(Received for publication, July 19, 1995; and in revised form, October 2, 1995)
From the
A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K),
is found in a large group of evolutionarily related membrane proteins
involved in the transport of small molecules across the membrane. This
motif is located within the cytoplasmic side of transmembrane domain 2
(TM-2) and extends through the hydrophilic loop that connects
transmembrane domains 2 and 3. The motif is repeated again in the
second half of the protein. In a previous study concerning the loop 2/3
motif (Jessen-Marshall, A. E., Paul, N. J., and Brooker, R. J.(1995) J. Biol. Chem. 270, 16251-16257), it was shown that the
conserved aspartate at the fifth position in the motif is critical for
transport activity since a variety of site-directed mutations were
found to greatly diminish the rate of transport. In the current study,
two of these mutations, in which the conserved aspartate was changed to
threonine or serine, were used as parental strains to isolate second
site suppressor mutations that restore transport function. A total of
10 different second site mutations were identified among a screen of 19
independent mutants. One of the suppressors was found within loop 1/2
in which Thr-45 was changed to arginine. Since the conserved aspartate
and position 45 are at opposite ends of TM-2, these results suggest
that the role of the conserved aspartate residue in loop 2/3 is to
influence the topology of TM-2. Surprisingly, the majority of
suppressor mutations were found in the second half of the permease. All
of these are expected to alter helix topology in either of two ways.
Some of the mutations involved residues within transmembrane segments 7
and 11 that produced substantial changes in side chain volume: TM-7
(Cys-234 Trp or Phe, Gln-241
Leu, and Phe-247
Val)
and TM-11 (Ser-366
Phe). Alternatively, other mutations were
highly disruptive substitutions at the ends of transmembrane segments
or within hydrophilic loops (Gly-257
Asp, Val-367
Glu,
Ala-369
Pro, and a 5-codon insertion into loop 11/12). It is
hypothesized that the effects of these suppressor mutations are to
alter the helical topologies in the second half of the protein to
facilitate a better interaction with the first half. Overall, these
results are consistent with a transport model in which TM-2 acts as an
important interface between the two halves of the lactose permease.
According to our tertiary model, this interaction occurs between TM-2
and TM-11.
The uptake of a variety of solutes, including sugars, amino
acids, and inorganic ions, is mediated by integral membrane proteins
known as cation/substrate cotransporters or symporters. The lactose
permease of Escherichia coli has provided a model system in
which to investigate the molecular mechanism of
symport(1, 2) . This protein is found in the E.
coli cytoplasmic membrane and couples the transport of
H and lactose. From the cloning and nucleotide
sequencing of the lacY gene, the lactose permease contains 417
amino acids with a molecular weight of 46,504 (Refs. 3 and 4).
Hydropathicity plots as well as genetic studies are consistent with a
secondary structural model in which the protein contains 12 hydrophobic
segments that traverse the membrane in an
-helical
manner(5, 6, 7) .
Evolutionary analyses
have indicated that the lactose permease is a member of a large group
of solute transporters that are homologous to each
other(8, 9, 10) . This superfamily includes
proteins that transport substrates such as sugars, Kreb's cycle
intermediates, and antibiotics. Interestingly, the superfamily includes
not only symporters but also uniporters and antiporters. It has been
referred to as the uniporter-symporter-antiporter (USA) superfamily and
as major facilitator superfamily(10, 11) . Secondary
structural models for members of the USA superfamily are commonly found
to predict a membrane protein with 12 transmembrane segments traversing
the membrane in an -helical manner. Furthermore, it has been
proposed that this arrangement of 12 segments has arisen by a gene
duplication/fusion event of an ancestral gene encoding a protein with
six transmembrane segments(12) . Based on the structural
characteristics of the members of the USA superfamily, we have recently
proposed a three-dimensional model that describes the orientation of
the 12 transmembrane segments(11) . In this model, eight of the
transmembrane segments act as channel-lining domains while the other
four perform a scaffolding function. The model also suggests that the
two halves of the protein are folded in a similar fashion and that they
associate with each other in a rotationally symmetric manner.
A
conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), is found
in all members of the USA
superfamily(8, 9, 10) . This motif is located
within the cytoplasmic side of transmembrane segment 2 and extends
through the hydrophilic loop that connects TM-2 ()and TM-3.
It is repeated again in loop 8/9. In our previous study with the
lactose permease and also in studies with the tetracycline antiporter,
it has been found that the negative charge in the fifth position of the
motif is critical with regard to transport
function(13, 14) . In the lactose permease,
substitutions of alanine, serine, threonine, tyrosine, asparagine,
histidine, and even glutamate exhibited transport activities that were
less than 5% the wild-type rate. In the current study, the serine and
threonine mutants were used as parental strains to identify second site
suppressor mutations that restore transport function.
Stock cultures of cells were grown in
YT media (16) 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
isopropyl-1-thio--D-galactopyranoside to induce the
synthesis of the lactose permease.
Figure 2:
Downhill lactose transport by wild-type (W.T.) and mutant strains. The uptake of
[C]lactose was measured as described under
``Materials and Methods.'' Downhill lactose uptake was
carried out in strain
HS4006/F`I
Z
Y
containing
the lacY plasmids described within the
figure.
Figure 1: Location of the loop 2/3 motif within the secondary structure of the lactose permease. This secondary model depicts 12 transmembrane segments connected by hydrophilic loops. Aspartate 68 is located within the loop 2/3 motif. This figure also illustrates the location of the suppressor mutations described in Table 3. These second site mutants are denoted by an &cjs3648;. A proposed three-dimensional arrangement of transmembrane domains is also shown in Fig. 4.
Figure 4: The three-dimensional arrangement of transmembrane domains was proposed by Goswitz and Brooker(11) . 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. The model suggests that two halves of the protein act as fairly rigid domains that move along the TM-2/TM-11 and TM-8/TM-5 interfaces to convert between the C1 and C2 conformation.
To explore the role of the conserved aspartate in the loop 2/3 motif, the Ser-68 and Thr-68 mutants were used as parental strains to isolate second site suppressor mutations that restore transport activity. These two parental strains were chosen because the serine and threonine codons require a two-base change back to the original aspartate codon. Therefore, it seemed likely that the use of these strains would favor our ability to identify single base substitutions at a ``second site'' within the gene that restores transport function.
The Ser-68 and Thr-68 strains were streaked on 1%
melibiose MacConkey plates as a method to identify suppressor mutants
that restore activity. It should be noted that the wild-type lactose
permease is able to transport melibiose (an -galactoside).
Therefore, when streaked onto 1% melibiose MacConkey plates, a strain
containing the wild-type permease forms red colonies since it can
transport (and ferment) the sugar. In contrast, the Ser-68 and Thr-68
strains were observed to form white or pink colonies (see Table 2). After several days, ``red flecks'' became
apparent within the primary streak. These red flecks were restreaked to
isolate individual red colonies. From 19 independent red colonies, the
plasmid DNA containing the lacY gene was then isolated and
transformed into a lacZ
/lacY
strain.
In all cases, the transformants exhibited a red phenotype on both 1%
lactose MacConkey and 1% melibiose MacConkey plates (see Table 2).
Surprisingly, all of the other mutations were in the second half of the protein. These mutants fell into two other categories. Several of the mutants were found within transmembrane domains (C234W, C234F, Q241L, F247V, and S366F). All of these mutants involved a dramatic change in side chain volume either by gaining or losing a large, hydrophobic side chain. Alternatively, other mutants were found at the edges of transmembrane domains or within hydrophilic loop regions. In all of these cases, the mutations are expected to be highly disruptive with regard to protein structure (G257D, A369P, V367E, and a 5-codon insertion). The most unusual example was a 5-codon insertion in loop 11/12 that arose as a small duplication of codons 372 through 376 and inserted between codons 376 and 377. Overall, both categories of mutations in the second half of the permease are expected to alter transmembrane topology. Assuming that the first site mutation disrupts TM-2, the suppressor mutations in the second half of the protein may act to restore the proper topology of helix 2 or create a more favorable interface between helix 2 and the second half of the protein. Interestingly, according to our tertiary model, TM-2 is expected to interact with TM-11. Most of the second site suppressors were found within or near TM-11 or were side chain volume changes within neighboring TM-7.
Figure 3:
Uphill lactose transport by wild-type (W.T.) and mutant strains. The uptake of
[C]lactose was measured as described under
``Materials and Methods.'' Uphill lactose uptake was carried
out in strain T184 containing the lacY plasmids described within the
figure.
The conservation of a motif within a large superfamily is indicative of an important functional and/or structural role. The results of the current study provide evidence that the aspartate within the GXXX(D/E)(R/K)XG(R/K)(R/K) motif is important in maintaining the conformation of the permease. In particular, it appears to be very important for the positioning of TM-2 within the tertiary structure of the protein. At the current time, it is not clear how the Ser-68 and Thr-68 substitutions exert an incorrect positioning of TM-2. One possibility is that the absence of an aspartate in loop 2/3 may allow a small perturbation in helix 2 toward the periplasm. If this is the case, the suppressor mutations may circumvent this perturbation in different ways. The T45R mutation introduces a positive charge in loop 1/2 that may counteract the movement of helix 2 toward the periplasm. In contrast, second site suppressor mutations in the second half of the protein are expected to exert their effects in a different way. For example, they may promote a stronger interaction between helix 11 and helix 2 that would prevent the repositioning of helix 2 toward the periplasm. This idea is consistent with the observation that most of the suppressors were found within or near TM-11 or were side chain volume changes within neighboring TM-7.
The transport of
H and lactose via the lactose permease requires
conformational changes that promote an interconversion between
outwardly accessible and inwardly accessible solute binding sites.
Within the entire superfamily, the conservation of the loop 2/3 and 8/9
motifs may be related to their abilities to maintain transporters in a
conformationally competent state. In our tertiary model, it is
interesting that TM-2 and TM-8 lie at the interface between the two
halves of the protein. Most of the suppressor mutations are located
near the TM-2/TM-11 interface, although one suppressor (i.e. G257D) is expected to be near the TM-8/TM-5 interface. Overall, it
is interesting to speculate that these interfaces form two
conformationally sensitive sites that are involved in the
interconversion between the C1 and C2 conformations (see Fig. 4). According to this model, the interhelical arrangements
of TM-2/TM-11 and TM-8/TM-5 form a critical juncture for protein
motion. The two halves of the permease are considered to be relatively
rigid domains that rotate at these two interfaces.
The motion of
proteins is usually facilitated by hinge motions or shearing
motions(19) . The model of Fig. 4can be explained by
small shear motions at the TM-2/TM-11 and the TM-8/TM-5 interfaces. In
other proteins that have been crystallized in both opened and closed
conformations, shear motions are commonly involved in conformational
changes that alter accessibility to the solute binding
site(19) . The motion does not involve the repacking of
interdigitating side chains at the interface (i.e. between
-helices). Instead, the side chains can alter their torsional
angles so that they move among conformational states of nearly the same
energy without crossing large energy barriers. However, it is important
to point out that the amount of rotation that is accomplished by this
type of shear motion is relatively small (i.e. typically a
maximum of 15°). Without further structural information, it is
difficult to assess whether a 15° rotation is sufficient to account
for the types of conformational changes described in Fig. 4.