(Received for publication, July 11, 1995)
From the
To determine the role in transmembrane signaling of the N-terminal peptide of the first transmembrane region of the aspartate receptor, it was subjected to extensive mutagenesis. Drastic changes did not alter the chemotactic ability of the receptor to aspartate significantly. Thus the cytoplasmic N terminus of the first transmembrane region does not play an essential role in transmembrane signaling, and the entire signal that is transmitted to the cytoplasmic domain must be sent through the second transmembrane region. This eliminates the models requiring an interaction of this N-terminal peptide with the remaining cytoplasmic portion of the receptor.
Receptor-mediated transmembrane signaling is important in all living organisms from bacteria to human beings. Most receptors function as either homodimers or heterodimers (for recent reviews see Refs. 1 and 2). The dimerization process is sometimes induced by ligand binding and sometimes totally independent of it. The human growth hormone receptors belong to the first category(3) . Others, such as human insulin receptor (4) and the bacterial chemotactic receptors(5) , do not change their oligomeric states upon ligand binding. These receptor families share similar transmembrane topology (one or two single transmembrane segments connecting an extracellular ligand binding domain and an intracellular signal domain in each subunit). They are believed to transmit signals by a common mechanism, because chimeric receptors containing the ligand binding domain of one receptor and the signaling portion of another are still capable of transmembrane signaling(6, 7) . Due to the detailed understanding of the biochemical pathways of the bacterial chemotaxis system and the relative ease in manipulating bacterial proteins, the aspartate receptor provides us with an excellent system to study the general principles underlining the signal transduction mechanism.
Each aspartate receptor subunit has a periplasmic ligand
binding domain, two transmembrane segments (transmembrane (TM) ()1 and TM 2), and one cytoplasmic domain. The native
receptor has two subunits that do not associate or dissociate during
signaling(5) . The aspartate binding sites, as seen in the
x-ray crystal structure, lie at the interface between the two
subunits(8) . Cross-linking experiments with introduced
cysteine residues at various locations of the receptor showed that the
signal is a conformational change of the receptor(9) . It can
be transmitted through one subunit of the functional
dimer(10) .
When the alanine residue (at position 19) in the middle of TM 1 was replaced by a lysine residue (A19K), the mutant receptor was nonfunctional, even though the binding affinity for aspartate remained similar to that of the wild type(11) . The pseudorevertants of this A19K mutant were largely found within a 40-residue region (residues 264-303) of the cytoplasmic domain. Thus there might be some interactions between the cytoplasmic domain and the TM 1, probably through the cytoplasmic extension of TM 1, a six-residue tail at the N-terminal end of the receptor sequence, and this interaction might be important for transmembrane signaling.
Short or long C-terminal tails exist in a number of receptors, but their role is still uncertain at the moment (for recent reviews see (12, 13, 14) ). The N-terminal tail of the aspartate receptor is highly conserved in the chemotactic receptor family (Table 1). In order to clarify its role in the signal transduction process, extensive mutations have been introduced into that region, and the cells with the modified receptors have been examined.
The minimal medium contains Vogel-Bonner citrate salt(16) , 1% glycerol, 100 µg/ml ampicillin, and 500 mg/liter of each of the following: L-histidine, L-methionine, L-leucine, L-threonine, and thiamine. The Luria broth contains 1% tryptone, 0.5% yeast extract, and 1% NaCl. The minimal plates have 0.3% agar in minimal medium. The aspartate plates are the same as the minimal ones except that aspartate was added to 100 µM. The tryptone plates have 1.3% tryptone, 0.6% NaCl, 0.3% agar, and 100 µg/ml ampicillin.
where B and F are the concentrations of bound
and free aspartate, respectively, B is the
maximal bound aspartate concentration, K
is the
dissociation constant, and n
is the Hill
coefficient.
For the methylation of purified receptors in a reconstituted system, partially purified receptor sample was added into the reconstitution buffer (50 mM NaPi, pH 7.0, 40% glycerol, 1/10 volume of null membrane, 0.5% OG, and 1 mM PMSF) and incubated at room temperature for 45 min and then the procedure above was followed.
Several mutants within the N-terminal cytoplasmic tail region
of the Salmonella typhimurium aspartate receptor were made (Table 2). Three of them were deletion mutants: Arg deleted (
R4), four residues deleted from Phe
to
Ile
(
2-5), and Ile
and Arg
deleted (
5-6). Four of them were point mutations that
converted Arg
to Gly (R4G), Glu (R4E), Lys (R4K), and Cys
(R4C), respectively. The last one (ICys2) was an insertion of a
cysteine residue between Met
and Phe
. Two
cysteine-containing mutants, R4C and ICys2, allowed us to cross-link
the receptor molecules with disulfides.
In order to optimize the
overexpression of the aspartate receptor, an EcoRI restriction
site was placed immediately upstream of the tar start codon so that the tar
gene could be
subcloned into another vector that has a strong promoter. It was
designed in the same oligonucleotide carrying each of the several
N-terminal mutations (class B mutants). This approach also made the
screening easier. Unfortunately, this EcoRI site altered the
putative ribosome binding site for the receptor synthesis(23) .
It indeed lowered the expression level of the receptor (data not
shown). These mutants were eventually subcloned into the plasmid
pBTac2, and the expression of these class C receptor mutants were much
better (data not shown). The oligonucleotides for the rest of the
mutants (class A) only contained the desired mutations, and tar
gene is under the control of its natural
promoter (Table 2).
We performed in vivo swarm assays to test the effects of the mutations on chemotaxis. As shown in Fig. 1, cells harboring each of the mutant receptor genes showed normal responses toward aspartate in the medium compared with those with wild type receptor genes, and the cells containing no receptor gene showed the same swarm rates in the presence and absence of aspartate. Fig. 2shows the swarm rates of the mutants on tryptone plates. The results agree with those in Fig. 1. Thus it seems that a wide variety of mutations at the N-terminal tail region do not impair the chemotactic ability of the cells significantly.
Figure 1: Swarm rates of the N-terminal mutants on minimal and aspartate plates. Swarm assays were performed at 30 °C on the minimal medium plate with or without attractant aspartate. Swarm rates (mm/h) were reported as the slopes of the linear fits of the time-course data points. For each receptor, the column on the left is the swarm rate in the absence of aspartate and the column on the right is that in the presence of aspartate. Top, class A mutants (see ``Results'' for details); middle, class B mutants; bottom, class C mutants. wt, wild type.
Figure 2: Swarm rates of the N-terminal mutants on tryptone plates. Swarm assays were performed at 30 °C on the tryptone plates. Swarm rates (mm/h) were reported as the slopes of the linear fits of the time-course data points. Top, class A mutants (see ``Results'' for details); middle, class B mutants; bottom, class C mutants. wt, wild type.
The exceptions were the R4G mutant of class B ( Fig. 1and Fig. 2) and the R4C mutant of class A ( Fig. 1and Fig. 2). The R4G mutant in class B was very poorly expressed. However, when it was subcloned into pBTac2, the expression level was comparable with other mutants, and it swarmed normally ( Fig. 1and Fig. 2). The R4C mutant showed lower swarm rates than most of the others on both aspartate and tryptone plates, but its rates were still higher than those of the negative controls. For each swarm assay, we did immunoblotting analysis to monitor the expression level of the receptor. The differences in swarm rates that did occur on minimal and aspartate plates of different classes could be traced to the differences in expression level (data not shown).
Because swarm assay is only a semiquantitative assay to measure chemotaxis and because several factors could affect swarm rates, we also did some in vitro biochemical studies on some of the mutants. First we did immunoblotting experiments on the membrane samples and found that the mutant receptors still associate with the membrane after high salt washes (data not shown), indicating that the mutated N-terminal tail region could still function as a leader peptide.
To determine whether the mutations caused any significant
change in the tertiary and quartenary structures of the receptor, the
aspartate binding affinities of the mutants were tested. Table 3listed the binding parameters of the wild type and some of
the mutants. The dissociation constants (K) were
within a 2-fold range of that of the wild type. The mutations at the
N-terminal cytoplasmic region did not alter the binding affinity for
aspartate of the receptor significantly. The index for cooperatively
(the Hill coefficient (n
)) of each mutant tested
(except for R4K) was within the previously published range,
0.6-0.8(20) , indicating that the binding behavior was
similar to that of the wild type.
We then used methylation assays as
a test of signal transduction in vitro. As shown in Fig. 3and 4 for the wild type and each of the mutants tested,
aspartate increased the methylation rates in a similar manner to wild
type. In the reconstituted system (Fig. 4), the addition of
aspartate increased the methylation rate of the wild type by 1.8-fold.
For the R4 mutant and
2-5 mutant, the increase was 1.9-
and 1.7-fold, respectively. The reactions with no purified receptor
showed minimal levels of methylation in the presence and the absence of
aspartate.
Figure 3: Methylation rate ratios of the N-terminal mutants in a membrane vesicle system. Methylation assays were performed at 37 °C in the presence and the absence of aspartate. The methylation rates were the slopes of the linear fits of the time-course data points. The ratio of each receptor was the value of the methylation rate in the presence of aspartate divided by that in the absence of aspartate. wt, wild type.
Figure 4: Methylation rates of the N-terminal mutants in a reconstituted system. Partially purified receptor samples were incubated with the null membrane at room temperature for at least 45 min prior to the methylation assays, which were performed at 37 °C in the presence and the absence of aspartate. The methylation rates were the slopes of the linear fits of the time-course data points. The rates were normalized to the methylation rate of the wild type receptor in the absence of aspartate. For each receptor, the column on the left is the rate in the presence of aspartate, and the column on the right is that in the absence of aspartate.
To probe the possible motion of TM 1 within each subunit (either a piston-like motion or a rotation), we compared the rates of disulfide bond formation of two cysteine mutants in the presence and the absence of aspartate. They looked virtually identical for each mutant, as shown in Fig. 5. Based on a previous cross-linking study(24) , residues 4 and 4` are at the dimer interface, with the closest distance among the N-terminal tail residues. The inserted cysteine residue of the ICys2 mutant is located at the position for methionine residue in the wild type receptor. The side chain of this residue faces the second transmembrane segment within the same subunit.
Figure 5: Cross-linking time-courses of the ICys2 mutant. Cross-linking reactions were performed in the presence of 1 mM copper-phenanthroline at 37 °C. Aliquots were removed at various times, and the reaction was quenched to yield the reaction coordinate, the fraction of the receptor that was cross-linked (cpm of the dimeric receptor divided by the sum of cpm of the dimeric and monomeric receptor). The filled circles were the points in the presence of aspartate, and the open circles were those in the absence of aspartate. The time-courses of the R4C mutant looked similar to those of the ICys2 mutant.
The results reported herein indicate that major changes can be made in the N-terminal peptide projecting into cytoplasm with very minor effects on either transmembrane signaling or aspartate binding ability. The mutants did not interfere with insertion into the membrane or the folding of the cytoplasmic domain.
Previous studies showed that a TM 1-less receptor (first 30 amino acid residues deleted) was not functional as determined by both swarm assays and methylation assays(25) . However, we found that deletion of four residues at the N-terminal end did not affect chemotaxis significantly. Thus the TM 1 transmembrane region is important, but the peptide extending from the TM 1 region into the cytoplasm is not. This suggests that the transmembrane helices (TM 1 and TM 1`) are important in maintaining the structural integrity of the whole receptor and probably the transmembrane signaling. A distortion of these interactions would lead to abolished function of the receptor, as shown by the A19K mutant in TM 1 (11) and the 204 mutants in TM 2(26) .
It has
also been found that when TM 1 and 1` were cross-linked by a
Cys-Cys
(24) ,
Cys
-Cys
(27) , or
Cys
-Cys
(9) disulfide bond,
the receptor could still signal as determined by methylation. This
observation leads to the conclusion that TM 1 and TM 1` do not change
position relative to each other during transmembrane signaling, and it
is confirmed by similar reactions of a
Cys
-Cys
/C
-Cys
double cross-linked receptor(28) .
If the interaction of the N-terminal hexapeptide with the cytoplasmic signaling domain is eliminated as a source of the indicated transmembrane conformational change, then the entire transmission of the signal that is delivered to the cytoplasmic domain must go through TM 2. Clearly that severely limits the mechanism for such transmembrane signaling. A rotation model, as suggested by Maruyama et al.(29) , seems unlikely as a sole contributor to the transmembrane signaling. Rotation of the two cytoplasmic subunits relative to each other would be excluded as a transmembrane signaling option by the results of Milligan and Koshland(10) , in which the cytoplasmic portion of one subunit could be eliminated with only a minor effect on signaling. Thus a piston model(30) , in which the cytoplasmic domain moves relative to the membrane, or a model involving relative motion of transmembrane segments TM 2 and TM 2` seems indicated. Such a model could also explain the transmembrane signaling of the epidermal growth hormone and low density lipoprotein receptors, which have one transmembrane region per receptor subunit.