(Received for publication, August 23, 1995; and in revised form, November 28, 1995)
From the
We synthesized a transportable diazirine derivative of D-glucose, 3-deoxy-3,3-azi-D-glucopyranose (3-DAG),
and studied its interaction with purified human erythrocyte
facilitative glucose transporter, GLUT1. 3-DAG was rapidly transported
into human erythrocytes and their resealed ghosts in the dark via a
mercuric chloride-inhibitable mechanism and with a speed comparable
with that of 3-O-methyl-D-glucose (3-OMG). The rate
of 3-DAG transport in resealed ghosts was a saturable function of 3-DAG
concentration with an apparent K of 3.2
mM and the V
of 3.2 µmol/s/ml. D-Glucose inhibited the 3-DAG flux competitively with an
apparent K
of 11 mM.
Cytochalasin B inhibited this 3-DAG flux in a dose-dependent manner
with an estimated K
of 2.4
10
M. Cytochalasin E had no effect. These
findings clearly establish that 3-DAG is a good substrate of GLUT1. UV
irradiation of purified GLUT1 in liposomes in the presence of 3-DAG
produced a significant covalent incorporation of 3-DAG into GLUT1, and
200 mMD-glucose abolished this 3-DAG incorporation.
Analyses of trypsin and endoproteinase Lys-C digestion of
3-DAG-photolabeled GLUT1 revealed that the cleavage products
corresponding to the residues 115-183, 256-300, and
301-451 of the GLUT1 sequence were labeled by 3-DAG,
demonstrating that not only the C-terminal half but also the N-terminal
half of the transmembrane domain participate in the putative substrate
channel formation. 3-DAG may be useful in further identification of the
amino acid residues that form the substrate channel of this and other
members of the facilitative glucose transporter family.
A family of structurally related intrinsic membrane proteins
known as facilitative glucose transporters catalyzes the movement of
glucose and other selected sugars across the plasma membrane diffusion
barrier in mammalian cells(1) . Six isoforms have been
identified in this family(2) ; these include GLUT1 or
erythrocyte type(3) , GLUT2 or liver/pancreatic beta cell
type(4) , and GLUT4 or muscle/adipose cell type(5) .
Hydropathy analyses of cDNA-deduced amino acid sequences together with
the biochemical information obtained from purified GLUT1 (1, 2, 3) suggest that this protein family
shows the common transmembrane topology composed of a highly conserved,
large (amounting to approximately 50% of protein mass) transmembrane
domain, with less conserved, grossly asymmetric, cytoplasmic and
exoplasmic (nonmembrane) domains. Evidence (1, 3, 6, 7, 8) further
suggests that the transmembrane domain is made of 12 transmembrane
-helices (TMHs, (
)(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) )
and accommodates a water-filled channel through which glucose and other
substrates move (glucose pathway). The cytoplasmic domain includes a
short N-terminal segment, a large cytosolic loop between TMHs 6 and 7
(central loop), and a large C-terminal segment. The exoplasmic domain
includes a large loop between TMHs 1 and 2 bearing a single N-linked oligosaccharide moiety. The fact that
isoform-specific amino acid sequences are found almost exclusively at
the cytoplasmic and exoplasmic domains suggests that they are
responsible for the tissue-specific regulation of the transporter
functions. The conserved transmembrane domain primary structure, on the
other hand, suggests that the substrate channel is basically identical
in its structure among isoforms of this protein family.
A number of recent observations have shown that the intrinsic activity of glucose transporters in vitro can be modulated by hormones and metabolites(9, 10) . Evidence also indicates that impaired glucose transporter intrinsic activity is in part responsible for insulin resistance seen in human diabetes and obesity(11) . The identification of possible molecular defects for the reduced glucose transporter intrinsic activity in these diseased states would be facilitated once we understand how transporters work at the molecular level. Detailed information on the tertiary structure of the transporter, particularly that of the putative glucose channel, would be an essential first step toward such an understanding.
Information
currently available on the transmembrane domain structure of GLUT1, the
best studied isoform in this protein family, is minimal. Circular
dichroism studies with purified GLUT1 reconstituted in liposomes (6) have revealed that more than 80% of the protein mass is in
the -helical structure. Since the transmembrane domain accounts
for not more than 50% of the protein mass, this finding strongly
suggests that the 12 transmembrane segments are largely if not totally
-helices. For the putative glucose channel structure, Mueckler et al.(3) have first noted the possible significance
of the presence of amphipathic TMHs, which may line an aqueous channel
for glucose movement and provide hydroxyl and amide hydrogens for
hydrogen bond formation with glucose. Conspicuously lacking in the
current literature is an effort to identify such a channel residue or
residues that interacts with glucose or other substrates (substrate
binding sites) in this protein.
The identification of GLUT1 inhibitor binding sites, in contrast, has been quite successful. Photolabeling with ATB-BMPA ({2-N- [4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine})(12) , cytochalasin B (13) , and forskolin (14) revealed that all of these inhibitors label exclusively at residues within the C-terminal half of the protein. Based on these findings, Holman and his co-workers (15) have proposed a model for GLUT1 structure suggesting that the N- and C-terminal halves are two separate domains and that only the C-terminal half is directly involved in substrate binding and translocation. This model implies that the C-terminal half alone forms a putative glucose channel.
The aim of the present study
is to identify experimentally the channel-forming transmembrane
-helices in GLUT1 by affinity labeling using a photoactive
substrate analog. We synthesized a diazirine derivative of D-glucose, 3-DAG, and demonstrated that this photoreactive
glucose analog is a good substrate of GLUT1. The analog was rapidly
transported by intact human erythrocytes and their ghosts, which was
inhibited by cytochalasin B but not by cytochalasin E. 3-DAG upon UV
irradiation was covalently incorporated into purified GLUT1 in
liposomes. Analyses of the trypsin and endoproteinase Lys-C cleavage
products of 3-DAG-labeled GLUT1 clearly revealed that both the N- and
C-terminal halves were affinity-labeled by this substrate analog. Based
on these findings, we propose that the putative glucose channel is
formed between the N- and C-terminal halves of the transporter rather
than exclusively within the C-terminal half of the transmembrane
domain.
Figure 1:
The time course of 3-DAG net uptake by
human erythrocytes. Erythrocytes were suspended (20% hematocrit) in a
balanced salt solution (18) of 50 mM Tris-HCl buffer,
pH 7.4, containing no cytochalasin B (control) or 10M cytochalasin B in a final volume of 1 ml. A small
aliquot of 3-DAG from a 10 mM stock solution was quickly
introduced to give a final concentration of 0.2 mM and
incubated under gentle magnetic stirring. After each specified
incubation time, 5 ml of prechilled (ice temperature) BSS buffer
containing 2 mM HgCl
was quickly added to the cell
suspension. Cells were separated from suspension medium by
centrifugation at 7 °C, and the 3-DAG content in cell lysates was
assayed as described under ``Experimental Procedures.'' The
amounts of 3-DAG in cells (in µg per 0.1 ml of packed cells) are
plotted as a function of incubation time, for control (solid
circles) and in the presence of cytochalasin B (solid
squares). Each data point represents an average of duplicate
measurements.
Resealed human erythrocyte
ghosts were suspended in 1:10 BSS (18) at a cytocrit of
approximately 65% and incubated in the presence of 0.2 mM 3-DAG in the dark. The concentration of 3-DAG inside the ghosts
measured after 10 min of this incubation was essentially identical to
that in the medium (not illustrated). The time course of 3-DAG net exit
from these preequilibrated ghosts suspended (10% cytocrit) in 1:10 BSS
buffer was then followed in the dark at 20 °C (Fig. 2). The
3-DAG exit from ghosts was also fast, being virtually completed within
10 s. This 3-DAG exit was sensitive to the presence of cytochalasin B;
the exit rate was reduced approximately 50% by 0.2 µM cytochalasin B and more than 80% by 1 µM cytochalasin
B but not at all significantly by 10 µM cytochalasin E (Fig. 2). More systematic assessment of this inhibition of 3-DAG
exit by increasing concentrations of cytochalasin B (not illustated)
revealed an apparent K (the concentration which
produced 50% of the maximal inhibition) of 2.4
10
M for this inhibition.
Figure 2:
The effects of cytochalasins B and E on
the time course of 3-DAG efflux from resealed erythrocyte ghosts.
Ghosts were suspended (60-65% cytocrit) in 1:10 BSS buffer (18) containing 0.2 mM 3-DAG for 10 min and recovered
free of the suspension medium by centrifugation (20,000 g for 20 min in Sorvall, RC5C). The 3-DAG-loaded ghosts
(approximately 10
ghosts) were then quickly resuspended in
1 ml of 1:10 BSS buffer containing no cytochalasin B (open
circles), 0.2 µM (open triangles) or 1
µM (solid circles) cytochalasin B, 10 µM cytochalasin E (solid triangles), or 2 mM mercuric chloride (open squares) at t = 0
and incubated for specified time intervals at 20 °C with gentle
stirring. At the end of the incubation, 5 ml of prechilled (7 °C)
1:10 BSS buffer containing 2 mM HgCl
was quickly
added, and the ghosts were separated as pellets by centrifugation as
above at 7 °C. 3-DAG content was assayed for each ghost pellet as
described under ``Experimental Procedures'' and expressed in
quantity relative to that measured at t = 0 (the 3-DAG
content in ghosts incubated in the presence of 2 mM HgCl
). The data were plotted as a function of
incubation time. Each data point represents an average of duplicate
measurements.
The rate of 3-DAG transport as
a function of 3-DAG concentration was studied by measuring the initial
3-s net 3-DAG uptake by intact erythrocytes suspended in medium
containing varying concentrations (0.2-5.0 mM) of 3-DAG.
The experiments were otherwise similar to those of Fig. 1.
Analysis of the results of these initial velocity measurements
indicated that the rate of 3-DAG uptake is a simple, saturable function
of 3-DAG concentration (Fig. 3) with an apparent K (the 3-DAG concentration at which the rate of
uptake was 50% of maximal) of 3.2 mM and the V
(maximal rate of uptake) of 3.2 µmol/s/ml.
The rate of [
H]3-OMG uptake by intact
erythrocytes as a function of 3-OMG concentration measured in parallel
(not illustrated) revealed an apparent K
of 18
mM with the V
of 3.1 µmol/s/ml.
Figure 3:
Kinetic analysis of the rate of 3-DAG
uptake by human erythrocytes as a function of 3-DAG concentrations.
Cells were suspended in BSS containing specified concentrations of
3-DAG at t = 0 as in the experiments of Fig. 1.
The initial velocities for 3-DAG uptake were measured by quantitating
the first 3-s uptake of 3-DAG by cells by arresting the flux with
HgCl and separating cells free of medium by centrifugation.
The results were plotted as k(S)/v versus (S) according to the relationship, (S)/v
= K
/V
+ (S)/V
, where v and (S) are initial velocity (in µmol/s) and 3-DAG
concentration in ghosts at the start (t = 0) of the
flux measurement, respectively, K
and V
are the Michaelis-Menten constant and maximal
velocity, respectively, and k is a composite constant and
equal to 1.81
V
Each data point
represents an average of triplicate measurements whose S.D. were less
than 10%.
The rate of 3-DAG uptake by erythrocytes was competitively inhibited
by D-glucose (Fig. 4) but not by L-glucose
(not illustrated). In these experiments, erythrocytes were incubated in
BSS containing a fixed concentration (either 0.3 or 3.0 mM) of
3-DAG and an increasing concentration (0-40 mM) of D-glucose, and the rates of 3-DAG uptake were assessed by
measuring the initial 3-s 3-DAG uptake as described under
``Experimental Procedures.'' Analysis of the data (Fig. 4) shows that D-glucose inhibited the 3-DAG
uptake as a saturable function of D-glucose concentrations
with an apparent inhibition constant (D-glucose concentration
at which 3-DAG uptake was inhibited 50%; K`). Two
distinct values for K
` of 11.2 and 18.4 mM were calculated for the inhibition for the uptake measured at 0.3
and 3.0 mM 3-DAG, respectively (Fig. 4). Further
analysis of the data indicated that D-glucose increases the
apparent K
` of 3-DAG uptake without changing the V
. These findings clearly demonstrate that 3-DAG
and D-glucose compete for the same transporter, namely GLUT1.
Figure 4:
Inhibition of 3-DAG uptake by erythrocytes
by D-glucose as a function of D-glucose
concentrations. Initial velocity measurements were as described for the
experiments of Fig. 3. Two fixed concentrations of 3-DAG, 0.3
mM (solid triangles) and 3 mM (solid
circles), were used. Data were plotted also as in Fig. 3and analyzed as a competitive inhibition with one-to-one
stoichiometry of the relationship, (S)/v = K/V
{[1
+ (S)/K
] + (I)/K
}, where (I)
and K
are inhibitor concentration and the
inhibitor constant, respectively, and k is a constant equal to
0.07
V
K
/K
.
Two straight lines represent least squares linear regression
analyses and correspond to apparent inhibition constants (K
`) of 11.2 and 18.4 mM for 0.3
and 3 mM 3-DAG, respectively.
Figure 5: Photoincorporation of 3-DAG into GLUT1. Purified GLUT1 (100 µg) in 3-DAG solution (1 mM, 100 µl) was photolyzed, as described under ``Experimental Procedures,'' in the absence (open circles) or in the presence (closed circles) of 200 mMD-glucose. After washing (three times) and treating with tritiated sodium borohydride, the protein was separated by SDS-PAGE on 12% polyacrylamide gel. The gel was sliced and radioactivity plotted against slice number. These results are reproduced in two other sets of experiments.
An extensive pepsin digestion (see ``Experimental Procedures'') of 3-DAG-photolyzed GLUT1 did not cause any significant reduction in the 3-DAG incorporation of GLUT1 protein (not illustrated). Since this pepsin digestion cuts all individual TMHs free of nonmembrane segments (26) , the finding indicates that 3-DAG was incorporated into TMH residues with little incorporation into nontransmembrane domain including loops.
A mild trypsin digestion of GLUT1 after photolysis with 3-DAG followed by SDS-PAGE and protein staining (Fig. 6a) revealed the two well known tryptic fragments(23) , namely a broad faintly stained band around 30 kDa of the glycosylated N-terminal half and a sharp 19-kDa band of the C-terminal half of the protein, respectively. A broad band at around 55 kDa was rather evident (Fig. 6a), which was identified in immunoblot (not illustrated) as an undigested GLUT1 monomer. Quantitation of 3-DAG labeling after blank subtraction (Fig. 6b) revealed a significant 3-DAG incorporation to each of these protein-staining bands. For mild trypsin-treated sample, consistent with incomplete digestion revealed by protein staining (Fig. 6a), the 30- and 19-kDa tryptic fragments accounted for approximately 50% of total protein label. Although the labeling of the 19-kDa fragment was sharp and noticeable, the overall level associated over the broad 30-kDa fragment was significant; it amounted to 64 ± 9% (n = 3) of the label associated with the 19-kDa fragment in each of three independent estimations. Treatment with N-glycosidase F sharpened the broad 30-kDa band to a 21-kDa band for protein staining and 3-DAG labeling (Fig. 6, a and b). This finding clearly demonstrates that both the N- and C-terminal halves of GLUT1 are labeled by 3-DAG, although labeling is significantly greater at the C-terminal half than at the N-terminal half. It is also important to note that 3-DAG labels the protein at more than one site. This would indicate that the transport process involves physical proximity of glucose to the channel residues at multiple sites. This is entirely possible as the length of the channel approximated by the thickness of the lipid bilayer is more than 3 times that of D-glucose.
Figure 6: SDS-PAGE separation of trypsin digestion products of GLUT1 after photolysis with 3-DAG. Purified GLUT1 (300 µg of protein) was photolyzed with 1 mM 3-DAG and digested with trypsin followed by N-glycanase F, as detailed under ``Experimental Procedures.'' Samples (100 µg of original GLUT1 protein equivalent each) of digested protein were separated by SDS-PAGE using 12% polyacrylamide. Panel a, Coomassie Blue staining of high molecular weight markers (lane 1) and photolyzed samples without (lane 2) and with (lane 3) N-glycosidase F treatment and with low molecular weight markers (lane 4). Positions of undigested GLUT1 (GT) and two trypsin fragments (30 kDa, 21 kDa when deglycosylated, and 19 kDa) are shown in the margin. Panel b, gel was sliced and assayed for 3-DAG incorporation before (closed circle) and after (open circle) treatment with N-glycosidase F, as detailed under ``Experimental Procedures,'' and radioactivities in gel slices were plotted against the gel slice number. These results were reproduced in two other sets of experiments.
Digestion of purified GLUT1 with endoproteinase Lys-C produced six cleavage fragments (fragments A-F in Table 1), all of which except fragment B were separately identifiable on SDS-PAGE (Fig. 7a). These fragments were individually eluted from the gel, and their relationship to individual TMHs was established based on partial N-terminal amino acid sequence determination (Table 1). Similar experiments using 3-DAG-incorporated GLUT1 (Fig. 7b) revealed at least three major 3-DAG-labeled peaks. Electrophoretic mobilities unequivocally identified the first two peaks as the Lys-C fragments A (residues 301-451) and C (residues 118-183), respectively. Assignment of the third labeled peak to Lys-C fragments was equivocal. The mobility of this labeled peak failed to match with either of the fragments D and E, although it was significantly closer to the fragment D, indicating that the label is largely at fragment D (residues 256-300), with a slight if any label at fragment E (residues 184-225). Although exact quantitation was not possible, relative intensities of 3-DAG label among identifiable Lys-C fragments were approximately 20, 30, and 50% for the fragments A, C, and D/E, respectively. The 3-DAG incorporation to the fragments A and D, which includes TMH8-12 and TMH7, respectively, demonstrates that the C-terminal half of the protein participates in glucose channel formation. Of particular interest is the significant 3-DAG incorporation in the fragment C, the peptide corresponding to the N-terminal half of the protein containing TMHs 4 and 5. This further supports the conclusion based on the results of tryptic digestion discussed above and demonstrates that the N-terminal half of the GLUT1 transmembrane domain, more specifically TMH4 and/or TMH5, is also directly involved in glucose channel formation. The intense labeling found over the fragments D and E (which includes TMH7 and TMH6, respectively) (Table 1) is mostly due to the C-terminal half or TMH7 and only in small part, if any, due to the N-terminal half or TMH6 labeling. A broad 3-DAG label peaked at an apparent molecular mass of 25 kDa (designated as O in Fig. 7b) may represent a mixture of incompletely cut fragments whose identity is yet to be established.
Figure 7: SDS-PAGE separation of endoproteinase Lys-C digestion products of photolyzed GLUT1 with 3-DAG. Photolysis of purified GLUT1 with 3-DAG was identical to that in Fig. 6, except that 500 µg of GLUT1 protein was applied in streak. Protein molecular weight markers (Promega, low range) are indicated (from the top): carbonic anhydrase, soybean trypsin inhibitor doublet, horse heart myoglobin, lysozyme, and myoglobin F1, -2, and -3, respectively. Lys-C digestion was carried out as described under ``Experimental Procedures.'' SDS-PAGE, Coomassie Blue staining, gel slicing, and radioactivity counting were as in Fig. 6. The protein staining pattern (panel a) and the 3-DAG incorporation pattern (panel b) are shown. The results were reproduced in two other independent sets of experiments.
Definitive assignment of the 3-DAG labeling
to individual TMHs and the amino acid residues was not possible in the
present study. The findings discussed above nevertheless strongly
indicate that the N-terminal half of the transmembrane domain is also
directly involved in the channel formation in GLUT1 and argue against
the model that only the C-terminal half of the protein is directly
involved in the putative glucose transport pathway(15) . The
findings, on the other hand, are consistent with an alternative model
of GLUT1 structure, where we (32) proposed that both the N- and
C-terminal halves of GLUT1 transmembrane domain form a glucose channel
at their interface. This alternative model emphasizes required physical
dimension and solvent accessibility of the channel in GLUT1 as well as
the -domain (28) structural motifs known in other
proteins. Five amphiphilic TMHs of either 3, 4, 7, 8, and 11 or 2, 5,
7, 8, and 11 are thought to form the glucose channel in this model.
Evidence indicates that all of the isoforms of the facilitative glucose transporter family have a common transmembrane domain structure, including that of the putative glucose channel. GLUT1 of human erythrocytes is the only glucose transporter isoform currently available as a pure and functional protein with which the transmembrane domain structure can be studied. The detailed description of this protein structure would provide insight into the basic molecular and subcellular mechanisms underlying the intrinsic transport activity and its regulation of not only this isoform but probably also of other isoforms.
Detailed protein structure may be best studied by x-ray crystallography. To obtain high quality crystals of the intrinsic membrane protein such as glucose transporters, however, is extremely difficult and not likely to be forthcoming in the near future. The biochemical approach to the structural determination of GLUT1 described here, on the other hand, is quite promising although the protocol for the separation of individual transmembrane segments requires further optimization. Preliminary results obtained in our laboratory already indicate that labeled residues can be individually identified by biochemical and biophysical methods. Improved methodology on protein chemistry together with the availability of covalently reactive GLUT1 substrates such as 3-DAG, 4-DAG, and 6-DAG would allow one to map the transmembrane glucose channel structure of not only facilitative glucose transporters but also many other hexose transporters including sodium-glucose cotransporter (30) and bacterial phosphotransferase-linked hexose transporters(31) .