From the Institut für Medizinische Mikrobiologie, Medizinische Hochschule, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
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ABSTRACT |
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Nucleotide sugar transporters form a family of
distantly related membrane proteins of the Golgi apparatus and the
endoplasmic reticulum. The first transporter sequences have been
identified within the last 2 years. However, information about the
secondary and tertiary structure for these molecules has been limited
to theoretical considerations. In the present study, an
epitope-insertion approach was used to investigate the membrane
topology of the CMP-sialic acid transporter. Immunofluorescence studies
were carried out to analyze the orientation of the introduced epitopes
in semipermeabilized cells. Both an amino-terminally introduced FLAG
sequence and a carboxyl-terminal hemagglutinin tag were found to be
oriented toward the cytosol. Results obtained with CMP-sialic acid
transporter variants that contained the hemagglutinin epitope in
potential intermembrane loop structures were in good correlation with
the presence of 10 transmembrane regions. This building concept seems to be preserved also in other mammalian and nonmammalian nucleotide sugar transporters. Moreover, the functional analysis of the generated mutants demonstrated that insertions in or very close to
membrane-spanning regions inactivate the transport process, whereas
those in hydrophilic loop structures have no detectable effect on the
activity. This study points the way toward understanding
structure-function relationships of nucleotide sugar transporters.
Nucleotide sugar transporters form a family of structurally
related multimembrane-spanning proteins of the Golgi apparatus and the
endoplasmic reticulum (ER).1
Their function resides in translocating activated sugars from the
cytosol into the lumen of the ER and Golgi apparatus (1-3). Transporters therefore provide essential components of the
glycosylation pathways in eukaryotic cells (for review, see Ref. 4).
Recently, the first nucleotide sugar transporters have been identified
at the molecular level. Complementation cloning in mutant cells lacking specific nucleotide sugar transport activities identified the mammalian
transporters (Tr) for CMP-sialic acid (5, 6), UDP-galactose (UDP-Gal)
(7, 8), and UDP-N-acetylglucosamine (UDP-GlcNAc) (9),
the yeast transporters for UDP-GlcNAc (10) and UDP-Gal (11), and the
Leishmania GDP-mannose transporter (12, 13). Additional
putative nucleotide sugar transporter sequences have been identified
using sequence homologies (8, 10, 13). Surprisingly high sequence
homology has been found between the mammalian transporters for
CMP-sialic acid, UDP-Gal, and UDP-GlcNAc (8, 9), whereas the
conservation between transporters of identical specificity in different
biological kingdoms can be low (9, 10).
As mentioned above, cloning of transporters was achieved by
complementation, and the cDNAs isolated were demonstrated to
correct the mutant phenotype. Transport activity, however, has only
been proven for the murine CMP-Sia-Tr, which could be functionally expressed in Saccharomyces cerevisiae (14). Because yeast
cells lack sialic acids, this result clearly demonstrates that the
cloned cDNA in fact encodes the CMP-Sia-Tr and not an accessory
protein required in the transport process. Moreover, the canine
UDP-GlcNAc-Tr, although only 22% identical to the yeast orthologue,
has been isolated by expression cloning in the UDP-GlcNAc-Tr negative
mutant of Kluyveromyces lactis. This result allows us to
speculate that structural elements involved in specific substrate
recognition are formed via the tertiary and/or quaternary organization
of the transport proteins.
Limited information is available on the regulation of CMP-sialic acid
translocation, or on how variations in the translocation rates
influence sialylation reactions in the Golgi lumen. It is well
established that lumenal CMP stimulates CMP-sialic acid uptake by Golgi
vesicles, indicating antiport of CMP and CMP-sialic acid (15). However,
CMP-sialic acid is also translocated in the absence of CMP (14, 15),
and CMP and derivatives of CMP-sialic acid have been shown to compete
with CMP-sialic acid translocation if added onto the cytosolic side of
the Golgi membrane (16). These reagents could therefore be used to
block the sialylation of cell surfaces. Artificial reduction of cell
surface sialylation via application of CMP-sialic acid derivatives has
been demonstrated to reduce growth and metastasis of tumor cells (17).
Moreover, because sialic acids provide recognition and receptor
structures for viral and bacterial pathogens (for review, see Ref. 18), reversible inhibition of cell surface sialylation has been discussed as
a perspective to protect healthy cells against these invasive organisms.
Understanding structure-function relationships of nucleotide sugar
transporters requires knowledge of the three dimensional organization
in the plane of the lipid bilayer. The first important step toward this
aim is the determination of the membrane topology. Hydrophobicity
analyses of the nucleotide sugar transporters cloned to date suggest
between 6 and 10 transmembrane domains and the use of secondary
structure prediction algorithms proposed models with eight
transmembrane domains for both CMP-Sia-Tr and UDP-galactose transporter
(6, 7).
This study was undertaken to develop a detailed topological model of
the CMP-Sia-Tr. An epitope-insertion approach was used to map membrane
orientation. This approach, in contrast to other methods
(e.g. methods using truncated proteins fused to
reporter proteins), takes into account that even small changes in
sequences surrounding transmembrane domains can alter the membrane
topology (19) and that it is therefore important to confirm unaltered membrane orientation of the analyzed protein by the remaining activity. The results of this study strongly suggest a 10-transmembrane domain topology in which amino and carboxyl termini are oriented toward
the cytosol.
Antibodies--
Monoclonal antibody (mAb) 12CA5, directed
against the hemagglutinin (HA) epitope YPYDVPDYASL, was purchased from
Boehringer Mannheim, and mAb M5, directed against the FLAG sequence
MDYKDDDDK, was from Eastman Kodak, New Haven. Polysialic acid
(PSA)-specific mAb 735 has been described (20). A rabbit antiserum
against the catalytic domain of Cell Lines and Plasmids--
Chinese hamster ovary (CHO) mutant
8G8 had been isolated from ethylmethane sulfonate-treated CHO-K1 cells
(22) and was found to belong to the Lec2 complementation group,
comprising cells with defects in the CMP-Sia-Tr gene (5, 23). CHO cells
were maintained in Ham's F-12 medium (Seromed) supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 100 units/ml
penicillin, and 100 µg/ml streptomycin. COS cells were grown in
Dulbecco's modified Eagle's medium (Seromed) supplemented with 5%
fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Construction of Insertion and Deletion Mutants--
Mouse
CMP-Sia-Tr, with carboxyl-terminal HA tag and amino-terminal FLAG
sequence, respectively, were generated as described previously (5, 6).
Using the FLAG-tagged construct as template, overlapping extension
polymerase chain reaction (24) was used to generate two series of
insertion mutants, the N constructs (N1-N15) and the HA constructs.
For the production of N construct, primers were designed that
introduced the sequence GGATCCAACGCTAGC at selected sites of the
CMP-Sia-Tr (see Table I). This sequence, which encodes the pentapeptide
GSNAS, introduces BamHI and NheI restriction
sites and harbors a potential N-glycosylation site. The
newly introduced restriction sites were then used to produce HA
constructs by ligating a linker encoding the HA epitope (HA1-HA15). The
linker was prepared by annealing the 5'-phosphorylated oligonucleotides HA sense (5'-GATCCTACCCTTATGACGTCCCCGATTACGCCAGCCTGG-3') and HA antisense (5'-CTAGCCAGGCTGGCGTAATCGGGGACGTCATAAGGGTAG-3').
In the final constructs, the peptide sequence
GSYPYDVPDYASLAS was inserted after the amino acid residue
indicated in Table I (the epitope of mAb 12CA5 is underlined).
Construct HA16 was generated by overlap extension polymerase chain
reaction, directly introducing the HA encoding sequence (see above)
between nucleotides 270 and 271 of the CMP-Sia-Tr coding sequence. A
carboxyl-terminal truncated version of construct HA12 was prepared by
digestion with NheI, fill-in with Klenow enzyme, and
religation, resulting in HA12STOP with a stop codon immediately
downstream of the HA epitope. All constructs were verified by DNA sequencing.
Transient Transfections--
Cells were seeded at 2.5 × 105 cells per 35-mm cell culture dish or at 1.5 × 106 per 10-cm dish. For immunofluorescence analysis, cells
were seeded onto glass coverslips. Epitope-tagged CMP-Sia-Tr cDNAs
were transfected using LipofectAMINE (Life Technologies, Inc.)
following the instructions of the manufacturer. Briefly, 1 µg
cDNA was mixed with 6 µl (24 µl) LipofectAMINE in 1 ml Opti-MEM
medium (Life Technologies, Inc.) added to the cells that had been
washed twice with Opti-MEM and incubated for 6-8 h. Transfections were
stopped by adding 2 volumes of Dulbecco's modified Eagle's
medium/Ham's F-12 medium (supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin), and cells were analyzed 48 h later.
Indirect Immunofluorescence--
Cells were washed twice with
PBS, fixed in 4% paraformaldehyde for 15 min, again washed in PBS, and
incubated for 20 min in 50 mM NH4Cl in PBS to
neutralize residual paraformaldehyde. Thereafter, cells were
permeabilized with 0.2% saponin, 0.1% BSA in PBS for 15 min and
incubated with the respective primary antibody for 2 h at room
temperature or overnight at 4 °C. Antibodies used were anti-FLAG mAb
M5 (5.4 µg/ml), anti-HA mAb 12CA5 (2.6 µg/ml), and rabbit
anti-
To selectively permeabilize the plasma membrane of transfected CHO
cells, we used low concentrations of digitonin (25). Cells were fixed
in paraformaldehyde as described above and washed three times with PBS,
and the plasma membrane was permeabilized by incubating the cells for
15 min at 4 °C in 5 µg/ml digitonin, 0.3 M sucrose,
0.1 M KCl, 2.5 mM MgCl2, 1 mM EDTA, and 10 mM Hepes, pH 6.9. Thereafter,
cells were washed three times with PBS, and nonspecific binding sites
were blocked with 1% BSA in PBS at room temperature for 30 min.
Control cells were treated in the same way, but 0.1% saponin was added
to the blocking solution to achieve complete permeabilization. For
staining, cells were processed as described above, but saponin was
omitted from solutions containing primary antibodies.
Western Blotting--
Microsomal fractions were isolated from
postnuclear supernatants of transiently transfected cells by
centrifugation for 1 h at 150,000 × g. Cells or
microsomal fractions were lysed in 20 mM Tris-HCl (pH 8.0),
150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride and mixed with an equal
volume of 120 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol,
5% Immunocytochemistry--
Two days after transfection, cells were
fixed in 50% methanol/50% acetone. Polysialic acid was detected by
sequential incubation with mAb 735 (10 µg/ml) and anti-mouse
Ig-alkaline phosphate conjugate in 2% nonfat dry milk in PBS, as
described for Western blotting. Bound secondary antibodies were
revealed using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl
phosphate in alkaline phosphatase buffer.
Structure Predictions--
Putative transmembrane-spanning
regions and helix ends of CMP-Sia-Tr, human and yeast UDP-Gal-Tr and a
related putative nucleotide sugar transporter of Caenorhabditis
elegans were estimated by using the programs PredictProtein (27)
and interfacial hydrophobicity analysis (28). The murine CMP-Sia-Tr
sequence was aligned to the human UDP-Gal-Tr, the
Schizosaccharomyces pombe UDP-Gal-Tr and the related
C. elegans protein ZK370.7 using CLUSTAL (29). Helix ends
were estimated by assuming that helix ends in corresponding transmembrane domains of different transporters are identical, if the
sequences surrounding the helix ends are highly conserved. When
different results were obtained for different transporter sequences or
with different algorithms, the most plausible distribution of charged
and polar residues was fitted to the experimental results.
Amino and Carboxyl Termini of CMP-Sia-Tr Are Oriented toward the
Cytosol--
The murine wild-type CMP-Sia-Tr, with either
amino-terminal FLAG or carboxyl-terminal HA tag, was transiently
expressed in the Lec2 clone 8G8 (5), and the orientation of the epitope tags was determined by immunofluorescence using digitonin-permeabilized cells. Low concentrations of digitonin selectively permeabilize the
plasma membrane because of its higher concentration of cholesterol compared with intracellular membranes (30). Cells were simultaneously stained with M5 and polyclonal Evidence for 10 Membrane-spanning Domains in the
CMP-Sia-Tr--
The orientation of amino and carboxyl termini as
evaluated in the first experiment was in agreement with the predicted
model of the CMP-Sia-Tr (6). Therefore, we used this model to select putative intermembrane loops for the generation of insertion mutants (Fig. 3). In the first set of mutants, a
short nucleotide sequence that encodes the pentapeptide GSNAS was
introduced at various positions of the amino-terminally FLAG-tagged
murine CMP-Sia-Tr. The resulting mutants received the name N
constructs, because the inserted pentapeptide contained the consensus
motif for N-glycosylation. The foreign nucleotide sequence,
in addition, introduced unique endonuclease restriction sites, which in
a second step could be used to ligate a linker that encodes the HA
epitope consisting of the amino acid sequence GSYPYDVPDYASLAS. The
latter constructs received the name HA constructs. All insertion
mutants are summarized in Table I.
To evaluate the membrane topology of the CMP-Sia-Tr, the HA constructs
were transiently expressed in CHO 8G8 cells (5), and the orientation of
the HA epitope was determined by immunofluorescence with the mAb 12CA5
in permeable (saponin-treated) and semipermeable (digitonin-treated)
cells. Simultaneous staining with the
The transporter variants HA9, HA3, HA15, HA11, HA8, and HA12 (see Figs.
3C and 8A) were undetectable in
immunofluorescence studies with mAb 12CA5; however, expression and
localization could be displayed with the anti-FLAG mAb M5 (examples are
displayed in Fig. 5). In the case of HA9,
only few transfected cells were detectable by immunofluorescence (see
Fig. 5), and the protein was not visible in Western blot (see Fig. 7).
All other variants were stained with mAb 12CA5 and migrated with an
apparent molecular mass of 31 kDa (see Fig. 7). The inaccessibility of
the HA tags HA9, HA3, HA15, HA11, HA8, and HA12 in immunofluorescence
suggests that these epitopes are inserted into transmembrane domains,
or, as in the case of construct HA3, they are in close proximity to the
membrane. Co-localization with
Neither the HA tag of construct HA8 nor that of HA12 could be detected
by immunofluorescence; therefore, it remained unclear whether the
hydrophobic regions VII and VIII (see Fig. 3C) transverse the membrane, form integral parts of the membrane, or are tightly membrane associated. To resolve this problem, construct HA12STOP was
generated by introducing a stop codon immediately after the HA sequence
in construct HA12. Immunofluorescence was then used to search for the
HA epitope in transiently transfected cells. As shown in Fig.
6C, the HA epitope was
still not detectable, although the correctly targeted protein was
stained with anti-FLAG mAb M5 (Fig. 6, A and B),
and a protein of the expected molecular mass was developed in Western
blot with mAb 12CA5 (Fig. 6D). In contrast, the HA
constructs obtained after insertion of the tag sequence after Ser-298,
Gly-302, and Ser-309 led to inactive proteins that were not detectable
in either immunofluorescence or Western blot (data not shown). These
results argue against membrane association and promote the assumption
that the hydrophobic regions VII and VIII are integral parts of
the Golgi membrane.
Activity of Insertion Mutants--
Even small sequence changes
near membrane-spanning domains can alter the membrane topology (19).
The model deduced from the epitope insertion study was therefore
verified by testing activity of the mutants. Although loss of transport
activity does not necessarily imply a changed topology, an active
transporter strongly indicates intact membrane topology. In the
activity tests, we took advantage of the fact that CHO wild-type cells
in contrast to Lec2 mutants express polysialic acid (5), detectable
with the mAb 735 (20). mAb 735 in different experimental systems detects polysialic acid with a much higher sensitivity than lectins (e.g. Maackia amurensis agglutinin)
(5).2
Constructs were transiently transfected into 8G8 cells, and the
expression of mutant CMP-Sia-Tr was analyzed by Western blotting with
the anti-FLAG mAb M5 and the anti-HA mAb 12CA5 (Fig.
7). Bands of approximately 31 kDa were
detected in transfectants expressing mutant or wild-type CMP-Sia-Tr. A
difference in the migration behavior that could be attributed to
N-glycosylation, however, was not found in the N constructs
(Fig. 7B and data not shown). The inability of the
pentapeptide sequence to serve as glycosylation site most probably
reflects insufficient spacing between the potential N-glycosylation site and the membrane. According to Nilsson
and von Heijne (32), a minimal distance of 12-14 amino acids is required.
Transporter mutants for which the HA epitope was undetectable by
immunofluorescence (HA9, HA3, HA11, HA15, HA8, and HA12) were unable to
correct the phenotype of the PSA-negative CHO mutant 8G8 (Fig.
7A), and transfection of the corresponding N constructs led
to the same results (Fig. 7B). On the other hand, only two of the HA epitopes inserted into hydrophilic loops, namely those in
constructs HA14 and HA7, inactivated the transporter, as shown by the
absence of polysialic acid after transfection into 8G8 cells (Fig.
7A). For the mutant pair N6/HA6, a gradual diminishing of
the ability to complement the 8G8 mutant was observed with the size of
the insert. Although N6 led to the same level of PSA expression as the
wild-type construct, complementation of 8G8 cells with construct
HA6 was less efficient. The level of PSA surface expression varied
between different experiments and was strongly dependent on the
transfection efficiency (data not shown), yet it never reached the
level obtained with N6. Interestingly, constructs HA10 and HA16, which
destroy the leucine zipper motif (amino acids 56-77), produced
fully active transporters.
The Membrane Topology of the CMP-Sia-Tr--
The above experiments
strongly suggest a 10 membrane-spanning domain topology for the
CMP-Sia-Tr, but the boundaries of transmembrane helices remain
undetermined. In order to propose a model of the CMP-Sia-Tr that can be
used to determine further structural details, we used different
algorithms (see under "Experimental Procedures") to predict
possible helix ends. The methods were applied to the CMP-Sia-Tr, to the
closely related UDP-Gal-Trs from human and yeast, and to the homologous
protein from C. elegans. The tertiary structure predictions
in combination with the experimental data discussed above allow the
deduction of the CMP-Sia-Tr model shown in Fig.
8A. The predicted
transmembrane domains were further analyzed by helical wheel
projection. The projections shown in Fig. 8B demonstrate the
presence of hydrophobic and hydrophilic faces in most transmembrane
helices. Conserved amino acids are thereby concentrated in the more
hydrophilic faces. In fact, only 16% of the hydrophobic residues are
conserved, whereas 33% of the nonhydrophobic amino acids are identical
in the four transporter sequences.
Complementation cloning in Lec2 cells identified the first
mammalian nucleotide sugar transporter (5). The highly hydrophobic protein was shown to be localized in the Golgi apparatus and to span
the lipid bilayer multiple times (5, 6). Additional nucleotide sugar
transporters were cloned from different species (5, 7, 9, 10, 12).
Depending on the algorithm used to predict structural features, and
depending on the transporter analyzed, the number of predicted putative
transmembrane domains varied considerably. Therefore, an epitope
insertion study was performed in the present study to elucidate the
membrane topology of the CMP-Sia-Tr. This experimental approach has
clear advantages over other strategies used to define the arrangement
of type III membrane proteins. The modifications introduced in the
primary sequence are small. Functional activity can be retained in many mutants and indicates correct molecular organization of the protein. Using epitope insertion and indirect immunofluorescence studies, we
provide strong evidence for 10 transmembrane passages (Fig. 8) in the
CMP-Sia-Tr.
Insertions in general bear the risk of changing the topogenic activity
of the adjacent membrane-spanning domains. Therefore, the activity of
the mutated proteins was assayed and taken as an indicator for intact
membrane organization. For this reason, we measured PSA expression in
the transiently transfected Lec2 mutant 8G8.
Indirect immunofluorescence in transiently transfected 8G8 cells
demonstrated that both the amino and carboxyl are oriented to the
cytosolic side of the Golgi membrane. These data indicate an even
number of transmembrane domains for the CMP-Sia-Tr. Moreover, the
epitope localization of the constructs HA1, HA10, HA13, HA4, HA5, and
HA6 confirmed the existence of TM1, TM2, TM5, TM6, and TM7. All
constructs used in this part of the study were functionally active (see
also Refs. 5 and 6). In contrast, HA tags introduced into HA14 and HA7
inactivated transport, but targeting of the proteins to the Golgi
apparatus suggested correct folding. These constructs therefore
confirmed TM3, TM4, and TM8.
We were unable to unequivocally demonstrate that TM9 and TM10
transverse the Golgi membrane, although the hydrophobic character of
this region suggests the presence of integral membrane domains. HA tags
inserted after the amino acid residues Ser-287 (HA8) and Gln-294 (HA12,
HA12STOP) were found in Western blot but were undetectable by
immunofluorescence, and further downstream insertion of the HA tag
(after Ser-298, Gly-302, and Ser-309) resulted in unstable proteins
that were not detectable neither in Western blot nor immunofluorescence
(data not shown). If the region TM9-TM10 were not an integral part of
the membrane but associated with the cytosolic side of the Golgi, we
would have expected to see the cytosolically oriented HA tag of
HA12STOP. The inaccessibility of the HA tag in the constructs (see Fig.
6) provides strong evidence that the hydrophobic regions VII and VIII
(Fig. 3C) are integral parts of the membrane. Moreover, all
structure prediction algorithms used so far suggest two membrane
integral helices in this domains. Additional studies using different
techniques (e.g. cysteine-modified proteins, as described in
Ref. 33) are required to find out whether a part of this region is
accessible from the lumenal site of the Golgi.
It will be interesting to determine whether all nucleotide sugar
transporters exhibit a common membrane topology. The high similarity of
the hydrophobicity plots and sequence comparisons of the different
transporters support this possibility. Ten hydrophobic domains can be
clearly distinguished in the GDP-mannose transporter of
Leishmania donovani (12), are compatible with the sequences of the K. lactis UDP-GlcNAc-Tr, the related proteins from
S. cerevisiae and C. elegans (10), and are in
accordance with the primary sequence of a putative human nucleotide
sugar transporter (8). In contrast, the S. pombe UDP-Gal-Tr,
although showing a strong sequence homology to CMP-Sia-Tr, lacks the
first transmembrane domain (11). A second exception from the
10-transmembrane model is a putative nucleotide sugar transporter
(ZK370.7) identified in C. elegans. In this protein the
region corresponding to the first two transmembrane domains in
CMP-Sia-Tr is absent. So far, no functional data on the C. elegans transporter are available; however, a mutant of the
hamster CMP-Sia-Tr has been identified in our laboratory that lacks the
first two transmembrane domains and is functionally inactive (34). In
contrast to the variability observed in the amino-terminal region,
significant sequence similarity between the different transporters has
been found in the carboxyl-terminal membrane regions (6, 9, 13). Taken
together, these data suggest that the majority of nucleotide sugar
transporters are composed of 10 transmembrane domains, although some
may lack the first or the first and second membrane-spanning domain.
Some transporter proteins form dimers (35-37), and based on data
obtained with the related adenosine 3'-phosphate 5'-phosphosulfate transporter (38), it has been proposed that nucleotide sugar transporters also function as dimers (39). The idea is further supported by the facts that (i) many Golgi resident proteins form dimers (40-42), and (ii) some nucleotide sugar transporters contain a
leucine zipper motif that could be involved in dimerization (4).
Introduction of a pentapeptide sequence or a hemagglutinin epitope
between the second and third (constructs N10 and HA10) or the third and
fourth leucine residues of the leucine zipper motif (construct HA16)
did not inactivate the CMP-Sia-Tr. These mutants were able to fully
correct the Lec2 phenotype. In agreement with these data, the
simultaneous change of the second and third leucine residue of the
zipper to alanine did not interfere with Golgi targeting or transport
activity.2 Although these data cannot answer the question
on the active transport unit, they clearly demonstrate that the leucine
zipper does not play an essential role in the formation and/or
stabilization of the active transporter.
With the exception of construct HA9, the insertion mutants were
correctly targeted to the Golgi apparatus. In the case of insertions
into lumenal or cytosolic loops, this observation fits well with the
present view that mainly the transmembrane domains of Golgi resident
membrane proteins determine the targeting process (43-45). The
insertion of 15 amino acids into a membrane-spanning helix was,
however, expected to perturb correct folding of the proteins. It was
therefore interesting to see that some of these constructs were
correctly targeted. In case of HA9, both ER and Golgi localization were
observed, most probably reflecting a decreased stability of HA9
(compare the Western blot in Fig. 7A). From these results we
conclude that not mistargeting, but perturbation of functional
organization is responsible for the loss of CMP-sialic acid transport
activity in insertion mutants. The second lumenal and forth cytosolic
loop form essential parts of the molecule, because the constructs HA14,
N14, HA7, and N7 are inactive. The functional importance of the fourth
cytoplasmic loop is further supported by the high conservation between
the mammalian transporters for CMP-Sia, UDP-Gal, and UDP-GlcNAc (9).
Also involved in the architecture of the transport unit is the forth
lumenal loop. Although insertion of a pentapeptide in N6 did not
interfere with activity, the insertion of the larger HA tag (HA6) led
to a drastic reduction of the PSA signal. In contrast, insertion of the
pentapeptide or of the HA tag into the first (HA1) and third (HA4)
lumenal and the first (HA10, HA16, and HA2), second (HA13), and third (HA5) cytosolic loop did not affect CMP-Sia-Tr activity.
Biochemical studies on nucleotide sugar transport showed that the
corresponding nucleoside monophosphates, but not the free sugars, are
efficient competitive inhibitors of transport activity (2, 46). These
data provide strong evidence that the specificity of translocation is
mediated by the nucleotide moiety, suggesting a specific nucleotide
binding site. In view of these results, it seems surprising that the
recently isolated canine UDP-GlcNAc-Tr exhibits only 22% amino acid
sequence identity to its orthologue isolated from K. lactis
(10), but 40% identity to the murine CMP-Sia-Tr sequence. However,
sialic acids appeared late in evolution, and in eukaryotes, they are
restricted to chordates and echinoderms (47). Thus, the relatively high
sequence homology of the mammalian nucleotide sugar transporters might
reflect a common evolutionary origin.
Hydrophobic moment and substitution moment often point to opposite
faces of helices of type III membrane proteins, suggesting that the
less conserved hydrophobic faces interact with membrane lipids, whereas
the hydrophilic faces interact with other transmembrane domains or with
substrate molecules (48-51). Helical wheel projections revealed an
asymmetric distribution of conserved and hydrophobic amino acids for
most transmembrane helices of the CMP-Sia-Tr (Fig. 8B).
Therefore, it seems likely that the less conserved, hydrophobic faces
are oriented toward the lipid phase of the membrane and that the more
conserved hydrophilic faces interact with other transmembrane domains,
or with the nucleotide sugar. TM4 exhibits the highest degree of
conservation (50% identity with the UDP-Gal-Tr). The conserved amino
acids are equally distributed and suggest that TM4 is less exposed to
the lipid phase. With respect to the different substrate molecules of
CMP-Sia-Tr and UDP-Gal-Tr, it is plausible to speculate that the highly
conserved polar residues are involved in the interactions between
adjacent membrane domains and not in substrate recognition.
Nevertheless, cytidine and uridine are chemically similar, and the
CMP-Sia-Tr is inhibited by both CMP and UMP (2). Conserved hydrophilic
residues could therefore participate in nucleotide sugar recognition
and binding. The generation of chimeric proteins and site directed
mutations of conserved amino acid residues provide promising strategies
to resolve these questions in the future. The model developed for the
CMP-Sia-Tr in the present study will greatly facilitate these studies
and provides a basis for investigating structure function relationships of nucleotide sugar transporters in general.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase II (21) was a kind gift of Dr. K. Moremen (University of Georgia, Athens, GA). Secondary antibodies anti-mouse Ig-alkaline phosphatase-conjugate, anti-mouse Ig
(DTAF)-conjugate and anti-rabbit Ig-tetramethylrhodamine isothiocyanate conjugate were from Dianova.
-mannosidase II antiserum (1:2000) in 0.2% saponin, 0.1% BSA
in PBS. After washing four times in 0.1% BSA/PBS, cells were incubated
with anti-mouse Ig-DTAF (1:100) and anti-rabbit Ig-TRITC (1:100) for
1 h at room temperature. The incubation was stopped by washing
three times in 0.1% BSA/PBS. After a final wash in PBS, slides were
briefly rinsed in water and mounted in moviol. Samples were visualized
under a Zeiss Axiophot Epifluorescence microscope.
-mercaptoethanol, and 0.01% bromphenol blue. Lysates were
subjected to SDS-polyacrylamide gel electrophoresis and Western
blotting onto nitrocellulose as described (26). In case
chemiluminescence detection was applied, samples were transferred onto
polyvinylidene difluoride membranes (Boehringer Mannheim). Membranes
were incubated with mAb 12CA5 (2.6 µg/ml), M5 (5.4 µg/ml), or 735 (10 µg/ml), in 2% nonfat dry milk in PBS. Primary antibodies were
detected with anti-mouse Ig-alkaline phosphate conjugate (Dianova)
using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in
alkaline phosphatase buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) as substrates. Alternatively, alkaline phosphatase was detected by chemiluminescence using
disodium-3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo-[3.3.1.13,7]decan}-4-yl)phenylphosphate
(Boehringer Mannheim).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase II antibodies or with
mAb 12CA5 and polyclonal
-mannosidase II antibodies. Because the
-mannosidase II antiserum recognizes the catalytic domain of the
enzyme exclusively (31), this staining could be used to control the
integrity of Golgi membranes after permeabilization. As shown in Figs.
1A and
2A, both mAb 12CA5 and mAb M5
recognize their epitopes in digitonin-treated cells, whereas no
-mannosidase II staining was detectable (Figs. 1B and
2B). After saponin permeabilization, all antibodies reached
their epitopes, but the intensity of the staining with mAbs 12CA5 and
M5 was not increased (Figs. 1, A and C, and 2,
A and C), demonstrating that HA and FLAG epitopes were fully accessible in semipermeabilized cells. These results clearly
demonstrate that CMP-Sia-Tr is composed of an even number of
transmembrane domains, with amino and carboxyl termini oriented toward
the cytosol.
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Fig. 1.
Orientation of the carboxyl terminus of
CMP-Sia-Tr. CHO cells on glass coverslips were transfected with
HA-tagged mouse CMP-Sia-Tr. Two days after transfection, cells were
fixed in paraformaldehyde and incubated in 5 µg/ml digitonin to
selectively permeabilize the plasma membrane (A and
B) or in saponin (C and D). Cells were
then subjected to indirect immunofluorescence using anti-HA mAb 12CA5
(A and C). Simultaneously, all cells were stained
with -mannosidase II antiserum (B and D)
exclusively directed against the catalytic domain. Bound primary
antibodies were visualized using anti-mouse Ig-DTAF and anti-rabbit
Ig-TRITC conjugates. Inaccessibility of the
-mannosidase II
antiserum to its antigen confirms integrity of the Golgi membrane in
digitonin-treated cells (A and B). Bar, 20 µm.
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Fig. 2.
Orientation of the amino terminus of
CMP-Sia-Tr. CHO cells were grown on glass coverslips and
transiently transfected with the amino-terminally FLAG-tagged mouse
CMP-Sia-Tr using LipofectAMINE. Two days after transfection, cells were
fixed in paraformaldehyde and incubated in 5 µg/ml digitonin to
selectively permeabilize the plasma membrane (A and
B) or in saponin (C and D). Thereafter
cells were subjected to indirect immunofluorescence using the
anti-FLAG mAb M5 (A and C). Simultaneously,
all cells were stained with -mannosidase II antiserum
(B D). Bound primary antibodes were visualized
using anti-mouse Ig-DTAF and anti-rabbit Ig-TRITC conjugates. Again,
inaccessibility of the
-mannosidase II antiserum to its
antigen revealed that the Golgi membrane was not permeabilized by
digitonin treatment. Bar, 20 µm.
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Fig. 3.
Position of HA epitope insertions analyzed in
this study. A, hydrophobicity plot of mouse CMP-Sia-Tr
using hydrophobicity values as described (52) and a window size of nine
amino acids. B, probability of transmembrane helices of
CMP-Sia-Tr as proposed by the neural-network based program
PredictProtein (27). C, hydrophobic segments (I-VIII)
predicted to form membrane-spanning helices. Note that segments IIIa
and IIIb were previously proposed to form a single transmembrane
domain. The positions of introduced HA epitopes are indicated.
Summary of the results obtained with HA epitope-tagged CMP-Sia-Tr
analyzed in this study
-mannosidase II antiserum
served as a control for the integrity of Golgi membranes in
digitonin-treated cells. The HA epitopes inserted at amino acid
positions 38 (HA1), 109 (HA14), 165 (HA4), and 233 (HA6) could be
detected after saponin but not after digitonin treatment of the cells
(Fig. 4, 12CA5), indicating
that the tags in these constructs are part of lumenal loops. In
contrast, the HA epitopes introduced in mutants HA2, HA13, HA5, and HA7
(insertions were at amino acid positions 83, 137, 202, and 266, respectively) were detectable in both semipermeabilized and
saponin-treated cells, demonstrating cytosolic orientation. Together,
these results identified the hydrophobic regions I, II, IIIa, IIIb, IV,
V, and VI (see Fig. 3C) as membrane-spanning domains.
Moreover, the cytosolic orientation of the HA epitope in construct HA2
and the lumenal orientation of the tag in construct HA14 clearly show
that amino acids between these regions, despite of the relatively weak
hydrophobic character, span the Golgi membrane. The co-localization of
the transporter mutants with
-mannosidase II (Fig. 4, compare
third and fourth columns) indicates that Golgi
targeting was not affected.
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Fig. 4.
Orientation and localization of HA epitopes
of HA-tagged CMP-Sia-Tr. CHO cells were transiently transfected
with the HA-tagged CMP-Sia-Tr mutants (indicated at left)
and simultaneously stained for HA epitopes using mAb 12CA5 and
-mannosidase II after digitonin or saponin permeabilization, as
described in the legend to Fig. 1. Data obtained for constructs HA10
and HA16 were identical to those for HA2. Bar, 20 µm.
-mannosidase II was found for all
constructs, but HA9 was partially retained in the ER. Correct targeting
of the transporter mutants is consistent with their correct
folding.
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Fig. 5.
Localization of HA-tagged CMP-Sia-Tr.
HA-tagged CMP-Sia-Trs that were undetectable with mAb 12CA5 were
transiently transfected into CHO cells. Two days after transfection,
cells were saponin permeabilized and subjected to immunofluorescence
analysis using anti-HA mAb 12CA5 or anti-FLAG mAb M5. All cells stained
with mAb M5 were simultaneously stained with -mannosidase II
antiserum. Results similar to those for HA11 and HA3 were obtained for
constructs HA15, HA8, and HA12 (data not shown). Bar, 20 µm.
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Fig. 6.
Expression of a carboxyl-terminally
truncated CMP-Sia-Tr. A carboxyl-terminally truncated
CMP-Sia-Tr with an HA tag at amino acid position 294, followed by a
stop codon (construct HA12STOP), was expressed in CHO 8G8 cells and
analyzed by immunofluorescence as described in Fig. 5 using anti-FLAG
mAb M5 (A), -mannosidase II antiserum (B), or
anti-HA mAb 12CA5 (C). D, cell lysates of 8G8
cells expressing HA12STOP (lane 2) or transfected with the
empty vector pcDNA3 (lane 1) were analyzed by Western
blotting using mAb 12CA5. A specific band with the expected molecular
mass of approximately 28 kDa was detectable (arrowhead).
Bands visible in both lanes are due to nonspecific binding of the
primary and secondary antibodies.
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Fig. 7.
Functional analysis of HA and N
constructs. Total cell lysates and microsomal fractions were
prepared from CHO 8G8 cells transiently transfected with the indicated
HA constructs (A) and N constructs (B) or with
the empty vector pcDNA3. Samples of total cell lysates (50 µg/lane) were resolved by 7% SDS-polyacrylamide gel electrophoresis
and transferred to polyvinylidene difluoride membranes. Functional
complementation of 8G8 cells was determined by probing the membranes
with polysialic acid specific mAb 735 (anti-PSA; upper
panels in A and B). Membrane fractions (50 µg/lane) were resolved by 15% SDS-polyacrylamide gel
electrophoresis, blotted onto polyvinylidene difluoride membranes, and
stained with anti-HA mAb 12CA5 (A, lower panel) and
anti-FLAG mAb M5 (B, lower panel). Bound antibodies were
revealed using anti-mouse alkaline phosphatase conjugates followed by
chemiluminescence detection. Bands of approximately 31 kDa are visible
in all lanes. Construct HA9 could not be detected by Western blotting.
The PSA-specific signal obtained with HA6 was always reduced compared
with the other PSA signals. Note that transfection of constructs HA12
and N12 lead to very weak yet still detectable PSA signal.
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Fig. 8.
Proposed membrane topology of mouse
CMP-Sia-Tr. A, topology model of CMP-Sia-Tr as deduced
from the results in the present study. The position of HA epitopes used
to deduced this model are indicated by arrowheads.
Filled arrowheads indicate HA tags that inactivated
CMP-Sia-Tr, whereas open arrowheads mark the position of HA
tags that did not inactivate CMP-Sia-Tr. Boundaries of
membrane-spanning segments were estimated using different algorithms as
described under "Experimental Procedures." B, helical
wheel projections of transmembrane helices. Hydrophobic residues are
circled. Amino acids conserved in mouse and hamster
CMP-Sia-Tr, human UDP-Gal-Tr, yeast UDP-Gal-Tr, and C. elegans ZK370.7 are shown with open
letters.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Michael Cahill for critical remarks on the manuscript, K. Moreman for kindly providing the mannosidase II antiserum, and D. Bitter-Suermann for continuous support.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft.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.
Supported by a postdoctoral fellowship of the Deutsche Forschungsgemeinschaft.
§ To whom correspondence should be addressed. Tel.: 49-0511-532-4359; Fax: 49-0511-532-4366; E-mail: rgs{at}mikrobio.h.shuttle.de.
2 M. Eckhardt, B. Gotza, and R. Gerardy-Schahn, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; CHO, Chinese hamster ovary; Tr, transporter; CMP-Sia-Tr, CMP-sialic acid transporter; HA, hemagglutinin; mAb, monoclonal antibody; PSA, polysialic acid; TM, transmembrane domain; UDP-Gal-Tr, UDP-galactose transporter; UDP-GlcNAc-Tr, UDP-N-acetylglucosamine transporter; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DTAF, dichlorotriazinylamino fluorescein.
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