These proteins appear to provide functions that were procured after the initial endosymbiont established itself in early eukaryotic cells, including protein Keywords detergent phase; mit
Trang 1membrane of Saccharomyces cerevisiae
Lena Burri1, Katherine Vascotto1,2, Ian E Gentle1,2, Nickie C Chan1,2, Traude Beilharz1,
David I Stapleton2, Lynn Ramage3,* and Trevor Lithgow1,2
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
structure with many strands of alternating
hydrophi-lic and hydrophobic residues and a high abundance
of aromatic residues that tend to be placed at the
start of the strands [2,5] These b-barrel proteins
are assembled in the bacterial outer membrane in a
process mediated by the integral membrane protein Omp85 [2,3,5–8]
Mitochondria also carry a member of the Omp85 family [8]: this protein, called Sam50, has been shown responsible for the assembly of b-barrel pro-teins in the mitochondrial outer membrane [9–11], and functions together with at least two other sub-units as part of a Sorting and Assembly Machine (SAM) complex [7,9–11] In addition to b-barrel teins, mitochondrial outer membranes also have pro-teins with a-helical transmembrane domains These proteins appear to provide functions that were procured after the initial endosymbiont established itself in early eukaryotic cells, including protein
Keywords
detergent phase; mitochondria; outer
membrane; transmembrane segments
Correspondence
T Lithgow, Department of Biochemistry and
Molecular Biology, University of Melbourne,
Parkville 3010, Australia
Fax: +61 39348 2251
Tel: +61 38344 4131
E-mail: t.lithgow@unimelb.edu.au
*Present address
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.
Trang 2translocation, 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-brane segments appear to be inserted into the outer
membrane by the translocase in the outer
mitochon-drial membrane (TOM) complex without assistance
from the SAM complex [16,17], but it has not been clear
whether the most abundant protein traffic into the outer
membrane of mitochondria comes from the b-barrel or
a-helical type integral membrane proteins
To identify and characterize a-helical type integral
membrane proteins, we fractionated outer membrane
vesicles from yeast mitochondria for analysis by
pro-tein sequencing and mass spectrometry We found
Tri-ton X-114 phase separation to be the most reliable
means of purifying integral membrane proteins from
the mitochondrial outer membrane, and of the 11 most
abundant integral proteins, 10 had a-helical
transmem-brane segments Of these 10 proteins, most had a
single transmembrane segment predicted at the N- or
C-terminus However, more complex topologies are
also seen, with the novel protein Om14 being an
integral membrane protein with three a-helical
trans-membrane segments that are assembled into the
mito-chondrial outer membrane
Results and Discussion
Integral membrane proteins in the mitochondrial outer membrane
The protein profile of mitochondrial outer membranes shows at least nine major proteins (Fig 1A, asterisks), with the three most abundant of these the 45 kDa pro-tein Om45 [18], the 29 kDa propro-tein Por1 [19] and the
14 kDa outer membrane protein we call Om14 At 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,
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 phospho-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-tify the major proteins in the detergent phase of the extracted outer membrane vesicles (see Table 1) Equivalent samples were analyzed by SDS ⁄ PAGE followed by transfer to PVDF membrane for N-terminal sequencing.
Trang 3and alkali extraction might not always be a reliable
indicator of whether a protein is integral in the outer
membrane [17,20–22]
As a distinct means to separate integral and
peri-pheral membrane proteins we undertook cloud point
extraction with Triton X-114 [23,24] This treatment
liberates three fractions from a solubilized membrane:
an aqueous phase containing peripheral membrane
proteins, a detergent-phase in which proteins with
a-helical transmembrane domains are soluble and a
‘phospholipid-enriched’ pellet fraction containing
pro-teins that either have lipid modifications or b-barrel
characteristics [25–29] After phase separation,
approximately 15 proteins partitioned exclusively into
the aqueous phase, while at least 19 integral membrane
proteins partitioned into the detergent phase: Om45
and Om14 clearly behaved as integral membrane
pro-teins with a-helical transmembrane segments,
partition-ing into the detergent phase (Fig 1B)
A combination of N-terminal sequencing and mass
spectrometry was used to identify the 11 major integral
membrane proteins (Table 1) As reported previously
and shown in Fig 1C, Tom20 and Tom22 migrate
closely together on this gel system [30] Tom70 and
Tom71 also migrate closely together on the gel, and
the relative intensity of Coomassie staining reflects
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 b5 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 which no N-terminal sequence could be determined Note for Gut2, the aspartate residue in position one of the determined sequence cor-responds to D 38 in the precursor protein, as previously predicted by Esser et al [32] For Tom22, the valine at position 1 corresponds to V 2
in the predicted protein sequence [31], suggesting removal of the N-terminal methionine.
Sequence coverage (%)
N-terminal sequence
Molecular mass (kDa) Apparent Predicted
import receptor, isoform 1
YHR117w TOM71 70 kDa protein import
receptor, isoform 2
YIL155c GUT2 Mitochondrial
glycerol-3-phosphate dehydrogenase
YML086c ALO1 D -Arabinono-1,4-lactone
oxidase
reductase
anion channel, isoform 1
YGR082w TOM20 20 kDa protein import
receptor
YNL131w TOM22 22 kDa protein import
receptor
Trang 4Ymr110c 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-cular chaperone previously reported localized to
mito-chondria [38] Xdj1 is likely to carry a C-terminal
prenylation that might be responsible for its location
in the pellet fraction: a closely related chaperone,
Ydj1, is prenylated and known to play a role in
pro-tein transport into mitochondria [39,40]
Om14 is a mitochondrial protein encoded from
an intron-containing gene
N-terminal sequencing showed Om14 to be encoded
from the YBR230c gene (Fig 2A) The protein sequence
we obtained confirmed that a 97-basepair intron predicted in the YBR230c gene [41] is spliced from the transcript encoding Om14 at the predicted splice sites between the sequences corresponding to H7 and D8 (Fig 2A) Introns are rare in the genome of
S cerevisiae [42] and Om14 is, to our knowledge, the first mitochondrial protein encoded in an intron-containing gene
Om14 was purified by anion-exchange chromatogra-phy from mitochondrial outer membranes solubilized
in the detergent octyl-POE (Ramage, Lithgow and Schatz, unpublished results) An antiserum raised in rabbits to purified Om14 was used in immunoblots on mitochondria derived from wild-type yeast cells and Dom14 yeast cells from which the YBR230c gene had been deleted confirm that Om14 is the product of the YBR230c gene (data not shown)
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 b2 (in the
mito-A
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 GFP-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 b2(Cytb2), the matrix-located Mdj1 or GFP (D) 100 lg purified mitochondria expressing GFP-Om14 was treated with 0.1 M Na2CO3 and centrifuged to separate solubilized proteins (S) from insoluble material (P) Proteins were then analyzed by immunoblotting after SDS ⁄ PAGE using antisera against the matrix-located mtHsp70, the membrane protein porin and GFP.
Trang 5chondrial intermembrane space) and Mdj1 (in the
mat-rix) were protected from the protease by the
outer membrane (Fig 2C) Alkali extraction of
puri-fied mitochondria showed only a partial extraction
of GFP-Om14 (Fig 2D), as seen for the untagged
pro-tein in Fig 1(A) Om14 is an integral outer membrane
protein with its N-terminus displayed in the cytosol
Transmembrane topology of Om14
Figure 3A shows a hydropathy analysis of the Om14
sequence that predicts two or three a-helical
transmem-brane segments, in support of the behavior of the
protein in Triton X-114 The transmembrane segments
corresponding to residues 71–90 and 104–119 are con-fident predictions and the corresponding sequences are highly conserved in orthologs of Om14 found in other yeasts, including Saccharomyces kluverii, Ashbya gossypiiand Candida albicans (Supplementary material) However, the region corresponding to residues 38–54 predicts relatively poorly as a transmembrane segment With the N-terminus of Om14 exposed in the cytosol, there are two possible models for transmembrane topology (Fig 3B) To distinguish between these two, mitochondria were isolated from yeast cells expressing Om14-GFP and shaved with trypsin The outer mem-brane protein Tom70 is degraded by trypsin, while cyto-chrome b2 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 outer membrane, and their steady-state levels are tightly coregulated [43] suggestive of some link in their function A previous report on changes to the mito-chondrial proteome during diauxic shift identified only
18 mitochondrial proteins whose steady-state levels change when yeast cultures are shifted from glucose- to glycerol-based growth media [43] Sixteen of the 18 pro-teins were known to function in metabolic pathways associated with respiration The only two proteins of unknown function to disappear when cells were grown
A
B
C
Fig 3 Topology of Om14 in the outer mitochondrial membrane.
(A) Hydropathy calculations for Om14 were made with DAS [52].
The solid line represents a high confidence limit and the dashed
line a medium confidence limit for predictions (B) Two potential
topologies for Om14 in the outer membrane Amino acid residues
that delimit the predicted transmembrane segments are numbered.
(C) Wild-type (lane 1) or Om14-GFP expressing (lanes 2–4) purified
mitochondria (100 lg) were treated with trypsin and 1% Triton
(where indicated, ‘+’) for 20 min at 4 C After precipitation in
tri-chloroacetic acid, proteins were analyzed by SDS ⁄ PAGE and
im-munoblotting with antisera recognizing the outer membrane protein
Tom70, the intermembrane space protein cytochrome b 2 (Cytb 2 ),
the matrix-located Mdj1 or GFP.
Trang 6on 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 nonferfer-mentable carbon sources at any
temperature between 14 and 37C (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 memmem-brane 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 transmemtransmem-brane 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
by the ancestral endosymbiont was a prime face
requirement to establish protein targeting to all
com-partments of the developing organelle
Experimental procedures
Purification of mitochondrial outer membrane
vesicles
The yeast strain D273–10B (Mata, ATCC 25657) was grown
in 30 L cultures of semisynthetic media with lactate as a
car-bon source to an A600of approximately 3 Cells were
harves-ted and mitochondria purified on 14–18% Nycodenz (Sigma,
Castle Hill, NSW, Australia) gradients at pH 6.0 (prepared
in 10 mm Mops, 0.6 m sorbitol) Mitochondrial outer
mem-brane vesicles were prepared and purified as previously
des-cribed [44]
Proteomics
Mitochondrial outer membrane vesicles were solubilized
essentially as described [37] in buffer containing 50 mm
Hepes (pH 8.0), 2% octyl-POE (N-octyl-polyoxyethylene,
Bachem, Bubendorf, Switzerland), 50 mm NaCl (to a
con-centration of 2–3 mgÆmL )1protein) and the solubilized pro-teins separated by chromatography on Mono-Q (HR5⁄ 5 column, Pharmacia), using the same buffer with 1.0 m NaCl
to develop the column Elution of the major integral membrane proteins from the column was monitored by SDS⁄ PAGE of every second fraction and subsequent Coo-massie staining of the gels
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 NH4HCO3⁄ 50% acetonitrile, dehydra-ted in acetonitrile for 5 min and then air dried Proteins were reduced with 50 mm dithiothreitol at 60C for 60 min and then alkylated with 50 mm iodoacetamide at room tem-perature in the dark Gel pieces were washed in 20 mm
NH4HCO3 ⁄50% acetonitrile, dehydrated as above and rehydrated in the presence of 250 ng proteomics grade tryp-sin (Sigma, Australia) in 20 mm NH4HCO3and the digest allowed to proceed overnight at 30C Tryptic peptides were analyzed by liquid chromatography-tandem MS
(LC-MS⁄ MS) Peptides were injected manually via a Rheodyne injector onto a Waters 3 lm, dC18 Atlantis column (300 lm· 100 mm) fitted with a Waters Atlantis Nanoease guard column that was connected to an Agilent 1100 XCT plus ion trap mass spectrometer Peptides were separated over a gradient of 3–40% acetonitrile (with 0.1% formic acid) during 25 min, followed by a rapid gradient to 70% acetonitrile (with 0.1% formic acid), with a total run time
of 40 min MS⁄ MS spectra were analyzed using the Mascot search engine [46]
Plasmids, yeast strains and media The DNA fragment corresponding to Om14 (YBR230c) was amplified from a preparation of S cerevisiae cDNA by
Trang 7PCR using primers that generated in-frame restriction sites.
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 30C 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 Na2CO3and
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
sequencing, Ian Dawes for the yeast cDNA
prepar-ation, Vasyl Demchyshyn for technical assistance and
Gottfried Schatz for very many contributions to the
early phase of our work We thank Agilent
Technol-ogies for their active collaboration with the Bio21
Molecular Science and Biotechnology Institute Thanks
also to Tony Purcell and Nick Williamson for critical
reading of the manuscript
This project was supported by an Early Career
Research Grant from the University of Melbourne (to
L.B.), Australian Postgraduate Awards (to K.V.,
I.E.G and N.C.C.) and a grant from the Australian
Research Council (to T.L.)
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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_ 208072.1), from Kluyveromyces lactis (CAG98233.1), from Saccharomyces cerevisiae (NP_009789.1), from Candida glabrata (CAG58159.1) Sequences were aligned with clustalw, using version 1.81 with default parameters (http://www.ebi.ac.uk/clustalw/) Amino acid residues are colored to show chemically similar residues (pink, basic; blue, acidic; green, polar; red, nonpolar) The three predicted transmembrane seg-ments are shaded grey and numbered TM1-TM3 Asterisks denote truncated sequences; with uncon-firmed introns breaking the open-reading frame in these genome sequences
This material is available as part of the online article from http://www.blackwell-synergy.com