From the
A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K),
has been identified among a large group of evolutionarily related
membrane proteins involved in the transport of small molecules across
the membrane. To determine the importance of this motif within the
lactose permease of Escherichia coli, a total of 28
site-directed mutations at the conserved first, fifth, sixth, eighth,
ninth, and tenth positions were analyzed. A dramatic inhibition of
activity was observed with all bulky mutations at the first-position
glycine. Based on these results, together with sequence comparisons
within the superfamily, it seems likely that small side chain volume
(and possibly high
An important mechanism for the uptake of solutes by living cells
involves membrane proteins known as cation/substrate cotransporters or
symporters. These types of transport systems are widely found in many
organisms including bacteria(1, 2) ,
fungi(3, 4) , plant(5, 6) , and animal
cells(7, 8) . During uptake, symporters simultaneously
couple the inwardly directed cation electrochemical gradient to the
transport of solute so that secondary active transport can be achieved
with regard to the solute(9, 10) . The lactose permease
of Escherichia coli has provided a model system in which to
investigate secondary active transport(11, 12) . This
protein is found in the E. coli cytoplasmic membrane and
couples the transport of H
Recently,
analyses of sequences among a variety of transporters has led to the
conclusion that a large and diverse group of solute transporters are
evolutionarily related to one
another(18, 19, 20) . At the functional level,
this superfamily includes proteins that transport a wide array of
substrates such as sugars, Kreb's cycle intermediates, and
antibiotics. For example, the lactose permease of E. coli, the
glucose transporter of red blood cells, and bacterial tetracycline
exporters are members. Mechanistically, this group is rather variable
and includes proteins that catalyze uniport, symport,
or antiport. This superfamily has been referred to as the
USA
When
comparing the sequences among distant relatives within the USA
superfamily, it is frequently the case that relatively little overall
homology is observed. Therefore, the superfamily has been divided into
several groups (see Refs. 19 and 20). It is extraordinarily striking,
however, that although members within different groups may bear little
overall sequence homology, a motif,
GXXXD(R/K)XGR(R/K), has been conserved throughout the
entire superfamily(19) . Within secondary structural models,
this peptide sequence is found in the hydrophilic loop between
transmembrane segments 2 and 3, and repeated again between
transmembrane segments 8 and 9. Although some variability is found
within this motif, it has been fairly well conserved among all members
of the superfamily. Indeed, it would appear that the presence of this
sequence within loop 2/3 and 8/9 in a membrane protein is indicative of
its appropriate placement within the superfamily. Upon careful
inspection of both loop 2/3 and 8/9 among more members of the
superfamily, it seems reasonable to suggest that the motif is D/E at
the fifth position and R/K at the ninth position.
Only a few
previous studies have involved perturbations in the structure within
the loop 2/3 motif of the lactose permease. McKenna et al.(23) inserted two or six histidine residues between Leu-72
and Arg-73 within the loop 2/3 motif. These insertional mutants
exhibited a marked defect in the downhill and uphill accumulation of
lactose. Very recently, the role of glycine residues within the lactose
permease has been investigated(24) . Even though none of the
glycine residues within the permease were found to be essential for
sugar transport, bulky substitutions at the glycine in the first
position of loop 2/3 were found to be inhibitory. Finally, Wrubel et al. (25) examined the ability of portions of the lactose
permease to be synthesized independently of each other and then
reassemble into a functional permease. A mutant lacking amino acids 4
through 69 was coexpressed with a truncated form of the permease
encoding amino acids 1 through 71. The results showed that these two
pieces of the lactose permease were able to reassemble and produce a
functional protein.
More detailed analyses of amino acids within
loop 2/3 have been conducted on the tetracycline antiporter of E.
coli (26-28). With regard to the conserved charged residues,
it has been found that the negatively charged residue at the fifth
position is critical for transport function. Of the three basic
residues, only the one at the ninth position in the motif appeared to
be important. The conserved glycines at the first and eighth positions
were both found to be important for function with the first glycine
being significantly more critical than the eighth. In the current
study, a similar approach has been undertaken to examine the functional
significance of the conserved amino acids within the loop 2/3 motif of
the lactose permease.
Plasmid DNA was isolated by
the NaOH-sodium dodecyl sulfate method (29) and introduced into
the appropriate bacterial strain by the CaCl
Stock cultures of cells
were grown in YT media (31) supplemented with tetracycline (0.01
mg/ml). For transport assays, cells were grown to midlog phase in YT
media containing tetracycline (0.005 mg/ml) and 0.25 mM isopropylthiogalactoside to induce the synthesis of the lactose
permease.
To determine if these mutations affect permease
levels of expression, the wild-type and mutant strains were analyzed by
Western blotting and densitometry. As shown in , the
majority of the mutants showed levels that were similar to the
wild-type strain. In most cases, it seems likely the variability among
the mutants is due to inaccuracies in this technique rather than actual
differences in the amount of permease. However, it was striking that
both of the aspartate substitutions at positions 64 and 71 showed very
low levels of permease proteins. Therefore, these mutants were not
analyzed with regard to transport activity.
As expected from the results of , most of the
first position mutants were very defective in both the downhill and
uphill transport of [
Very different results were obtained at the eighth-position glycine.
With regard to downhill lactose transport, all of the site-directed
mutants at this position were observed to have transport levels that
are similar to the wild-type strain (see I). These results
indicate that, under the conditions of our transport assays (37 °C,
pH 7.0), a glycine at the eighth position is not particularly necessary
for transport activity per se. Likewise, Gly-71 does not appear
essential for the proper insertion and/or stability of the lactose
permease since the levels of expression of the position 71 mutants were
found to be similar to wild-type (see ). These observations
seem particularly surprising considering the extremely high degree of
conservation that occurs at the eighth position. However, it remains a
possibility that an eighth-position glycine may be important for
transport activity and/or expression under conditions that are
different from those described in this study. Also, it should be
pointed out that some of the mutants, such as Cys-71, Pro-71, and
Leu-71, exhibited uphill accumulation levels that were less than 40%
that of the wild-type value indicating that substitutions at this
position can affect the transport process.
The acidic residue at the
fifth position appears to be critical for maximal transport activity.
As shown in I, all of the mutants at this position were
found to have less than 5% of the downhill rate of lactose transport.
The results obtained with the Glu-68 mutant seem particularly striking
because it retains the negative carboxyl group and a glutamate is
commonly found at the fifth position in the analogous loop 8/9 motif.
Nevertheless, the results obtained with the lactose permease indicate a
rather strict requirement for an aspartate residue at position 68.
Finally, the role of the basic residues was examined by making
single changes at all three basic residues. None of the basic residues
were found to be required for normal downhill transport function. These
results are quite different from those obtained with the tetracycline
antiporter where it was found that the basic residue at the ninth
position (analogous to Arg-73 in the lactose permease) is essential for
transport activity(27) . Mutations at the basic residues in the
lactose permease were shown to have only moderate defects in their
uphill accumulation ranging from 50% for the Gly-69 strain to 83% for
the Gly-73 mutant (see I).
The conservation of a motif within a large superfamily is
indicative of an important functional and/or structural role. With
regard to the lactose permease, the experiments described in this study
support this notion. The most dramatic results were obtained from
mutations at the first-position glycine and the fifth-position
aspartate. A variety of single amino acid substitutions were shown to
result in substantial or complete loss of transport activity. By
comparison, the eighth-position glycine and the three basic residues
seem to be less essential although many single mutations at these
locations showed significant decreases in the uphill accumulation of
lactose. However, the possibility should be considered that these
residues may be important for transport activity and/or expression
under conditions that are different from the current study.
The role
of glycine residues within loop 2/3 may be related to different
features of the glycine side chain. In particular, this side chain has
the smallest volume and exhibits a high
At the eighth
position, the role of the glycine side chain remains unclear. Mutations
with large side chains and/or low
All
substitutions of the aspartate at position 68, including the
conservative glutamate substitution, produced permeases with
substantially lower levels of transport activity. However, it is clear
that an acidic residue at position 68 is not essential for sugar
transport. The Thr-68, Tyr-68, Ser-68, and Ala-68 mutants were able to
catalyze the downhill and uphill accumulation of sugars to a low, but
significant level. Likewise, an acidic residue at this position does
not appear to be necessary for cation recognition since the
nonionizable Ala-68 and Thr-68 mutants were directly shown to transport
H
Since
the GXXX(D/E)(R/K)XG(R/K)(R/K) motif is conserved
among proteins that transport a variety of different kinds of solutes,
it seems unlikely that it is directly involved with solute recognition.
Instead of substrate binding, alternative roles for the
GXXX(D/E)(R/K)XG(R/K)(R/K) motif can be postulated.
For example, since this motif is found along the cytoplasmic domain of
transporters, it is interesting to speculate that this region of the
protein may be a critical domain that allows access of the solute into
the cytoplasm. Along these lines, the conserved motif may be important
in facilitating global conformational changes following solute binding.
As shown in Fig. 3, the C1 conformation of the permease provides
access to solute binding from the outside of the cell while in the C2
conformation the solute has access to the cytoplasm. The loop 2/3 motif
may be important in promoting the interconversion between C1 and C2.
Another possibility may be that loop 2/3 and/or loop 8/9 may be acting
as a gate for channel opening and closing.
[
-turn propensity) may be structurally important
at this position. The acidic residue at the fifth position was also
found to be very important for transport activity and even a
conservative glutamate at this location exhibited marginal transport
activity. In contrast, many substitutions at the eighth-position
glycine, even those with a high side chain volume and/or low
-turn
propensity, still retained high levels of transport activity.
Similarly, none of the basic residues within the motif were essential
for transport activity when replaced individually by nonbasic residues.
However, certain substitutions at the basic residue sites as well as
the eighth-position glycine were observed to have moderately reduced
levels of active transport of lactose. Taken together, the results of
this study confirm the importance of the conserved loop 2/3 motif in
transport function. It is suggested that this motif may be important in
promoting global conformational changes within the permease.
and lactose. From the
cloning and nucleotide sequence of the lacY gene, the lactose
permease contains 417 amino acids with a molecular weight of 46,504
(Refs. 13 and 14). Hydropathicity plots as well as genetic studies are
consistent with a secondary structural model in which the protein
contains 12 hydrophobic segments which traverse the membrane in an
-helical manner(15, 16, 17) .
(
)superfamily and as MFS (major facilitator
superfamily)(20, 21) . Like the lactose permease,
secondary structural models for members of the USA superfamily are
commonly found to depict a membrane protein with 12 transmembrane
segments traversing the membrane in an
-helical manner. It has
been proposed that this arrangement of 12 transmembrane segments may
have arisen by a gene duplication/fusion event of an ancestral gene
encoding a protein with six transmembrane segments(22) . More
recently, we have proposed a three-dimensional model based on the
structural characteristics of transmembrane domains, hydrophilic loops,
putative helical interactions in the lactose permease, and rotational
symmetry between the two halves of the protein(21) .
Reagents
Lactose (O--D-galactopyranosyl-[1,4]-
-D-glucopyranose)
and melibiose (O-
-D-galactopyranosyl-[1,6]-
-D-glucopyranose)
were purchased from Sigma. [
C]Lactose was
purchased from Amersham Corp. The remaining reagents were analytical
grade.
Bacterial Strains and Methods
The bacterial
plasmids are described in .
transformation
procedure of Mandel and Higa(30) .
Sugar Transport Assays
For the experiments of I, midlog cells were washed in phosphate buffer, pH 7.0,
containing 60 mM KHPO
and 40 mM KH
PO
, and resuspended in the same buffer
to a density of approximately 0.5 mg of protein/ml. Cells were then
equilibrated at 37 °C and radioactive sugar (final concentration
= 0.1 mM) was added. At appropriate time intervals,
0.2-ml aliquots were withdrawn and filtered over a membrane filter
(pore size = 0.45 µm). The external medium was then washed
away with 5-10 ml of phosphate buffer, pH 7.0, by rapid
filtration. For the uphill transport experiments, 10 mM HgCl
was included in the wash buffer to rapidly
inhibit the lactose permease and thereby minimize sugar efflux during
the removal of the extracellular medium. As a control, the lacY
strain,
HS4006/F`I
Z
Y
/pACYC184,
was also assayed for radiolabeled sugar uptake in order to obtain an
accurate value for nonspecific sugar uptake.
H
For the
HTransport
transport experiments of Fig. 2, cells were
grown to midlog phase and washed twice with 120 mM KCl. The
cells were then suspended in 120 mM KCl and 30 mM potassium thiocyanate to a density of approximately 5 mg of
protein/ml. 2.5 ml of cells were placed in a closed vessel with a lid
containing tight-fitting openings for the insertion of a pH electrode,
the introduction of nitrogen, and the insertion of (gas-impermeable)
Hamilton syringes. Cells were made anaerobic under a continuous stream
of nitrogen for at least 30 min. To initiate sugar-induced H
transport, an anaerobic solution containing lactose was added to
a final concentration of 10 mM. The change in external pH was
measured with a Radiometer pH meter (PHM82) and electrode (GK2401C).
Changes in pH were continuously recorded on a Radiometer chart recorder
which had been modified to expand the scale of pH changes to a range
where a 0.1-unit pH change caused a 10-cm deflection in the chart
recording.
Figure 2:
Sugar-induced H transport. H
transport was measured in
the strain T184 containing the designated plasmids as described under
``Materials and Methods.''
Site-directed Mutagenesis
The plasmid, pTE18 (Ref.
32) was digested with EcoRI to yield a 2300-base pair fragment
containing the entire lacY gene. This fragment was ligated to
the vector M13 mp18 (Ref. 33) in such a way that the antisense strand
of the lacY gene was colinear with the plus-strand of the
viral DNA. Site-directed mutagenesis was then performed by the method
of Zoller and Smith (34) as modified by Kunkel et al. (35) using oligonucleotide primers producing the desired base
change. Clones containing the appropriate mutation were identified by
DNA sequencing (see below). The double-stranded replicative form DNA
was then isolated and digested with EcoRI to produce the
2300-base pair fragment containing the lacY gene. This
fragment was then ligated into the EcoRI site of pACYC184
(Ref. 36). Following transformation of E. coli strain
T184, cells harboring hybrid plasmids with a lacY insert were
identified by their loss of chloramphenicol resistance. The hybrid
clones were restriction mapped to verify the orientation of the lacY gene on the plasmid. Only those clones which contained
the lacY gene and the tetracycline resistance gene in the
opposite transcriptional direction were used. The mutant plasmids were
sequenced throughout the entire lacY coding sequence to verify
the presence of the mutation and to be certain that no other secondary
mutations had occurred. At least two independent clones for each mutant
type were saved for further study.
DNA Sequencing
Single-stranded viral DNA was
sequenced by the Sanger dideoxy method (37) using
oligonucleotide primers which anneal within the lacY gene.
Double-stranded plasmid DNA was isolated using Magic Minicolumns from Promega and sequenced according to Kraft et
al.(38) .
Membrane Isolation and Immunoblot Analysis
For
Western blot analysis, T184 cells containing the appropriate plasmid
were grown as described above for the sugar transport assays. Ten ml of
late log cells were pelleted by low speed centrifugation, quick frozen
in liquid N, and thawed at room temperature. Cells were
resuspended in 800 µl of MTPBS (150 mM NaCl, 16
mM, Na
HPO
, 4 mM NaH
PO
). Cells were quick frozen in liquid
N
and thawed twice. Cells were then sonicated three times
for 20 s each. Membranes were harvested by centrifugation and
resuspended in 100 µl of MTPBS. 100 µg of total membrane
protein was subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. Proteins were electroblotted to nitrocellulose and
then probed using an antibody that recognizes the carboxyl terminus of
the lactose permease. The amount of lactose permease was then
determined by laser densitometry.
Isolation of Site-directed Mutants
As noted
above, a conserved motif is found within hydrophilic loop 2/3 and 8/9
among members of the USA superfamily. Fig. 1describes the loop
2/3 motif in 65 members of the USA superfamily. According to an
extensive hydropathicity analysis(21) , the first three or four
amino acid residues are commonly predicted to lie within transmembrane
domain 2. At the first and eighth positions, a glycine residue (small
side chain volume, high -turn propensity) is highly conserved
although amino acids with a small side chain volume (i.e. alanine or serine) or amino acids with a high or moderate turn
propensity (proline or tyrosine) are found in a few cases. Hydrophobic
amino acids are found at the second, third, fourth, and seventh
positions. An acidic residue is frequently present at the fifth
position while basic residues are conserved at the sixth, ninth, and
tenth position. It should be noted that an extra amino acid is
occasionally ``inserted'' into this decapeptide motif
sequence. In the case of the lactose permease, an extra leucine residue
is found between the glycine at the eighth position and the arginine at
the ninth position.
Figure 1:
Alignment of the loop 2/3 domains of
the lactose permease and selected members of the USA superfamily. The
end of transmembrane segment 2 and the beginning of transmembrane
segment 3 are shaded in gray. The members of the superfamily are
identical to those described in Ref. 21.
As mentioned above, evidence from the
tetracycline antiporter has shown that the conserved glycine residues,
acidic residue, and one of the basic residues, are very important for
transport activity(26, 27, 28) . To explore
whether these conserved residues are also important for the function of
the lactose permease, we have made the site-directed mutants described
in .
Phenotype on MacConkey Plates
The phenotype of
bacterial colonies on MacConkey plates can provide qualitative
information concerning the functionality of transport. Those cells that
are able to appreciably transport and subsequently metabolize the added
sugar form red colonies, whereas a defect in this process results in
the formation of white colonies. In general, a lactose permease mutant
must be greatly defective in transport to prevent a red colony
phenotype. When the wild-type and mutant strains were plated on
MacConkey plates containing lactose (a -galactoside) or melibiose
(an
-galactoside), some general trends were observed. As shown in , mutations at the first glycine (i.e. Gly-64 in
the lactose permease) or at the fifth acidic residue (Asp-68) were
found to be highly defective in sugar transport. A few mutations at the
seventh glycine (Gly-71) were slightly defective. Surprisingly, none of
the mutants at the basic residues (e.g. Lys-69, Arg-73, or
Lys-74) appeared to be defective even though nonbasic substitutions
were made at these sites.
In Vitro Galactoside Transport
The experiments of indicate that the first- and fifth-position mutants are
severely defective in their ability to transport galactosides while the
other mutants are generally less defective or exhibit a wild-type
phenotype. In the experiment of I, in vitro transport assays were carried out to provide a more quantitative
description of the uptake process. In I, the wild-type and
mutant strains were analyzed for their ability to transport
[C]lactose. In ``downhill'' transport
assays, plasmids containing the wild-type lacY gene or mutant
genes were introduced into an E. coli strain that is lacZ
(i.e.
-galactosidase
positive). Upon entry into the cell, lactose is rapidly metabolized so
that the extracellular lactose concentration remains higher than the
intracellular concentration(39) . Therefore, under these
conditions, lactose transport is downhill or with its concentration
gradient. In the ``uphill'' transport assays the plasmids
were transformed into a bacterial strain that is
-galactosidase
negative.
C]lactose (see I). Only the Ala-64 mutant was similar to the wild-type
strain in downhill transport and had approximately 50% uphill
accumulation. Besides the Ala-64 substitution, the next highest values
were observed in the Ser-64 strain which showed transport activities
that were less than 10% the wild-type level. These results are
consistent with the conservation of residues at this position. Aside
from glycine, alanine and serine are the two residues that are most
likely to be at the first position in this motif (see Fig. 1).
H
Since the
wild-type residue at position 68 is acidic, it was of interest to test
whether the position 68 mutants are able to couple HTransport
and sugar transport. To accomplish this, a pH electrode was used
to measure H
transport upon the addition of lactose.
These results are shown in Fig. 2. When lactose was added, the
wild-type strain exhibited a rapid alkalinization of the medium due to
the cotransport of H
and lactose into the bacterial
cytoplasm. Similarly the Ala-68 and Thr-68 mutant strains also
exhibited a significant alkalinization. As expected, the magnitude of
the mutant's alkalinization was less than that of the wild-type
strain since these mutants have a diminished rate of lactose transport
(see I).
-turn propensity. Either
or both of these features may be important with regard to transport
function. At the first position, there is evidence to suggest that both
features may play a role. In the current and other studies, it appears
that only amino acids with a smaller side chain, such as alanine and to
a lesser extent serine, are able to substitute for glycine. These
results are consistent with the idea that side chain volume plays an
important role. This conclusion also correlates with the observation
that alanine and serine are the most likely amino acids, other than
glycine, to occur at the first position among members of the USA
superfamily. However, it is also worthwhile to point out that proline
(high
-turn propensity) and tyrosine (moderate
-turn
propensity) are occasionally found at the first position even though
these residues have significantly larger side chain volumes. In loop
8/9 of the lactose permease, a proline is found in the first position
of this motif. Therefore, it is interesting to speculate that a
combination of side chain volume and
-turn propensity may
contribute to the effects of amino acid substitutions at the first
position. In some cases, such as the Pro-64 mutant in the lactose
permease, the negative effect of a large side chain volume may override
the positive effect of high
-turn propensity.
-turn propensity, showed
relatively high transport activity. Also, the position 71 mutants with
low uphill transport activity (e.g. cysteine, proline, and
leucine, see I), do not correlate with high side chain
volume or low
-turn propensity. Therefore, based solely on these
transport experiments, the results do not suggest that side chain
volume or
-turn propensity are critical at the eighth position.
This conclusion seems very unusual considering that the glycine at the
eighth position is the most highly conserved amino acid in the loop 2/3
motif and it is 100% conserved in the loop 8/9 motif.
upon the addition of lactose. Among the USA
superfamily, an aspartate is not always conserved in the loop 2/3
motif. However, it is rather striking that an acidic residue (e.g. aspartate or glutamate) is nearly always present at the fifth
position of this motif in, at least, one of the two repeated motifs.
The only exception is a gene designated LTP from Leishmania(40) . In any case, it is intriguing that a
negative charge in one of the two repeated motifs has been highly
conserved throughout evolution. Future work will be necessary to
determine whether this negative charge is always an important
requirement for transport among this superfamily of proteins.
Figure 3:
Hypothetical role of the loop 2/3 motif in
conformational changes of the lactose permease. The three-dimensional
arrangement of transmembrane domains has been proposed in Ref. 21. The
channel-lining segments are TM-1, TM-2, TM-4, TM-5, TM-7, TM-8, TM-10,
TM-11, while the scaffolding segments are TM-3, TM-6, TM-9, and TM-12.
The two halves of the protein exhibit rotational symmetry around a
central axis. The C1 conformation has its solute binding site
accessible from the outside while the same site in the C2 conformation
is accessible from the inside.
Table: Expression levels in mutant strains
Table: Phenotype on MacConkey plates
Table: Lactose
transport activities of wild-type and mutant strains
C]Lactose was measured as described under
``Materials and Methods.'' Downhill lactose uptake was
carried out in strain
HS4006/F`I
Z
Y
(Ref. 42)
containing the lacY plasmids described within the table.
Uphill accumulation was carried out in strain, T184 (Ref. 41), that is
-galactosidase negative. The values are presented as the % of wild
type activity.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.