spectrometry in mitochondria isolated from wild
type (WT) and ? phb1 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 ).
593GENETIC INTERACTOME OF PROHIBITINS • Osman et al.
ing of the dynamin-like GTPase Mgm1, which is required for
membrane fusion and cristae formation ( Meeusen et al., 2006 ).
Under these conditions, prohibitins are essential to maintain
inner membrane integrity. Thus, residual Mgm1 processing
suffi cient to maintain mitochondrial cristae at decreased PE
levels appears to critically depend on prohibitins. Notably, we
have recently identifi ed the processing of the mammalian
Mgm1 homologue OPA1 as the central process controlled by
prohibitins in mouse fi broblasts ( Merkwirth et al., 2008 ),
which indicates that the same processes depend on prohibitin
function in evolutionary distant organisms. Therefore, pheno-
typic differences associated with the loss of prohibitins in
yeast and mammals likely refl ect differences in the phospho-
lipid profi le of mitochondrial membranes or the lipid depen-
dence of Mgm1/OPA1 processing itself.
Although the absence of CL did not inhibit Mgm1 process-
ing in our experiments, we do not exclude a role of CL for proteo-
lytic cleavage under certain growth conditions in yeast or
in other organisms. Variations of the PE content of the inner
membrane may mask the dependence of Mgm1 processing on
CL. Accordingly, differences in the relative content of PE and
CL may explain why the loss of Ups1 was observed previously to
inhibit Mgm1 processing ( Sesaki et al., 2006 ). It is therefore an
intriguing possibility that impaired processing of the mammalian
Mgm1 homologue OPA1 causes the disturbed formation of mito-
chondrial cristae, which was observed in lymphoblasts of Barth
syndrome patients or yeast cells lacking the CL transacylase ta-
fazzin ( Acehan et al., 2007 ; Claypool et al., 2008 ). The identifi -
cation of Gep1-like proteins as regulators of CL and PE now
allows for the direct examination of the role of an altered phos-
pholipid composition for mitochondrial dysfunction in disease.
Gep1-like proteins and prohibitins acting as membrane
organizers may affect various membrane-associated processes,
which are known to depend on nonbilayer phospholipids.
Mitochondrial fusion requires phospholipase D, which hydrolyzes
CL in the outer membrane and generates the fusogenic lipid
phosphatidic acid ( Choi et al., 2006 ). CL and PE affect also the
insertion and oligomerization of the proapoptotic Bcl2-family
member Bax in the outer membrane ( Lucken-Ardjomande
et al., 2008 ). A CL defi ciency in the inner membrane impairs
the activity of mitochondrial enzymes ( Jiang et al., 2000 ), de-
creases the stability of respiratory chain supercomplexes and
of mitochondrial DNA ( Pfeiffer et al., 2003 ), and accelerates
apoptosis by facilitating the release of cytochrome c from the
intramitochondrial storage compartments ( Choi et al., 2007 ).
It is conceivable that the assembly of the F 1 F O ATP-synthase and
respiratory complexes is yet another process dependent on de-
fi ned functional domains within mitochondrial membranes.
This is suggested by the synthetic interaction of prohibitins with
genes coding for assembly factors of inner membrane complexes
( Osman et al., 2007 ), which do not drastically affect mitochon-
drial PE or CL levels when deleted.
Interestingly, mutations in the majority of GEP genes, which
show a synthetic interaction with prohibitins, cause decreased
levels of nonbilayer lipids. This includes MDM10 and MMM1 ,
which were originally identifi ed as genes required for mito-
chondrial inheritance and DNA stability ( Boldogh et al., 1998 )
In agreement with an inhibitory role of Gep1 for PE export from
mitochondria, we observed in the absence of Gep1 a slight in-
crease of PC, which is generated by methyl transferases in ER
membranes using mitochondrial PE ( Kodaki and Yamashita,
1989 ). Contact sites between inner and outer mitochondrial mem-
branes have been discussed as sites of phospholipid transport
( Simbeni et al., 1990 ). It will be therefore of interest to examine
whether Gep1-like proteins locate to these sites.
We identify the morphogenesis of cristae as one process
critically dependent on mitochondrial PE. Decreased PE lev-
els in Gep1- or Psd1-defi cient cells compromise the process-
Figure 8. Prohibitins and mitochondrial inner membrane organization.
(A) Genetic interaction of prohibitins with PE and CL biosynthetic path-
ways. Synthetic lethal interactions between CHO1 and PGS1 and between
PSD1 and CRD1 have been described previously ( Janitor et al., 1996 ;
Gohil et al., 2005 ). (B) Hypothetical model for the role of prohibitins as
membrane organizers. The maintenance of putative functional membrane
domains containing CL and PE (gray dots) depends on prohibitin ring com-
plexes or a high level of CL and PE in the inner membrane.
JCB • VOLUME 184 • NUMBER 4 • 2009 594
H 2 O (1:1, vol/vol). Samples were then dried under a constant stream of air.
Lipids were dissolved in chloroform, phosphate concentration was deter-
mined ( Rouser et al., 1970 ), and samples were subjected to TLC analysis.
TLC plates (HPTLC; Merck & Co., Inc.) were developed with chloroform/
methanol/25% ammonia (50:50:3, vol/vol/vol) when not stated otherwise,
allowing the separation of PC, PE, and CL from other mitochondrial phos-
pholipids (Fig. S1, available at http://www.jcb.org/cgi/content/full/
jcb.200810189/DC1). TLC plates were stained with 470 mM CuSO 4 in
8.5% o -phosphoric acid and subsequently incubated for 10 min at 180 ° C.
Quantifi cation of PE and CL by mass spectrometry. Mitochondrial lipids
were extracted and phosphate concentration was determined according to
Rouser et al. (1970) . For mass spectrometric analysis, 1.5-nmol phospho-
lipids of mitochondrial fractions were extracted in the presence of CL
(CL56:0; Avanti Polar Lipids, Inc.) and PE standard (50 pmol each) as de-
scribed previously ( Br ü gger et al., 2006 ). Dried lipids were redissolved in
10 mM ammonium acetate in methanol.
Quantifi cation of PE was performed by neutral-loss scanning, select-
ing for a neutral loss of 141 D as described previously ( Br ü gger et al.,
2006 ). Quantifi cation of CL was performed in negative ion mode on a
quadrupole time-of-fl ight mass spectrometer (QStar Elite, Applied Bio-
systems). 10 μ l of lipid extracts was diluted 1:2 with 0.1% piperidine in metha-
nol and automatically infused (Triversa Nanomate; Advion Biosciences).
The ionization voltage was set to ? 0.95 kV and gas pressure was set to
0.5 psi. CLs were detected as single charged molecules. CL species (all
combinations of fatty acids 16:0, 16:1, 16:2, 18:0, 18:1, and 18:2) were
analyzed by targeted product ion scanning. The peak areas of CL-derived
fatty acid fragments were extracted from the respective product ion spectra
via the “ Extract Fragments ” script (Analyst QS 2.0; Applied Biosystems).
Isotope correction for M +2 ions was performed manually, and values were
corrected for response factors of standards.
Determination of Psd1 activity. Mitochondria were resuspended in
assay buffer B (0.1 M Tris/HCl, pH 7.4, 10 mM EDTA, and 2 μ M PS-C6-NBD
[Avanti Polar Lipids]) to a fi nal concentration of 5 mg/ml and incubated at
25 ° C. At the time points indicated, mitochondria (500 μ g) were removed
from the reaction mixture, and phospholipids were extracted and analyzed
by TLC (developing solvent: chloroform/methanol/H 2 O/triethylamine;
30:35:7:35 vol/vol/vol/vol). NBD signals were detected and quantifi ed
by fl uor imaging (TyphoonTrio; GE Healthcare).
In vivo labeling of phospholipids. Logarithmically growing yeast cells
were harvested and resuspended in medium supplemented with [ 3 H]serine
(12.5 μ Ci/ml) to a fi nal OD 600 of 5. After incubation at 30 ° C for 0, 20, 40,
or 60 min, cells corresponding to 10 OD 600 were harvested and stored in
liquid nitrogen. Phospholipids were extracted and analyzed by TLC. PE spots
were recovered from the TLC plates and mixed with 400 μ l H 2 O and 8 ml of
scintillation cocktail. Labeled PE was quantifi ed by liquid scintillation counting.
The stability of PE was monitored in pulse chase experiments. Yeast cells
were labeled for 10 min as described above. Cells were then harvested, re-
suspended in medium containing 20 mM of unlabeled serine, and incubated
at 30 ° C for 0, 30, 60, 90, 120, or 150 min before PE was quantifi ed.
Online supplemental material
Fig. S1 documents the resolution of the TLC system applied in this study. Table S1
lists yeast strains used in this study. Online supplemental material is available
We thank Stefan Geimer and Rita Grotjahn for help with electron microscopy,
Martine Collart and Justus Ackermann for their support at different stages of this
project, and Mark D ü rr for helpful discussions. We would like to thank Iris
Leibrecht and Timo Sachsenheimer for technical assistance.
This work was supported by grants from the Deutsche Forschungs-
gemeinschaft to T. Langer (SFB635/C4) and B. Westermann (WE2174/4-1),
and support from the German-Israeli Project (DIP grant F.5.1) and the European
Research Counsel to T. Langer.
Submitted: 31 October 2008
Accepted: 13 January 2009
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Materials and methods
Yeast genetic procedures
The SGA using ? phb 1 cells was performed as described previously ( Tong
et al., 2001 ). Synthetic genetic interactions with ? phb 1 were confi rmed
by sporulation and tetrad dissection. In agreement with the functional inter-
dependence of Phb1 and Phb2, the same interactors were identifi ed in SGAs
using ? phb2 cells. To identify multicopy suppressors of synthetic lethal inter-
actions, ? phb1 ? gep1 [ PHB1 ] cells were transformed with a genomic Yep13
high-copy library. After growth for 1 d at 30 ° C on SC-Leu, plates were rep-
licated on plates containing 5 ? fl uoroorotic acid (1 mg/ml). Clones were
analyzed after 2 d, and the suppressor gene was identifi ed by subcloning.
The genotypes of yeast strains used are described in Table S1 (available at
Fluorescence and electron microscopy was performed as described previ-
ously ( D ü rr et al., 2006 ) using a microscope (Axioplan 2; Carl Zeiss, Inc.)
equipped with a Plan-Neofl uar 100 × /1.30 NA Ph3 oil objective lens (Carl
Zeiss, Inc.). Images were recorded with a monochrome camera (Evolution
VF Mono Cooled; Intas) and processed with Image-Pro Plus 5.0 and Scope
Pro4.5 software (Media Cybernetics). For the determination of the cristae/
mitochondrial contour ratio, ultrathin sections were viewed in a transmission
electron microscope (JEM-2100; JEOL Ltd.) and images were taken with a
digital camera (Erlangshen ES500W, model 782; Gatan Inc.). Approx-
imately 100 micrographs of mitochondria were taken for each strain at the
same magnifi cation. For each image, the numbers of cristae were counted
and the contour of each mitochondrion was measured in micrometers by
using the measure function of ImageJ. For each strain the numbers of cristae
were put into relation to the sum of the mitochondrial contours. Processing
of Mgm1 was monitored by immunoblotting using Mgm1-specifi c antiserum,
provided by A. Reichert (University of Frankfurt, Frankfurt, Germany).
TLC analysis. Mitochondria were isolated from cells grown on yeast-peptone
(YP) medium containing 2% galactose and 0.5% lactate. The mitochondrial
fraction was washed, resuspended in buffer A (0.6 M sorbitol and 5 mM
MES, pH 6), and loaded on a continuous sucrose gradient (20 – 50% in buf-
fer A). Mitochondria were harvested from the lower third of the gradient and
diluted 1:5 in buffer A, pelleted, washed once in SEM buffer (250 mM su-
crose, 10 mM MOPS, pH 7.2, and 1 mM EDTA), and fi nally resuspended
in SEM buffer. The purity of the mitochondrial fraction and absence of con-
taminations by vacuolar and ER membranes was assessed by immuno-
blotting using Sec61- and Vac8-specifi c antisera, provided by T. Sommer
(Max-Delbr ü ck-Centre, Berlin, Germany) and C. Ungermann (University of
Osnabr ü ck, Osnabr ü ck, Germany), respectively. Purifi ed mitochondria (1 mg)
were mixed with 1.5 ml chloroform/methanol (1:1, vol/vol) and vigor-
ously shaken for 60 min. 300 μ l H 2 O was added and samples were vor-
texed for 60 s. After centrifugation (1,000 rpm, 5 min), the aqueous phase
was removed and the solvent phase was washed with 250 μ l methanol/
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