From the Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden
Received for publication, January 7, 2003
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ABSTRACT |
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We provide experimentally based topology models
for 37 integral membrane proteins from Saccharomyces
cerevisiae. A C-terminal fusion to a dual Suc2/His4C
topology reporter has been used to determine the location of the C
terminus of each protein relative to the endoplasmic reticulum
membrane, and this information is used in conjunction with theoretical
topology prediction methods to arrive at a final topology model.
We propose that this approach may be used to produce reliable
topology models on a proteome-wide scale.
The topology of a membrane protein, i.e. a
specification of its transmembrane segments and their in/out
orientation relative to the membrane, is a basic structural
characteristic that is a very powerful guide to experimental studies
when no three-dimensional structure is available. Most proteins
for which experimentally derived topology models exist are from
bacteria; only a handful of topologies are known for yeast membrane
proteins. As an example, the widely used MPtopo data base (1) currently
holds a total of 92 experimentally determined topology models, of which
only 3 are for yeast proteins.
The preponderance of known topologies for bacterial proteins is mainly
a result of the relative ease with which they can be determined
experimentally by making series of C-terminal-truncated versions of the
target protein fused to topology reporters such as PhoA, LacZ, Bla, or
green fluorescent protein (2, 3). This approach often yields
clear-cut results, and reliable topology models can be proposed.
Although a couple of topology reporters have also been developed for
the yeast Saccharomyces cerevisiae (4), they seem to yield
less definitive results than can be obtained in bacterial systems (5)
and have not been much used.
We have recently shown that highly reliable topology predictions can be
obtained for a subset of membrane proteins for which many different
topology prediction methods (five in our case) give the same prediction
(6, 7). When some limited experimental information (such as the in/out
location of the C terminus of a protein) is available, consensus
predictions are even more useful, and a combination of C-terminal
reporter fusions and consensus predictions has been shown to make it
possible to rapidly produce reliable topology models for
Escherichia coli inner membrane proteins (3). We have
further shown that this basic approach can be even more widely applied
by using the experimentally determined location of the C terminus as a
constraint on the topology predictions produced by the TMHMM
program (8).
Considering that topology mapping based on reporter fusions to
truncated target proteins may be problematic in yeast, it appeared to
us that approaches based on a combination of C-terminal fusions to the
full-length protein, which should be minimally disruptive to the
structure of the proteins, and theoretical topology prediction should
be particularly useful for yeast membrane proteins. In this pilot
study, we report results for C-terminal reporter fusions to 40 S. cerevisiae membrane proteins. We find that only one of the 40 proteins that we cloned initially cannot be expressed using either of
two vectors that we have tried, that the dual Suc2/His4C reporter (9)
that we have used yields consistent results for 37 of the 39 expressed
proteins, and that the location of the C terminus as predicted by our
consensus method (6) is correct for 31 proteins of these 37. One of the
two proteins for which the experimental results are inconsistent is a
mitochondrial protein. We have also used the experimentally determined
C-terminal locations to constrain the predictions from the TMHMM
method, and we report TMHMM reliability scores for all proposed
topology models. Our results suggest that large-scale topology mapping
strategies where limited but reliable experimental information is
combined with topology prediction will be successful in yeast.
Construction of Plasmids--
All plasmids were
constructed by homologous recombination
(10). Plasmid pJK90,1 which contains the
OST4 gene fused to three hemagglutinin
(HA)2 epitopes, a part of the
SUC2 gene, and the HIS4 gene, was treated with
SmaI to linearize the vector between the end of TPI promoter and the start codon of OST4. The 5'-end homologous
recombination region was selected to match the 3'-end of
SmaI-digested pJK90, and the 3'-end homologous region was
chosen to match the linker between the end of OST4 and the
start of the HA sequence. Each homologous region comprised 35 nucleotides, (5'-AGGTGGTTTGTTACGCATGCAAGCTTGATATCGAA-3' and
5'-GATGGTCTAGAGGTGTAACCACTTGAGTTCTTAGG-3'). A gene of interest was
amplified by PCR using genomic DNA as a template and two primers, a
5'-end primer complementing the start codon of the gene with the
homologous region sequence and a 3'-end primer complementing the end of
the gene excluding the stop codon with the homologous region sequence.
Genomic DNA was isolated as described (11) from W303-1a (MATa,
ade2, can1, his3, leu2, trp1, ura3) and from W303-1
For construction of plasmids with an inducible Gal promoter, the
fragment carrying the gene, HA epitope, SUC2, and
HIS4C was amplified by PCR using pJK92(YEL059C) or
pJK92(YJL028W) as a template. The two primers used in this PCR carried
the homologous region sequences with the EcoRI-digested
424GALS (ATCC, Manassas, VA). The PCR product and the linearized
424GALS plasmid were transformed into strain STY50, and transformants
were selected on Preparation of Whole-cell Lysates--
Yeast transformants
carrying TPI promoter plasmids were grown to OD600 0.8 to 1 in 10 ml of SD Deglycosylation by Endo H Digestion--
Whole-cell lysates were
supplemented with a final concentration of 80 mM potassium
acetate, pH 5.6, and 2 µl of Endo H (1 unit/200 µl, Roche Molecular
Biochemicals) was added. Samples were incubated at 37 °C for 1 to
2 h. Mock samples were treated and incubated in the same way but
without Endo H.
Western Blot Analysis--
Solublized proteins were separated on
7.5% SDS-polyacrylamide gels, transferred onto nitrocellulose
membranes, and probed with anti-HA antibody (Babco, Richmond, CA).
Growth Assay--
Transfomants carrying each fusion construct
were streaked on SD Computational Methods--
All predicted S. cerevisiae open reading frames (15) were downloaded from
genome-ftp.stanford.edu (version June 29, 2001). TMHMM1.0 (16) was used
to identify putative membrane proteins with a minimum of two predicted
transmembrane helices. From this set, 55 proteins were selected for
which five different topology prediction methods, TMHMM1.0, HMMTOP2.0
(17, 18), MEMSAT1.8 (19), PHD2.1 (20), and TOPPRED1.0 (21, 22), all
gave the same predicted topology. Three genes carrying introns
(YDR376W, YML052W, YMR292W) were removed from this set, as were seven
genes annotated as questionable open reading frames (YCL023C, YDR526C, YFL032W, YGL024W, YGL204C, YGR228W, YNL266W). A gene encoding a known
mitochondrial protein (Q0275) was also excluded. The remaining 44 genes
were cloned into the expression vectors described above.
The 37 proteins for which the location of the C terminus could be
determined experimentally (Table I) were further analyzed using
a new version of TMHMM (8) that calculates a reliability score for the
predicted topology and also allows any part of the topology to be fixed
to a given location a priori. The experimentally determined
C-terminal locations were used as constraints in these predictions.
Selection of Target Proteins--
To select S. cerevisiae membrane proteins for this study, we first searched all
predicted open reading frames in the yeast genome (15) for membrane
proteins for which five prediction methods (TOPPRED, TMHMM, HMMTOP,
MEMSAT, and PHD) all give the same predicted topology. From our
previous work (6, 7), we anticipated that the predicted topologies
should be correct for a high proportion of these proteins. We further
required that the TMHMM method predict at least two transmembrane
helices in each protein because currently available bioinformatics
tools cannot reliably distinguish between N-terminal signal-anchor
sequences and cleavable signal peptides and thus may mistakenly
identify secreted proteins as single-spanning, N-terminal anchored
membrane proteins. This initial screen produced a list of 55 proteins.
Three genes carrying introns (YDR376W, YML052W, YMR292W) were excluded
from the original list, as were seven genes annotated as questionable
open reading frames (YCL023C, YDR526C, YFL032W, YGL024W, YGL204C,
YGR228W, YNL266W). A gene encoding a known mitochondrial protein
(Q0275) was excluded because the glycosylation assay cannot be used for
mitochondrial proteins. Five additional proteins could not be analyzed.
YNL323W was not amplified by PCR, the cloned sequence of YOL137W turned
out to be different from the expected sequence, the cloned sequences of
YDL196W and YNL101W contained frameshifts relative to the published
sequences, and protein expression of YJL028W was not detected. We
successfully made and expressed C-terminal reporter fusions to the
remaining 39 proteins (Table I).
Experimental Determination of C-terminal Locations--
For this
study, we chose a 125-kDa dual Suc2/His4C topology reporter (4) to
determine the location of a protein's C terminus in either the cytosol
or the endoplasmic reticulum lumen (9, 12). The histidinol
dehydrogenase activity of the His4C moiety converts histidinol to
histidine only when it is localized in cytosol. Thus, only cells
expressing fusion proteins with the reporter domain in the cytosol can
grow on histidine-free media supplemented with histidinol. The part of
the SUC2 gene that is present in the reporter encodes a
segment of invertase containing eight N-glycosylation
acceptor sites. When this domain is localized in the lumen of the
endoplasmic reticulum, the fusion protein becomes heavily glycosylated.
The cytosolic/non-cytosolic location of the C terminus of each of the
39 Suc2/His4C fusion proteins was determined by Endo H treatment (to
identify a glycosylated, lumenally
oriented reporter) Fig. 1 and growth on histidine-free media
containing histidinol (to identify a cytosolically oriented reporter),
Fig. 2. We did not observe any general
growth defects of the yeast transformants carrying these fusion
constructs, indicating that the addition of the reporter domain to the
target proteins had no obvious harmful effects.
For 35 of the 39 fusion proteins, the results from the glycosylation
and histidinol growth assays were entirely consistent (Table I). Some
of these proteins are known to be localized to the membranes of
secretory organelles and the plasma membrane, but most have no known
localization or function annotated in the Saccharomyces
genome data base (14) or in MIPS (23). Because these 35 fusion proteins
were either glycosylated or had histidinol dehydrogenase activity, it
is reasonable to assume that their natural locations are in the
membranes along the secretory pathway, although we cannot completely
rule out that some of the unglycosylated proteins are located in
mitochondria with their C terminus facing either the cytosol or the
intermembrane space.
The initial results from the glycosylation and histidinol growth assays
were inconsistent for four proteins. Growth on histidinol was seen for
YGR290W, despite the fact it was efficiently glycosylated. A small
amount of unglycosylated protein possibly representing molecules where
the reporter is cytosolically oriented was evident, however, and given
the high level of expression seen for this protein this may be enough
to allow growth on histidinol. We thus conclude that YGR290W has its C
terminus in the endoplasmic reticulum lumen. We further found that
YEL059W was not expressed from the constitutive TPI promoter. However,
a low level of expression was seen when the inducible Gal promoter was
used, Fig. 2. The fusion protein was sensitive to Endo H digestion,
indicating that the C terminus of the protein was glycosylated and
located in the lumen of the endoplasmic reticulum. Because YEL059W was
only expressed from the inducible Gal promoter, growth on histidinol could not be assayed.
Finally, in the case of YKR065C and YER185W, the fusion proteins were
expressed, but neither became glycosylated nor allowed growth on
histidinol. We considered that a possible explanation for this observation might be that the proteins are localized to
mitochondria with their C termini in the matrix space. YKR065C is
strongly predicted to have an N-terminal mitochondrial targeting peptide both by TargetP (24) and a predictor specifically developed for
yeast proteins (25). Indeed, YKR065C has recently been identified as a
mitochondrial inner membrane protein with a cleavable, matrix-targeting presequence.3 The location of
YER185W is so far unknown.
We also roughly estimated the relative expression levels based on
Western blotting with HA antibodies (Table I). It appears that small
fusion proteins with few transmembrane helices tend to be better
expressed than large proteins, but the correlation is not very strong.
Topology Models--
As shown in Table I, the consensus
predictions for the location of the C termini of the 37 proteins for
which this could be deduced from the fusion protein data matched the
experimental results in 31 cases (84%). Thus, for six proteins the
consensus prediction does not yield a good topology model.
As a more direct way of integrating the experimental results into the
final predictions, we took advantage of a recent improvement to the
TMHMM program that allows predictions to be constrained by experimental
information (8). This new version of TMHMM also calculates a
reliability score for each prediction that correlates strongly with
prediction accuracy. The TMHMM results with inclusion of the
experimentally determined C-terminal location are shown in Table I,
together with the corresponding reliability score and the estimated
probability that the prediction is correct (the "expected
accuracy").
In contrast to the situation for bacterial inner membrane
proteins, experimentally derived topology models are available for only
a handful of yeast membrane proteins. This lack of data is aggravated
by the fact that theoretical topology prediction methods seem to
perform less well on yeast membrane proteins than on both bacterial and
mammalian ones (8).4
In this study, we have applied and extended a strategy initially
proposed for bacterial inner membrane proteins (3) to a set of 39 predicted membrane proteins from the yeast S. cerevisiae. The strategy is based on the premise that reliable topology models can
be produced rapidly by combining limited experimental information with
topology predictions. The experimental information generated is the
cytosolic/non-cytosolic location of the C terminus of the target
proteins, and we show that this can be easily and reliably obtained by
fusion of the full-length protein to a C-terminal, dual topology
reporter (9) composed of a hemagglutinin tag for immunodetection, a
part of Suc2p that contains eight acceptor sites for
N-linked glycosylation, and the His4p enzyme that converts histidinol to histidine. Because N-linked glycosylation can
only be carried out in the endoplasmic reticulum lumen and histidinol cannot be transported into this compartment, the
cytoplasmic/non-cytoplasmic location of the reporter (and thus of the C
terminus of the target protein) is easily assayed by checking whether
Endo H can digest any N-linked glycans and whether
his4 cells expressing the target protein-reporter fusion can
grow on histidinol-containing media lacking histidine.
38 of the 39 fusion proteins could be expressed from the constitutive
TPI promoter in amounts sufficient for analysis; one protein could be
expressed only from the inducible Gal4 promoter (which precludes use of
the histidinol growth assay). Only one protein that was included in our
initial set could not be expressed from either promoter. It thus
appears that nearly all membrane proteins in S. cerevisiae
can be analyzed by our procedure.
The results from the two topology assays were entirely consistent for
35 of the 39 proteins, and the location of the reporter could be
reliably inferred also for 2 of the remaining 4. In only two cases did
we fail to observe both glycosylation and growth on histidinol; a
possible explanation is that these proteins are not targeted to the
endoplasmic reticulum but rather are imported into the mitochondrial
inner membrane and have their C termini in the matrix space. In fact,
one of the two proteins (YKR065C) has recently been identified as being
located in the inner mitochondrial membrane,5 although its
membrane topology is not yet known.
When combined with theoretical predictions, the experimentally mapped
C-terminal locations allow us to propose what we consider are reliable
topology models for 37 yeast proteins for which no such information was
previously available. To this end, we have used two approaches.
First, all our target proteins were selected from the full set of
predicted yeast open reading frames in such a way that five different
topology prediction methods all gave the same prediction for each
protein. We have previously shown that such consensus predictions are
highly reliable for bacterial inner membrane proteins (6, 7). The
experimentally determined C-terminal location was the same as the
predicted one for 31 of the 37 proteins, and we thus regard the
topology models for these proteins as very likely to be correct.
Second, we constrained the TMHMM predictions by fixing the C-terminal
end of each protein to the experimentally determined location, because
this is known to substantially increase the prediction accuracy (8). We
further calculated a reliability score for each predicted topology,
both without and with a fixed C-terminal location, Table I. Because
there is an approximately linear relationship between the reliability
score and the probability that a particular prediction is correct (8),
such probability values (expected accuracy) were also
calculated. Most of the 37 proteins have high scores compared with the
score distribution calculated for all predicted S. cerevisiae membrane proteins (8) (data not shown). This was
expected, because the proteins in our set were selected based on the
requirement that five different topology prediction methods should all
give the same predicted topology. We also note that the changes in the
reliability score for the 37 proteins seen after the inclusion of the
experimentally determined C-terminal location in the prediction have a
distribution that is very similar to the one derived for the much
larger set of bacterial inner membrane proteins analyzed previously (8) (data not shown).
It is interesting to compare the 37 proteins studied here with TMHMM
predictions for the whole S. cerevisiae membrane proteome (16). The overall distribution of proteins with different numbers of
transmembrane helices is roughly the same for the set of 37 proteins
and the whole proteome, with peaks at 2 helices and 10-12 helices.
Proteins with an even number of predicted transmembrane helices are
1.8-fold more numerous than proteins with an odd number of helices
among the 37 proteins and are 1.7-fold more numerous in the whole
proteome (excluding the single-spanning proteins). There are 1.8 times
more proteins with Cin as compared with Cout orientation in our set (1.7 times more in the whole proteome) and 4.3 times more proteins that are predicted to have Nin as compared with Nout orientation in our set (1.6 times more
in the whole proteome). The proteins analyzed here thus seem to
represent a rough cross-section of the whole proteome, except that
their topologies are easier to predict and have higher TMHMM
reliability scores than the proteome as a whole.
In summary, we have shown that reliable topology models for S. cerevisiae membrane proteins can be produced on a reasonably large
scale by a combination of C-terminal reporter fusion analysis and
theoretical prediction. This approach not only reduces the experimental
efforts required but also avoids the pitfalls inherent to fusions
between a truncated target protein and topology reporters.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(MAT
, ade2, can1, his3, leu2, trp1, ura3). A yeast strain STY 50 (MATa, his4-401, leu2, -3, and -112, trp1-1, ura3-52, HOL1-1, SUC2::LEU2) (12) was
transformed with the linearized pJK90 vector and the PCR product
carrying the gene of interest flanked by the homologous region
sequences. Transformation was carried out by the lithium acetate
protocol (13). Transformants were selected on synthetic medium lacking
uracil (SD
Ura). Plasmids were isolated and verified by PCR
analysis and DNA sequencing. Plasmids were named as pJK92 (gene
name) using gene names from the Saccharomyces genome
data base (14).
Trp plates. The correct construction of the plasmid
was confirmed by yeast colony PCR.
Ura. Harvested cell pellets were washed with 5 ml of
dH2O and left at
20 °C for at least 1 h. Frozen
cells were resuspended in 200 µl of SDS sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5%
-mercaptoethanol, 0.5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, protease inhibitor mixture (Roche
Molecular Biochemicals), 0.0025% bromphenol blue), incubated at
60 °C for 10 to 15 min and centrifuged for 10 min at 13,000 rpm in
an Eppendorf microfuge. Soluble fractions were transferred to new tubes
and subjected to Endo H digestion. Transformants carrying the GALS
promoter were grown to OD600 1 to 2 in 5 ml of
Trp
media. Cells were harvested by centrifugation and diluted to
4-fold with
Trp media supplemented with galactose instead of glucose
as carbon source and grown for 5 h at 30 °C. Cell lysates were
prepared as described above.
Ura medium lacking histidine but containing 6 mM histidinol. Plates were incubated at 30 °C for 3 to 4 days.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Summary of the results for the 39 proteins analyzed in this study
View larger version (34K):
[in a new window]
Fig. 1.
Endo H treatment of the 39 fusion proteins
analyzed in this study. Proteins were expressed as detailed under
"Materials and Methods," and the samples were either treated (+) or
not treated ( ) with Endo H to remove N-linked glycans.
After separation by SDS-PAGE, gels were blotted with an anti-HA
antibody. Proteins for which Endo H treatment results in a reduction of
the Mw are indicated by underlining.
View larger version (77K):
[in a new window]
Fig. 2.
Growth of his4 cells expressing
the 39 fusion proteins analyzed in this study on a medium lacking
histidine but including histidinol. Strains that grow well are
indicated by underlining.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Johan Nilsson (Stockholm Bioinformatics Center) for bioinformatics analyses.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Research Council and the Swedish Cancer Foundation (to G. v. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 46-8-16-25-90;
Fax: 46-8-15-36-79; E-mail: gunnar@dbb.su.se.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M300163200
1 Kim et al., submitted for publication.
3 N. Pfanner, personal communication.
4 K. Melén, A. Krogh, and G. von Heijne (2003) J. Mol. Biol., in press.
5 N. Pfanner, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
HA, hemagglutinin;
Endo H, endo--N-acetylglucosaminidase;
SD
Ura, synthetic medium
lacking uracil.
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