Integral membrane proteins in the mitochondrial outer
membrane of Saccharomyces cerevisiae
Lena Burri
1
, Katherine Vascotto
1,2
, Ian E. Gentle
1,2
, Nickie C. Chan
1,2
, Traude Beilharz
1
,
David I. Stapleton
2
, Lynn Ramage
3,
* and Trevor Lithgow
1,2
1 Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Australia
2 Bio21 Molecular Science and Biotechnology Institute, Parkville, Australia
3 Biozentrum, University of Basel, Switzerland
Mitochondria were derived from endosymbiotic bac-
teria and the mitochondrial outer membrane shares
many features with bacterial outer membranes [1,2].
Polypeptides embedded in the outer membrane of
Gram-negative bacteria are either lipoproteins
anchored by a covalently linked lipid, or have a
b-barrel structure with eight or more antiparallel
b-strands hydrogen bonded into a cylindrical barrel
[3,4]. The b-barrel proteins have an unusual primary

Hoffmann-La Roche Ltd., CH-4070 Basel,
Switzerland
(Received 30 October 2005, revised 29
January 2006, accepted 9 February 2006)
doi:10.1111/j.1742-4658.2006.05171.x
Mitochondria evolved from a bacterial endosymbiont ancestor in which the
integral outer membrane proteins would have been b-barrel structured
within the plane of the membrane. Initial proteomics on the outer mem-
brane from yeast mitochondria suggest that while most of the protein
components are integral in the membrane, most of these mitochondrial pro-
teins behave as if they have a-helical transmembrane domains, rather than
b-barrels. These proteins are usually predicted to have a single a-helical
transmembrane segment at either the N- or C-terminus, however, more
complex topologies are also seen. We purified the novel outer membrane
protein Om14 and show it is encoded in the gene YBR230c. Protein sequen-
cing revealed an intron is spliced from the transcript, and both transcription
from the YBR230c gene and steady-state level of the Om14 protein is dra-
matically less in cells grown on glucose than in cells grown on nonfermenta-
ble carbon sources. Hydropathy predictions together with data from limited
protease digestion show three a-helical transmembrane segments in Om14.
The a-helical outer membrane proteins provide functions derived after the
endosymbiotic event, and require the translocase in the outer mitochondrial
membrane complex for insertion into the outer membrane.
Abbreviations
DAS, dense alignment surface; PVDF, poly(vinylidene difluoride); TOM, translocase in the outer mitochondrial membrane.
FEBS Journal 273 (2006) 1507–1515 ª 2006 The Authors Journal compilation ª 2006 FEBS 1507
translocation, mitochondrial fission, and the contri-
butions made by mitochondria to cellular redox
metabolism and programmed cell death [12–15].
Integral membrane proteins with a-helical transmem-

least six other major proteins are present, as judged
from the Coomassie-stained protein profile. In order to
characterize the proteins of the outer membrane, we
set out to determine what proportion was integral and
to identify the major protein species.
To determine whether the major outer membrane
proteins were integral or peripheral, we initially used
alkali extraction. Extraction of the membrane vesicles
with alkali sodium carbonate releases several proteins
that might be peripheral components of the membrane
(Fig. 1A). Under these conditions, Om14 and Om45
are partially extracted by alkali, as are several other
outer membrane proteins. Om45 is known to be
anchored by a single a-helical transmembrane segment
[16]. Mitochondrial outer membrane proteins have
amphipathic character in their transmembrane segments,
A
BC
Fig. 1. Biochemical characterization of proteins present in purified mitochondrial outer membranes. (A). Outer membrane vesicles were puri-
fied and subject to treatment with 0.1
M Na
2
CO
3
. A sample of total vesicle proteins (100 lg) was analyzed by SDS ⁄ PAGE (‘T’) and compared
with the proteins resistant to alkali extraction (‘P’) and those extracted into the supernatant (‘S’). The marker protein sizes and the positions
of Om45 and Om14 on the Coomassie-stained gel are shown. (B). Outer membrane vesicles were subject to cloud-point extraction with Tri-
ton X-114, and SDS ⁄ PAGE used to determine the proteins present in the aqueous extract (‘Aq’), the detergent phase (‘D’) and the phospho-
lipid-rich pellet (‘P’). Arrowheads designate the size of proteins enriched in the lipid-rich pellet including the major 29 kDa protein Por1 and
Tom40, identified by mass spectrometry and immunoblotting. (C). After in situ digestion with trypsin, mass spectrometry was used to iden-

previous immunoblot analysis that suggested an
10 : 1 ratio of Tom70 to Tom71 [31].
The mitochondrial glycerol-3-phosphate dehydroge-
nase Gut2 behaves as an integral protein, though the
precise topology of Gut2 remains unclear. Our protein
sequencing confirms a predicted processing site [32],
and our predictions with the dense alignment surface
(DAS) algorithm (see Experimental procedures) agree
with the previous proposal that Gut2 has two trans-
membrane segments [33]. Previously, Gut2 had been
assumed located on the outer surface of the mitoch-
ondrial inner membrane. However, since we identified
no other inner membrane proteins in our vesicle pre-
paration, we suggest that Gut2 is an outer membrane
protein with its N-terminal FAD-binding domain
facing the intermembrane space. This topology would
enable Gut2 to transfer of electrons to Nde1 in the
intermembrane space [32].
Alo1 catalyses the final step in d-erythroascorbic
acid biosynthesis [34], was previously located to mito-
chondria [34,35], and has a predicted a-helical trans-
membrane segment from residue 174 to residue 191.
The cytochrome b
5
reductase, Mcr1, is an outer mem-
brane protein with a single N-terminal, a-helical trans-
membrane segment [36], and assists d-erythroascorbic
acid biosynthesis [14].
Table 1. Major proteins identified in the Triton X-114 detergent phase from the mitochondrial outer membrane of Saccharomyces cerevisiae.
The details of mass spectrometry and N-terminal sequencing are provided in the Experimental procedures section. ND refers to proteins for

22 MSRLSRSHSKALPIALGTV 34.137 34
YNL055c POR1 Voltage-dependent
anion channel, isoform 1
66 MSPPVYSDIS 30.428 29
YGR082w TOM20 20 kDa protein import
receptor
30 ND 20.317 22
YNL131w TOM22 22 kDa protein import
receptor
22 VELTEIKDDVVQLDEPQFSR 16.790 22
YBR230c OM14 Unknown ND MSATAKHDSNAS 14.609 14
L. Burri et al. Integral proteins in the mitochondrial outer membrane
FEBS Journal 273 (2006) 1507–1515 ª 2006 The Authors Journal compilation ª 2006 FEBS 1509
Ymr110c was previously identified as a mitochond-
rial protein [35] and the protein encoded from the
YMR110c gene fused to GFP shows punctate intra-
cellular localization similar to the outer membrane
proteins Mmm1 and Mmm2 [37]. The DAS predictor
suggests Ymr110c would have a single transmembrane
segment, from residues 134–152, which would anchor
it in the mitochondrial outer membrane.
As reported for bacterial b-barrel proteins and lipid-
modified proteins [27–29], Por1, Tom40, and nine
other proteins (of relative molecular masses 27, 34, 35,
50, 55, 65, 70, 85 and 105 kDa) precipitate out of the
Triton X-114 detergent phase (Fig. 1B, arrowheads).
None of the proteins so far identified in this fraction is
predicted to have a a-helical transmembrane segment.
Of interest, the 50 kDa protein band includes peptides
(8% sequence coverage) derived from Xdj1, a mole-

To be sure that Om14 is located exclusively in the
mitochondria, GFP fusions were constructed and
expressed in yeast. Confocal fluorescence microscopy
of live cells revealed the N-terminal fusion (GFP-
Om14) localizes exclusively to cortical structures that
costain with the mitochondria-specific dye Mitotracker
(Fig. 2B). The C-terminal GFP fusion (Om14-GFP)
gave identical profiles (Fig. 2B). Mitochondria were
isolated from cells expressing the N-terminal GFP-
Om14 fusion protein and treated with trypsin. The
GFP domain was released with protease, while trypsin-
sensitive proteins like cytochrome b
2
(in the mito-
A
BCD
Fig. 2. Identification and characterization of Om14. (A) The determined N-terminal sequence of Om14 (Table 1) is shown in bold. Basic resi-
dues, representing sites for trypsin cleavage, are circled and predicted transmembrane segments boxed. (B) Yeast cells expressing GFP-
Om14 or Om14-GFP were co-stained with the fluorescent dye Mitotracker Red and viewed by confocal microscopy. Filters selective for the
green fluorescence of GFP (left panel) or the red fluorescence of Mitotracker Red (middle panel) were used. Green and red fluorescence pic-
tures merged is shown in the right panel. (C) Wild-type (lane 1) or GFP-Om14 expressing (lanes 2–4) purified mitochondria (100 lg) were
treated with trypsin and 1% Triton (where indicated, ‘+’) for 30 min at 4 °C. After precipitation in trichloroacetic acid, proteins were analyzed
by SDS ⁄ PAGE and immunoblotting with antisera recognizing the outer membrane protein Tom70, the intermembrane space protein cyto-
chrome b
2
(Cytb
2
), the matrix-located Mdj1 or GFP. (D) 100 lg purified mitochondria expressing GFP-Om14 was treated with 0.1 M Na
2
CO

chrome b
2
is protected in the intermembrane space
(Fig. 3C). An antibody recognizing the GFP tag at the
C-terminus of Om14-GFP shows the electrophoretic
mobility of the fusion protein is reduced, indicating a
2 kDa mass difference. The C-terminal GFP epitope
is protected within the intermembrane space, and the
proteolysis must therefore represent a loss of 2 kDa
from the N-terminus. Given there are seven arginine
and lysine residues spread through the N-terminal
stretch of Om14, the three transmembrane segment
model of Om14 is the only one consistent with our data.
Om14 is not a subunit of the TOM, SAM or
morphology-related complexes
Proteins closely related in sequence to Om14 were
found in all budding yeast for which genome sequence
data is available (Supplementary material), but we
were unable to find closely related proteins from other
fungi, or even in the fission yeast Schizosaccharomyces
pombe. Both the lack of obvious orthologs in other
fungi and the abundance of Om14 in the outer mem-
brane would argue against it having a fundamental
role in mitochondrial biogenesis.
While the function of Om14 remains unclear, it may
be related functionally to another major protein in the
outer membrane, Om45. No detailed data on the stoi-
chiometry of these proteins exists, however, the Coo-
massie-stained profile of membrane proteins suggests
Om45 and Om14 are present at similar levels in the

L. Burri et al. Integral proteins in the mitochondrial outer membrane
FEBS Journal 273 (2006) 1507–1515 ª 2006 The Authors Journal compilation ª 2006 FEBS 1511
on glucose were Ybr230c (i.e. Om14) and Om45. Like
Dom45 cells [18], Dom14 cells have no obvious defects
in mitochondrial morphology or inheritance (data not
shown) and show no obvious defects in growth on fer-
mentable or nonfermentable carbon sources at any
temperature between 14 and 37 °C (data not shown).
Topography of the mitochondrial outer
membrane
The data presented here show that most of the protein
associated with the yeast mitochondrial outer mem-
brane in yeast is integral in the membrane and most of
the integral outer membrane proteins behave as if they
have a-helical transmembrane segments, partitioning
into the detergent phase after Triton X-114 extraction
of the outer membrane. This includes a number of
proteins whose submitochondrial location was not
known. While many of these proteins have a single
transmembrane segment at the N- or C-terminus, some
like Gut2 are predicted to have multiple transmem-
brane segments. Three transmembrane segments were
demonstrated for the newly identified protein Om14.
Thus, most of the newly synthesized protein flux into
the mitochondrial outer membrane consists of integral
proteins with a-helical transmembrane segments, and
the outer membrane is versatile enough to assemble a
large mass of protein with this topology. As the TOM
complex is needed to insert these a-helical transmem-
brane segments, the development of a TOM complex

To raise mono-specific antisera, samples of the outer
membrane proteins eluted from the column were resolved
by SDS ⁄ PAGE. After briefly Coomassie-staining the gels,
bands representing proteins of interest were excised and
mounted into an electroelution chamber, with a BT2 mem-
brane (Schleicher & Schuell, Bottmingen, Switzerland) at
one end. Electroelution proceeded for 6 h at 70 V, with two
changes of the eletrophoresis buffer (25 mm Tris, 192 mm
glycine, 0.025% SDS). The eluted protein sample was preci-
pitated with nine volumes of ice-cold ethanol and lyophi-
lized for injection into rabbits.
For direct protein sequencing, outer membrane proteins
(500 lg of total protein per lane) were separated by
SDS ⁄ PAGE, blotted to polyvinylidene difluoride (PVDF;
Immobilon-P; Millipore, Australia) and stained briefly with
Coomassie blue [45]. Slivers of PVDF-carrying protein were
excised and loaded into the sample cartridge of a Perkin-
Elmer HPG1005A Sequenator, and N-terminal sequences
read for up to 20 cycles.
For identification by mass spectrometry, proteins were
excised from a dried gel using a protein-free blade and
rehydrated in water for 15 min. Gel pieces were diced and
destained in 100 mm NH
4
HCO
3
⁄ 50% acetonitrile, dehydra-
ted in acetonitrile for 5 min and then air dried. Proteins
were reduced with 50 mm dithiothreitol at 60 °C for 60 min
and then alkylated with 50 mm iodoacetamide at room tem-

The PCR product was cloned into a centromeric plasmid
(with the URA3 gene for selection) to encode GFP-Om14
or Om14-GFP. These plasmids were used for transforma-
tion into the om14 strain (MATa, leu2, ura3, trp1, his3?,
om14::kanMX). Yeast was grown at 30 °C on YPAD (2%
(w ⁄ v) glucose, 1% (w ⁄ v) yeast extract, 2% (w ⁄ v) peptone
supplemented with adenine sulfate).
Characterization of membrane protein insertion
and topology
Mitochondria were isolated according to [47] and trypsin
treatments were performed as described [48]. Membranes
were extracted by resuspension in 0.1 m Na
2
CO
3
and incu-
bation for 30 min on ice with gentle vortexing. Soluble and
insoluble proteins were separated by centrifugation at
100 000 g in a Beckman Airfuge (Beckman Coulter,
Gladesville, NSW, Australia) [49]. For Triton X-114 extrac-
tions, samples of outer membrane vesicles corresponding to
100 lg protein were treated with detergent as described
[50]. Samples of mitochondrial protein (100 lg) were separ-
ated by Tris-glycine SDS ⁄ PAGE and western blots were
carried out according to published methods [48]. Fluores-
cence microscopy was as previously described [51]. Trans-
membrane domains were predicted from protein sequence
using DAS [51].
Acknowledgements
The authors thank Rosemary Condron for protein

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Supplementary material
The following supplementary material is available
online:
Fig. S1. Alignment of Om14 from various yeast
species. Draft form sequence data for Saccharomyces
paradoxus, Saccharomyces kudriazevii, Saccharomyces
kluveryii and Naumovia castellii were accessed via the
Genome Sequence Center (University of St Louis;
http://genomeold.wustl.edu/). Final sequences, publicly
available through GenBank, have the following acces-
sion numbers: Om14 from Ashbya gossypii (NM_