Download Membrane Topology of the Mammalian CMP

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 274, No. 13, Issue of March 26, pp. 8779 –8787, 1999
Printed in U.S.A.
Membrane Topology of the Mammalian CMP-Sialic Acid Transporter*
(Received for publication, July 24, 1998, and in revised form, December 14, 1998)
Matthias Eckhardt‡, Birgit Gotza, and Rita Gerardy-Schahn§
From the Institut für Medizinische Mikrobiologie, Medizinische Hochschule, Carl-Neuberg-Strasse 1, 30625 Hannover,
Germany
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),
* 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. This 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.: 49-0511-5324359; Fax: 49-0511-532-4366; E-mail: [email protected].
1
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.
This paper is available on line at http://www.jbc.org
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 CMPsialic 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 CMPsialic 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 CMPsialic 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
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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.
8780
Topology of the CMP-Sialic Acid Transporter
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.
EXPERIMENTAL PROCEDURES
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 a-mannosidase II antibodies or
with mAb 12CA5 and polyclonal a-mannosidase II antibodies.
Because the a-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 per-
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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 a-mannosidase II (21) was a
kind gift of Dr. K. Moremen (University of Georgia, Athens, GA). Secondary antibodies anti-mouse Ig-alkaline phosphatase-conjugate, antimouse Ig (DTAF)-conjugate and anti-rabbit Ig-tetramethylrhodamine
isothiocyanate conjugate were from Dianova.
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 mg/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 mg/ml streptomycin.
Construction of Insertion and Deletion Mutants—Mouse CMP-SiaTr, 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 59phosphorylated oligonucleotides HA sense (59-GATCCTACCCTTATGACGTCCCCGATTACGCCAGCCTGG-39) and HA antisense (59-CTAGCCAGGCTGGCGTAATCGGGGACGTCATAAGGGTAG-39). 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 3 105 cells per
35-mm cell culture dish or at 1.5 3 106 per 10-cm dish. For immunofluorescence analysis, cells were seeded onto glass coverslips. Epitopetagged CMP-Sia-Tr cDNAs were transfected using LipofectAMINE
(Life Technologies, Inc.) following the instructions of the manufacturer.
Briefly, 1 mg cDNA was mixed with 6 ml (24 ml) 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 mg/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 mg/ml), anti-HA mAb
12CA5 (2.6 mg/ml), and rabbit anti-a-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.
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 mg/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 3 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% b-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
mg/ml), M5 (5.4 mg/ml), or 735 (10 mg/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-4chloro-3-indolyl phosphate in alkaline phosphatase buffer (100 mM TrisHCl, 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-3,29-(59-chloro)tricyclo-[3.3.1.13,7]decan}-4-yl)phenylphosphate (Boehringer Mannheim).
Immunocytochemistry—Two days after transfection, cells were fixed
in 50% methanol/50% acetone. Polysialic acid was detected by sequential incubation with mAb 735 (10 mg/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.
Topology of the CMP-Sialic Acid Transporter
8781
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
mg/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
a-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 a-mannosidase II antiserum to its antigen confirms integrity of
the Golgi membrane in digitonin-treated
cells (A and B). Bar, 20 mm.
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 a-mannosidase II (Fig. 4, compare third and fourth
columns) indicates that Golgi targeting was not affected.
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 a-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.
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
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meabilization. As shown in Figs. 1A and 2A, both mAb 12CA5
and mAb M5 recognize their epitopes in digitonin-treated cells,
whereas no a-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.
Evidence for 10 Membrane-spanning Domains in the CMPSia-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 (saponintreated) and semipermeable (digitonin-treated) cells. Simultaneous staining with the a-mannosidase II antiserum served as
a control for the integrity of Golgi membranes in digitonintreated 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 saponintreated cells, demonstrating cytosolic orientation. Together,
these results identified the hydrophobic regions I, II, IIIa, IIIb,
8782
Topology of the CMP-Sialic Acid Transporter
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 mg/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 a-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 a-mannosidase
II antiserum to its antigen revealed that
the Golgi membrane was not permeabilized by digitonin treatment. Bar, 20 mm.
Construct
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.
N1
HA1
N10
HA10
HA16
N2
HA2
N9
HA9
N14
HA14
N3
HA3
N15
HA15
N11
HA11
N13
HA13
N4
HA4
N5
HA5
N6
HA6
N7
HA7
N8
HA8
N12
HA12
HA12STOP
Insertion after
amino acid
HA epitope
orientation
Ala-38
Lumenal
Gly-68
Ala-75
Cytosolic
Cytosolic
Gly-83
Cytosolic
a
Asn-102
ND
Leu-109
Lumenal
Ala-114
ND
Gln-118
ND
Lys-124
ND
Asn-137
Cytosolic
Ala-165
Lumenal
Ser-202
Cytosolic
Ile-233
Lumenal
Tyr-266
Cytosolic
Ser-287
ND
Gln-294
Gln-294
ND
ND
Complementation
of CHO 8G8 cells
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yesb
No
No
No
No
No
No
No
Subcellular
localization
Golgi
Golgi
Golgi
Golgi
Golgi 1 ER
Golgi 1 ER
Golgi
Golgi
Golgi
Golgi
Golgi
Golgi
Golgi
Golgi
Golgi
Golgi
Golgi
Golgi
a
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
2
M. Eckhardt, B. Gotza, and R. Gerardy-Schahn, unpublished
observations.
ND, not detectable.
complementation of 8G8 cells by transient transfection of construct
HA6 was less efficient and varied between different experiments,
strongly depending on transfection efficiency.
b
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
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TABLE I
Summary of the results obtained with HA epitope-tagged CMP-Sia-Tr
analyzed in this study
Topology of the CMP-Sialic Acid Transporter
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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 a-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 mm.
8784
Topology of the CMP-Sialic Acid Transporter
FIG. 6. Expression of a carboxyl-terminally truncated CMPSia-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),
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.
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
Downloaded from www.jbc.org at MHH-BIBLIOTHEK, on October 11, 2012
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 a-mannosidase II antiserum. Results similar
to those for HA11 and HA3 were obtained for constructs HA15, HA8, and HA12 (data not shown). Bar, 20 mm.
Topology of the CMP-Sialic Acid Transporter
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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.
DISCUSSION
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;
Downloaded from www.jbc.org at MHH-BIBLIOTHEK, on October 11, 2012
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 mg/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 mg/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.
8786
Topology of the CMP-Sialic Acid Transporter
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 39-phosphate 59phosphosulfate 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 resi-
due 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
Downloaded from www.jbc.org at MHH-BIBLIOTHEK, on October 11, 2012
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.
Topology of the CMP-Sialic Acid Transporter
Acknowledgments—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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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.
8787