Sites of Deamidation and Methylation in Tsr, a Bacterial Chemotaxis ...

THEJOURNALOF BIOLOGICAL CHEMISTRY (0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 15, Issue of May 25, pp. 9746-9753,1991 Printed in U.S.A.

Sites of Deamidation and Methylationin Tsr,a Bacterial Chemotaxis Sensory Transducer* (Received for publication, August 6, 1990)

Margaret S. Rice and Frederick W. DahlquistS From the Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

The sensory transducer proteins in bacterial chemotaxis undergo two covalent modifications, deamidation and reversible methylation, in response to attractants and repellents. Oligonucleotide-directed mutagenesis was used to alter putative methylation and deamidation sites in one of the transducers to further define these sites and their role in chemotaxis. The mutations, in combination with peptide maps and Edman analysis, have clarified the sites of covalent modification in Tsr. Tsr contains six specific glutamates and glutamines that serve as methyl-accepting sites. An arginine-containing tryptic peptide ( R l ) has two sites, one at glutamate 493 and anewly located site atglutamate 502. A lysine-containing peptide ( K l ) has four methyl-accepting sites. Two of the lysine peptide sites are glutamates and can accept methyl groups without deamidation. The other two sites are glutamines and two methyl-accepting sitesare created by two distinct deamidations. Both deamidations can occur on the same polypeptide chain. Single glutamate mutants have shown that one deamidation (at glutamine 311) proceeds rapidly, while the other deamidation (at glutamine 297) has a half-life of approximately 60 min under our experimental conditions.

Methyl groups are added by CheR, the methyltransferase (Springer and Koshland, 1977; DeFranco et al., 1979), and removed by CheB, the methylesterase (Stock and Koshland, 1978; Hayashi et al., 1979). Attractants increase the net level of transducer methylation, while repellents decrease methylation. Methylation is necessary for adaptation (Goy et al., 1977; Weis and Koshland, 1988; Hazelbauer et al., 1989) to stimuli which are sensed by Tsr, Tar, Tap, and Trg. Each transducer has two highlyconserved tryptic fragmentswhich contain the methyl-accepting sites (Chelsky and Dahlquist, 1980,1981; Kehry and Dahlquist, 1982b). The two fragments are called K1, which designates the methyl-accepting lysine peptide, and R1, the methyl-accepting arginine peptide. The methylaccepting sites areusually specific glutamate residues (Kleene et al., 1977; Van Der Werf and Koshland, 1977) within these peptides. However, in each transducer, two of these sites are translated as glutamines, which are irreversibly deamidated by CheB to glutamate (Rollins and Dahlquist, 1981; Sherris and Parkinson, 1981; Kehry et al., 1983, a and b, see Fig. 1). These deamidated glutamines then function as methyl-accepting glutamates. Several previous studies have characterized the locations of covalent modification of the transducers (Kehry et al., 1983c; Nowlin et al., 1987) and of Tsr in particular(Kehryand Chemotaxis is an integrated system of sensory transduction Dahlquist, 1982, a and b).Earlier experiments with Tsr were and behavioral response. In Escherichia coli, sensory trans- unable to determine the exact sites of deamidation and methduction is mediated by four closely related proteins: Tsr, Tar, ylation occurring on Tsr. To delineate more clearly where Trg, and Tap. (For recent reviews, see Stewart and Dahlquist, methylation and deamidation occur in the Tsr transducer, we 1987 or Hazelbauer, 1988.) Each of these inner-membrane- have generated mutations that alterputative deamidation spanningtransducers (Ridgway etal., 1977)recognizes a sites (3 glutamine residues in the K1 peptide) to glutamate different set of attractants and repellents (Silverman and and changed the established R1 methyl-accepting glutamate Simon, 1977; Springer et al., 1977; Kondoh et al., 1979). A to alanine. Comparisons of the mutated and wild type forms change in attractant or repellent bound to a transducer pro- of Tsr in various cell backgrounds have demonstrated that duces an excitatory signal, which alters the phosphorylation both Q297 and Q311 can be deamidated in the wild type of other chemotaxis proteins (Borkovich et al., 1989; Bourret transducer. Deamidation occurs more rapidly at Q311 than at et al.,1989; Hess et al., 1987, 1988). Phosphorylation of these Q297. The slow rate of deamidation at Q311 suggests possible proteins leads to transientchanges in the swimming behavior roles for deamidation in chemotaxis. of the bacterium (Sanders et al., 1989). EXPERIMENTALPROCEDURES’ The ability toadaptto a wide concentration range of attractants and repellents is critical to chemotaxis. AdaptaRESULTS tion is accomplished by altering the number of methyl groups Our experiments made use of host strains with mutations present on the transducers (Goy et al., 1977; DeFranco and in either the cheR or cheB genes (encoding the methyltransKoshland, 1980; Boydand Simon, 1980).Each transducer can accept four to six methyl groups in its cytoplasmic domain. ferase and methylesterase/deamidase) to obtain proteins and peptides that were methylated and deamidated (wild type), * This work was supported by National Institutes of Health Grant unmodified (cheRB-), or either methylated (cheB-) or deamGM33677 (to F. W. D.), National Science Foundation Grant BBS- idated (cheR-) only. Comparisons of proteins and peptides in 8714102 to the University of Oregon Biotechnology Laboratory, and grants from the Murdock Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore behereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $. To whom correspondence and reprint requests should be sent.

Portions of this paper (including “Experimental Procedures” and Tables 1 and 2) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

9746

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*

FIG. 1. Amino acid sequence of the methyl-accepting peptides. These sequences are given in the one-letter code; E represents glutamate, and Q is glutamine. The methyl-accepting sites are starred, and the methyl-accepting amino acid residues are numbered for Tsr. Residues which differ from Tsr are underlined (Nowlin et al., 1987).

different backgrounds allowed us to determine which peptides may be methylated and deamidated. Arginine-containing TrypticPeptides Are Not Deamidated

A previous study (Kehry and Dahlquist, 1982b) compared the retention timesof Tsr arginine-labeled peptides incheRBand cheR- cells using HPLC3 peptide mapping, which can detect changes in retention times produced by deamidation or methylation events. Kehry and Dahlquist (1982b), observed a peptide that had an increased retention timein cheR- cells. Receptors produced in a cheRB- background are unmodified, while receptors in a cheR- host may be deamidated, but not methylated. Therefore,the observed change in retention time suggested a possible deamidation site in an arginine-containingtryptic peptide. This peptide was not identified. Two techniques,a new HPLC ion-pairinggradient that offers superior resolution and an inducible Tsr plasmid that facilitates highly specific labeling of Tsr were combined to further investigate this issue. A comparison of Tsr labeled with [3H] arginine in cheRB- cells and with [14C]argininein cheR- cells is shown in Fig. 2. The peptide maps are identical in both backgrounds; none of the peptides has a change in retention timethat would indicate that deamidation occurred. The resolution of thesemaps is much improved over previous maps, and we conclude that none of the arginine peptides contains deamidation sites.

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A similar comparison of lysine-labeled tryptic peptides in cheRB- and cheR- cells was performed to identify possible deamidated peptides. As shown in Fig. 3, only one peptide had an altered appearance. The last peak in the cheRB- map appears as three peaks in the cheR- map. The first peak in the cheR- map has the same retention time as the peak in the cheRB- map and also corresponds to the peptide previously identified as a methyl-accepting peptide, K1. The two additional peptidesproduced in thecheR- cells have retention times and levels of radioactivity that are consistent with deamidation of K1 to produce the two new peptides. One site of deamidation, Q297, has been identified in the K1 peptide (Kehry e t al., 1983, a and b). Inconclusive data suggested that Q311 might be deamidated, and the deamidation of a third glutamine, Q298, could not be ruled out. We mutated glutamines 297, 298, and 311 to glutamate to deter-

’’The abbreviations used are: HPLC,high performance liquid chromatography; IPTG, isopropyl-1-thio-@-D-galactopyranoside;SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

50

100

150

200

250

300

Fractionnumber

FIG. 2. Peptide maps of arginine-labeled tryptic peptides. Plasmid pMSR141 (wild type) was labeled with [3H]argininein strain D349 (Atsr, Atar-cheRB) and with [’*C]arginine in strain D227 (cheR-). Tsr tryptic peptides were produced as described under “Experimental Procedures,” and these peptides were separated with a HPLC reverse phase ion-pairing gradient. The peptides produced in D349 and D227 were combined and run on the same HPLC gradient. The 3H and I4C radioactivity in each fraction was determined by liquid scintillation counting. The 3H radioactivity from the cheRBpeptides (unmodified by either deamidation or methylation) is plotted in the upper map, and the 14Cradioactivity from the cheR- peptides (which may be deamidated but not methylated) is plotted in the lower map.

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FIG. 3. Peptide maps of lysine-labeled tryptic peptides. Plasmid pMSR141 (wild type) was labeled with [3H]lysine in D349 (Atsr, Atar-cheRB) and D227 (cheR-) hosts. Tryptic peptide maps were prepared as described in Fig. 2; however, the two maps were run separately. The upper map displays unmodified peptides produced in strain D349, whilethe lower map is peptides which maybe deamidated but not methylated from strain D227. The lysine-containing peptide with an altered retention time is marked with arrows. The first peak in the cheR- panel is coincident with the last peak in the cheRBmap and also corresponds to K1, a previously identified methylaccepting peptide.

mine if these residues could serve as deamidation sites. Comparison of M u t a n t and Wild Type Protein MigrationWe changed residues Q297, Q298, and Q311 to glutamate to determine which of the 3 residues normally serves as substrate for CheB. A number of experiments suggest that the Q298E mutant protein behaves differently than thewild type protein after its normaldeamidation and that Q297E and Q311E behave similarly to wild type. Fig. 4A shows a Western blot of an SDS-PAGE ofwild

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= FIG. 4. Western blots of the glutamate mutants. Panel A is a Western blot of Tsr proteins in strain D349 (Atrs, Atar-cheRB).The lanes are unmodified Tsr produced by plasmids carrying the following mutations: lane 1 , 6297,298,3113; 2, 6298,3113; 3, 6297,2983; 4, Q2983; 5,Dl11 (Atar-cheB);6,wild type; 7,62973; 8,Q311E; and 9, Q297.311E from left to right. The lower band is produced by Trg, which cross-reacts with the Tsr antibody. Panel B is a Western blot of plasmid-encoded Tsr proteins produced in D414 (tsr-, cheR-) or D349 (Atsr, Atar-cheB). Receptors produced in the cheR- strain can be deamidated, which decreases their migration, while receptors produced in the cheRB- strainare unmodified. The lanes are 1, Q297,311E in D414; 2, 6297,3113 in D349, and 3, wild type in D349, from left to right.

type, each single glutamine to glutamate mutant (Q297E, Q298E, Q311E), doublemutants, and the triple mutant proteins produced in a cheRB- strain. Conversion of glutamine residues to glutamate results in slower migration of the Tsr variants. However, the changes in migration differ depending on the site of mutation. The reasons for these migration changes are unknown, but theyprovide a powerful analytical procedure to monitor deamidation. It is instructive to consider the nature of the products formed when CheB acts on the Tsr variants. In cases where more than one protein band is observed, the slowest migrating form is most deamidated.The most deamidated form of wild type and mutants containing 6297, Q311, or both Q297 and 6311, but not 6298, all have the same mobility in a cheRhost. This suggests that the slowest migrating forms of Tsr contain glutamate residues at both positions 297 and 311 and not at Q298.Fig. 4B compares the migration of a double glutamate mutant (Q297,311E)in cheR- and cheRB- strains. Only this double mutant and the triple mutant (Q297, 298, 311E) show the same migration on SDS-PAGE in both cheRand cheRB- strains. When positions 297 and 311 are both converted to glutamate, no SDS-PAGE migration changes are observed upon exposure to CheB. We concludethat Q297 and Q311 are substrates for CheB, and that Q298 is not a site of deamidation. Peptide Maps of Mutant and Wild Type Tsr-These conclusions are strengthened by examination of HPLC-derived peptide maps of the Tsr variants. Peptide maps provide a higher resolution picture of the changes produced by deamidation and methylation. In cheRB+ cells, where additional deamidation and methylation can occur, the correct glutamate mutants should produce peptides with retention times identical with wild type. Fig. 5 E shows a tryptic peptide map of Tsr which has incorporated tritiated methyl groupswhen incubated with labeledmethionine in cheRB+ cells. One peak is produced by methylation of the R1 peptide, called T4 in correspondence to previous work (Kehry and Dahlquist, 1982b), whilethe other peptides, T3, T5, and T6 are produced

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FIG. 5. Comparison of methyl-labeled Tsr wild type and Q298E mutant peptide maps. Wild type and mutant plasmid encoded Tsr were labeled with [methyl-'H]methionine in D311 (cheRB+),and the resulting bands were cut out of the low crosslinked polyacrylamide gel and treated with trypsin. The tryptic peptides were separated on an ion-pairing gradient and detected by liquid scintillation counting. Only the relevant portion of the map is shown; the remainder had no radioactivity except a wash through peak at the beginning. Panel A is the peptides produced by the 6297,298,3113 mutant, panel B is the 6298,311E mutant, panel C is the 6297,2983 mutant, panel D is the Q298E mutant, andpanel E is wild type. The peaks are labeled T4 through T6 in wild type in correspondence with previous work (Kehry and Dahlquist, 1982a).

by methylation of the K1 peptide. Mutants with the Q298E change produce the wild type R1 peptide, but have K1 peptides with retention times that differ from the wild type peptides. Deamidation and methylation both increase the retention time of peptides in thisHPLC gradient, andthe Q298E peptides have increased retention times from wild type, suggesting that thissite is not normally deamidatedin wild type Tsr. The altered migration of the mutantpeptides is consistent with an additional glutamine to glutamate change as compared to wild type. In contrast to the Q298E mutants, the single Q297E and Q311E mutants each produce methyl-labeled peptides that correspond to theT3, T4, T5, and T6 peaks produced by wild type Tsr (Fig. 6). The double mutant 6297,3113 also produces these peptides. This suggests that Q297 and Q311 are the normal deamidation sites and the two deamidationscan occur together, because the double Q297,311E double mutant produces the wild type peptides. Additional evidence that Q297 and Q311 are the deamidation sites is shown inFig. 7. These are methyl-labeled peptide maps produced in cheB- cells.The wild type protein produces two methyl-labeled peptides, T1 (actually a doublet, see below) and T4. The single Q297E and Q311E mutants each

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"' Fraction number FIG. 6. Peptide maps of glutamatemutantsin D311 (cheRB+).The glutamate mutants containing the Q297E and Q311E mutations were methyl-labeled and mapped as described in Fig. 5. Panel A is the peptides produced by the double Q297,311E mutant, panel B is the Q311E mutant, panel C is Q297E, and panel D is wild type peptides. Thepeaks are labeled T3 through T6 in correspondence with previous work (Kehry and Dahlquist, 1982a).

produce peptides with longer retention times, corresponding to more deamidated and methylated peptides, but do not produce the most deamidated and methylated peptides observed in wild type cells in Fig. 6. The Q297,311E double mutant in a cheB- host does produce a peptide with the same migration as the most deamidated and methylated peptides, observed for wild type Tsr in a cheRB+ host. This strongly suggests that Q297 and6311arethe two and only two deamidation sites, andthat theycan occur on the same molecule. Q297 Is Deamidated Slowly-The single glutamate mutants have enabled us to assign bands in SDS-PAGE and peaksin peptide maps to individual deamidation sites. The gels and maps discussed above revealed that the two single mutants Q297E and Q311E produce proteins and peptides with different mobilities. This is somewhat surprising since the single mutant proteins and peptides are isomers. The single mutant proteins produced in a cheRB- background have different mobilities on a Western blot, as shown in Fig. 4A. The Q297E mutantprotein runs only slightly above the wild type form, while Q311Emigrates above Q297E. The change in migration of the double mutant is approximately the sum of the two single mutant mobility changes. We therefore conclude that the lower band is produced by remains glutamine (the deamidation of Q311,whileQ297 same situation as the Q311E mutant). The higher band is produced by deamidation of both Q297 and Q311. Therefore, Q311 is deamidated rapidly (the lower protein band is not present), while Q297 is deamidated slowly, and produces the

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Fraction number FIG. 7. Peptide maps of the Q297E and Q3llE mutants in D269 cheB-). Plasmid-encoded Tsr variants with mutations at Q297 or Q311 were labeled with [3H]methionine in D269 (tsr-, AcheB) to produce methyl-labeled tryptic peptides, as described in Fig. 5. The peptides were separated by HPLC as described under "Experimental Procedures."Panel A is the peptides produced by Q297,311E, panel B is Q311, panel C is Q297E, and panel D is wild type peptides. The peaks labeled TI and T4 are usually produced by wild type Tsr in cheB- cells (Kehry and Dahlquist, 1982a). The methylation and deamidation state of each peak is also labeled; Mdenotes methylation, D is deamidation.

middle band. Alternatively, the deamidation of Q297 requires deamidation at Q311.

Methyl-accepting Sites in Tsr Previous work tentatively located four methyl-accepting sites in Tsr, but also indicated that Tsr might contain up to six sites (Kehry and Dahlquist, 1982b). To locate the additional sites, we combined peptide maps and Edman degradation of methyl-labeled tryptic peptides, similar to a method employed previously (Kehry et al., 1983a). The radioactive methyl labeling procedure is a newlydeveloped invitro method which produces highly labeled Tsrand does not require methyl-accepting sites to turnover to be labeled. The methylation reactions are carried out on membrane fragments containing Tsr overproduced using a plasmid expression system (see "Experimental Procedures"). The R l Peptide Contains Methyl-acceptingSites atPositions 493 and 502-Our previous sequenator studies had failed to identify the second methyl-accepting site in the R1 peptide although earlier reports concluded that two sites were present. We were concerned that turnover might be slow at one site, and hence the incorporation of new labeled methyl groups at such a site might be minimal. Unpurified tryptic peptides of wild type Tsr which had been methyl-labeled using our in vitro method were used for sequenator analysis. The results are shown in Fig. 8A. This profile shows that cycle11,

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Cyclenumber FIG. 8. Sequenator profiles of arginine peptides. Panel A, purified Tsr produced by pCS20 (wild type plasmid) was methyllabeled with S-[3H]adenosylmethioninei n vitro as described. The unpurified tryptic peptides were cleaved by automated Edman degradation. Radioactivity detected at each residue is plotted. Panel B, the E493A Tsr mutantmethyl-labeled in a cheB- host, which reduces the methylation of the K1 peptide, and tryptic peptides obtained as described in Fig. 5. The A2 peak was isolated and subjected to sequenator analysis as described under “Experimental Procedures.” The plot is radioactivity detected at each residue. Only 12 cleavages were made.

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Fractlon number FIG. 9. Methyl-labeled peptide maps of wild type versus the E493A mutant. E493A and wild type plasmid-encoded Tsr was labeled with [3H]methioninein D269 (tsr-, AcheB) to produce methyllabeled tryptic peptides that are methylated but not deamidated, as described in Fig. 5. Panel A is the E493A mutant peptides, and panel B is wild type peptides. Panel C is the mutant andwild type peptides combined. The wild type cells have the two usual peaks, T1 and T4. The E493A mutant has the T1 peak and two new peaks, labeled AI and A2.

corresponding to E493, is methyl-labeled, as previously observed. Cycle20, corresponding to E502, also containsa 4000 significant amount of radioactive methyl groups. We conclude that the R1 peptide contains two potential methyl-accepting 2 3000 sites at positions E493 and E502. a In order to look for low levels of methyl group incorporation 0 2000 at other sites,we blocked position E493 by an alanine substiI 4 tution. Methyl-labeled peptide maps of the E493A mutant in cheB- cells show three peaks: the doublet produced by T1 and two new peaks designated A1 and A2 (Fig. 9). These peaks both contain arginine (data not shown) and replace the T4 r,l 0 0 o 50 o 100 150 0 200 j peptide. A sequenator analysis of the HPLC-purified A2 peak is shown in Fig. 8B. Cycle 10, which corresponds to E492, has Fraction number significant radioactivity, suggesting that in the E493A mutant FIG. 10. Peptide map of peptides produced in cheB- cells. E492 can accept methyl groups. The change in retention time Plasmid pMSR141 (wild type) was labeled with [3H]methionine in between A1 and A2 is consistent with methylation of A1 to D269 (tsr-, AcheB). The tryptic peptides were produced and separated produce A2, suggesting that the R1 peptide in the E493A as described in Fig. 5. Previously only two peptides, T1 and T4, were mutant can accept two methyl groups, with the second site observed (Kehry and Dahlquist, 1982a). This map exhibits three peaks, T4,and a doublet of the T1 peak, marked T l a and Tlb. probably corresponding to E502. The K l Peptide Contains Four Methyl-accepting Sites-A previous description of methylation sites (Kehry and Dahlqu- mutant has only two deamidations, the T6 peak must have ist, 1982b) deduced that the K1peptide was methylated four an additional methylation and can be designated D2M4. We conclude that the K1peptide can accept four methyl groups. times. The peptide maps of the double glutamate mutant Contrary to our previous reports, the K1 peptide has two (Q297,311E) strengthen this conclusion. Peptide maps of the double mutant in a cheB- background have the T3, T5, and methyl-accepting sites before it is deamidated. The higher T6 peaks, which means that these peaks produced by the resolution provided by the ion-pairing HPLC gradient has Q297,311E mutant all contain two and only two deamidations. enabled us to resolve two forms of the T1 peptide in cheBThe T3 and T5 peaks have been previously identified by base cells (Fig. 10). Kehry and Dahlquist (Kehry and Dahlquist, hydrolysis as having two and threemethylations, respectively. 1982b) concluded that the T1 peptide accepted only one They can be designated D2M2 and D2M3 for two deamida- methyl group because it did not produce a second peak upon tions and two or three methylations, respectively. The T6 base hydrolysis, but they were unable to resolve the two peak was tentatively identified as having four methylations methylated forms of T1. These could represent isomeric forms and has a retention time change from T5 that is consistent of the monomethylated species, but it is also likely that the with one more deamidation or methylation. Since the double more slowly migrating form is doubly methylated. In either

Sites of Deamidation and Methylation in Tsr

9751

case, two distinct methyl-accepting sites exist before deami- T6 peptide is methylatedfour times. However, the sequenator runs have notidentified the fourth methyl-accepting site. The dation. The two deamidations a t Q297 and Q311 create two methyl- Q298 residue has been ruled out as a methyl-accepting site, accepting sites at these positions, and E304 was previously and E310 can accept methyl groups when Q311 is not deamestablished as a methyl-accepting site. The fourth site must idated. We cannotpositively identify a fourth methyl-accepting site; one or all three of the remaining glutamates, E296, be at one of the remaining glutamines or glutamates in the K1 peptide. The only remaining glutamine is Q298, but we E303,or E310,may accept methyl groups. have shown above that Q298 is not deamidated and therefore DISCUSSION cannot accept methyl groups. There are additional glutamates at residues 296, 303, and 310. These three glutamates are The results presented here identify thetwo sites of deamipossible methyl-accepting sites. dation (Q297and Q311) and five of the six methyl-accepting The tryptic peptide Sequenator analysismay reveal the siteof the fourth meth- sites in the chemotaxis transducer Tsr.K1 major sitesat ylation. We choose t o methyl-labelthe Q297,311E double contains four methyl-acceptingsites,three and 311,and one minorsite. Two of these mutant for sequenator analysisbecause it iscompletely deam- positions 297, 304, methyl-accepting sites (residues 297 and 311) are glutamines idated and may accept more methyl groups than partially deamidated wild type protein. HPLC-purifiedK1 peptide was deamidated to glutamate. Two methyl-accepting sites exist in the K 1 peptide without deamidation, as shown by the doublet subjected to sequenator analysis with the results shown in 297,304,and produced by the K1 peptide incheB- cells. The two additional Fig. 11A. The profile confirms that amino acids methyl-accepting sites are created by the two deamidations, 311 (cycles 3, 10,and 17) are methylated. The radioactivity at amino acids 298, 305,306, and 312 is most likely the result and thedouble mutant Q297,311E demonstrated that thetwo polypeptide. There is of sequenator lag (only 298 is a glutamate or glutamine in any deamidations can occur on the same some evidence that deamidation can change the reactivityof E296, E303, case). The three possible fourth methylation sites, shown by the sequenator and E310 (cycles 2, and 9, 16) are all labeled poorly, if a t all. the other methyl-accepting sites, as profile in Fig. 11. In this profile of the single Q297E mutant, Similarresults were obtainedwith wild typeTsrearlier E310 can accept methylgroups when Q311 is not deamidated, (Kehry et al., 1983,a and b). Another sequenator study was performed on the Q297E but we do not observe methylation at this site with either the mutant,shownin panel B of Fig. 11. HPLC-purified K1 double mutant or wild type protein (where Q311 is quickly peptide was methyl-labeled in a cheB- background and ana- deamidated). The locationof the fourth methyl-accepting site in theK1 lyzed to confirm that only residues 297 and 304 were methand 310 were peptide is still not certain. There areseveral explanations for ylated. Instead, it was found that 297, 304, the difficulty in defining the fourth site. One possibility is E310 canbe methylated.When Q311 isnotdeamidated, that methyl groups on the first glutamateof a pair are more detectably methylated in theQ297E mutant version of Tsr. In summary, the peptide maps have demonstrated that the labile than those on the second glutamate. The previously detected methylation sites are all on the second of the pairof glutamates, while the additional site is most likely on the first 1000 PANEL A glutamate of the pair. Another possibility is that there is a 0297,311 E limited amount of ordered methylation, which has been proI 750posed from other experiments (Springer et al., 1982; Stock CL 0 group is and Koshland, 1981). In this case, the fourth methyl 500 only acquired in an extremely small fraction of Tsr molecules I F3 and may be the first to be removed. Therefore, only a very 250 small percentage of the fourth site would be methylated and will not be detected in the sequenator analysis. Alternatively, perhaps the fourth site does not turnover very quicklyand is not methyl-labeled well enough to be detected. Another possibility is that the fourth methyl group can placed be on any PANEL B of the three glutamates which are candidates for the fourth Q297E peptide methylation site. Methyl groups would therefore be spread among the three sites, and their presence on any one site would be more difficult to establish. The sequenator profile from the Q297E mutant shows thatE310 can be methylated when Q311 is not deamidated, which makes this site a good candidate for a fourth methyl-accepting site although others may also exist. All the methyl-accepting sites are pairs of glutamate-glutamate or glutamate-glutamine residues. Canonical sequences such as -[A(S)]-X-X-E-[E/Q]-X-[A(S/T)]-A-[S/T(A)](Xis FIG. 11. Sequenator profile of the K 1 peptide. Panel A , plasany amino acid, and the residues in parentheses are less mid pMSR208 (Q297,311E) was transformed into D311 (receptor-) published and labeled with ['HJmethionine. The methyl-labeled protein was prevalent) for the methyl-accepting sites have been digested with trypsin, and the peptides were separated by HPLC, as (Terwilliger et al., 1986;Nowlin et al., 1988).Our resultsabove in Fig. 5. The T2, T3, T5, and T6 peptides which correspond to suggest that the major methyl-accepting sites occur on the differently methylated forms of the K1 peptide were combined and 2nd residue of each pair, whether theresidue is glutamine or used for sequenator analysis. The radioactivity detected in each amino glutamate. The minor sites, those which infrequently accept acid cycle is plotted above. Panel B , plasmid pMSR206 (Q297E) was labeled and prepared for sequenator analysis as described above. The methyl groups, may be located on the first glutamate of the major methylDIM1 and D1M2 peaks were isolated and used for sequenator analy- pair.For example, the R1 peptidehasone sis. The radioactivity detected in each cycle is plotted. accepting site at E493, and one minor site at E502, which is

-

h

Sites of Deamidation and Methylation in

9752

methylated at approximately 5% of the level of E493. When E493 is inactivated (in this case by mutation to alanine), another minor site, E492, can accept methyl groups, at a level comparable tothe minor site at E502. This low level of methylation makes the methyl-accepting site at E502 difficult to detect. The glutamate single mutants have also revealed that the deamidation reaction does not proceed as quickly as previously thought. Kehry and Dahlquist (Kehry and Dahlquist, 1982a; Kehry et al., 1983a) observed a cheR- gel protein banding pattern with two bands, both with slower migration than the unmodified Tsr protein. This earlier report attributed these bands to two complete, single deamidations at different sites. The gel pattern of the Q297E and Q311E mutants incheRB- cells (Fig. 4A) reveals that thelower band observed in the cheR- cells has the same migration as the Q311E mutant protein, and the upper band has the same migration as the Q297,311E protein. The cheR- wild type bands produced are therefore the doubly deamidated protein and a band from the single deamidation of Q297. The deamidation reaction at Q311 proceeds rapidly, since the lowest band, which would be produced by the Q297E mutant, is not observed. The two cheR- bands areof roughly equal intensity, giving a half-life for deamidation of Q297 of approximately 60 min. Repellents increase deamidation (Rollins and Dahlquist, 1981) and therefore this half-life may vary depending on the experimental conditions. The determination of deamidation and methylation sites in Tsr reveals that Tsr has two sites of deamidation, in agreement with the other transducers for which the number of deamidations is known. Tsr can accept six methyl groups, which ismore than Tar with four sitesor Trg with five methyl-accepting sites (Tenvilliger et al., 1986; Nowlin e t al., 1987). Only Tsr has two methyl-accepting sites on the R1 peptide (Kehry andDahlquist, 1982; Nowlinet al., 1987). Tsr is themajor transducer, comprising approximately half of the transducers in wild type cells (Engstrom e t al., 1983), and responds to a wide variety of stimuli, including temperature, pH, amino acids, and weak acids (Kleene e t al., 1979; Kihara and MacNab, 1981; Hedblom and Adler, 1983; Imae e t al., 1987; Leee t al., 1988). The six methyl-accepting sites may be necessary to provide the appropriate adaptational sensitivity to all these stimuli. The role of deamidation in bacterial chemotaxis is still not well understood. The rate of deamidation is increased by repellents and may be a primitive response mechanism that was superseded by reversible methylation. Deamidation reactions may also be necessary to create newly translated transducers in a neutral signaling form. If 2 glutamines are present, then two of the six methyl-accepting sites are “neutralized” and do not signal such an undermethylated state. The glutamate mutants described in this paper have already shown that deamidation reactions occur moreslowly than previously thought andwill behelpful in determining the role of deamidation in bacterial chemotaxis. Acknowledgments-We thank J. S. Parkinson, G. L. Hazelbauer, and M. I. Simon for the kind gift of strains used in this work, C. Sprecher for creating some of the glutamate mutants, and D.R. Graham for proteins and antibodies. REFERENCES Borkovich, K. A., Kaplan, N., Hess, J. F., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A . 8 6 , 1208-1212 Bourret, R. B., Hess, J. F., Borkovich, K. A., Pakula, A.A., and Simon, M. I. (1989) J. Biol. Chem. 264,7085-7088 Boyd, A., and Simon, M. I. (1980) J. Bacteriol. 143,809-815

Tsr

Callahan, A. M., Frazier, B. L., and Parkinson, J. S. (1987) J . Bacteriol. 1 6 9 , 1246-1253 Chelsky, D., and Dahlquist, F. W . (1980) Proc. Natl. Acad. Sci. U. S. A . 77,2434-2438 Chelsky, D., and Dahlquist, F. W . (1981) Biochemistry 2 0 , 977-982 Clark, D. J., andMaaloe, 0.(1967) J. Mol. Biol. 2 3 , 99-112 DeFranco, A. L., and Koshland, D. E., Jr. (1980) Proc. Natl. Acad. Sci. U. S. A. 7 7 , 2429-2433 DeFranco, A. L., Parkinson, J. S., and Koshland, D. E., Jr. (1979) J. Bacteriol. 1 3 9 , 107-114 Engstrom, P., Nowlin, D., Bollinger, J., Magnuson, N., and Hazelbauer, G. L. (1983) J. Bacteriol. 1 5 6 , 1268-1274 Goy, M. F., Springer, M. S., and Adler, J. (1977) Proc. Natl. Acad. Sci. U. S. A . 74,4964-4968 Hayashi, H.,Koiwai, O., and Kozuka, M. (1979) J. Biochem. (Tokyo) 85,1213-1223 Hazelbauer, G. L. (1988) Can. J. Microbiol. 3 4 , 466-474 Hazelbauer, G. L., Park, C., and Nowlin, D.M. (1989) Proc. Natl. Acad. Sci. U. S. A . 86,1448-1452 Hedblom, M. L., and Adler, J. (1983) J. Bacteriol. 155, 1463-1466 Hess, J. F.,Oosawa,K., Matsumura, P., and Simon, M. I. (1987) Proc. Natl. Acad. Sci. U. S. A . 8 4 , 7609-7613 Hess, J. F., Bourret, J. P., and Simon, M. I. (1988) Nature 336,139143 Imae, Y., Oosawa, K., Mizuno, T., Kihara, M., and Macnab, R. M. (1987) J. Bacteriol. 1 6 9 , 371-379 Kehry, M. R., and Dahlquist, F. W . (1982a) Cell 2 9 , 761-772 Kehrv, M. R., and Dahlquist, . F. W . (1982b) J. Biol. Chem. 257, 10378-10386 Kehrv. M. R.. Dahlouist. F. W . . and Bond. M. W . (1983a) in Mobilitv and kecogn’ition in Ceil Biology: Proceedhgs (Sund, H.,’andVeege;, C., eds) pp. 533-549, Walter de Gruyter & Co., New York Kehry, M. R., Bond, M. W . , Hunkapiller, M. W., and Dahlquist, F. W . (1983b) Proc. Natl. Acad. Sci. U. S. A . 8 0 , 3599-3603 Kehry, M. R., Engstrom, P., Dahlquist, F. W . , and Hazelbauer, G. L. (1983~)J. Biol. Chem. 258, 5050-5055 Kihara, M., and MacNab, R. M. (1981) J. Bacteriol. 145, 1209-1221 Kleene, S. J., Hobson, A.C., and Adler, J. (1979) Proc. Natl. Acad. Sci. U. S. A . 76,6309-6313 Kleene, S. J., Toews, M. L., and Adler, J. (1977) J. Biol. Chem. 252, 3214-3218 Kondoh, H., Ball, C. B., and Adler, J. (1979) Proc. Natl. Acad. Sci. U. S. A . 7 6 , l l - 1 5 Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1988) Methods Enzymol. 1 5 4 , 367-382 Lee, L., Mizuno, T., and Imae, Y. (1988) J. Bacteriol. 1 7 0 , 47694774 Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY Muchmore, D. C., McIntosh, L. P., Russell, C. B., Anderson, D. E., and Dahlquist, F. W . (1989) Methods Enzymol. 177, 44-73 Nowlin, D. M., Bollinger, J., and Hazelbauer, G. L. (1987) J . Biol. Chem. 262,6039-6045 Nowlin, D. M., Bollinger, J., and Hazelbauer, G. L. (1988) Proteins 3,102-112 Osborn, M. J., and Munson, R. (1974) Methods Enzymol. 31A, 642653 Ridgway, H. F., Silverman, M., and Simon, M. I. (1977) J. Bacteriol. 132,657-665 Rollins, C., and Dahlquist, F. W . (1981) Cell 25, 333-340 Russell, C. B., Thaler, D. S., and Dahlquist, F. W . (1989) J. Bacteriol. 171,2609-2613 Sanders, D. A., Gillece-Castro, B. L., Stock, A. M., Burlingame, A. L., and Koshland, D. E., Jr. (1989) J. Biol. Chem. 2 6 4 , 2177021778 Sherris. D.. and Parkinson. J. S. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,6051-6055 Silverman, M., and Simon, M. (1977) Proc. Natl. Acad. Sci. U. S. A . 74,3317-3321 Springer, W . R., and Koshland, D. E., Jr. (1977) Proc. Natl. Acad. Sci. U. S. A . 74,533-537 Springer, M. S., Goy, M. F., and Adler, J. (1977) Proc. Natl. Acad. Sci. U. S. A . 74,3312-3316 Springer, M. S., Zanolari, B., and Pierzchala, P. A. (1982) J. Biol. Chem. 257,6861-6866

Sites of Deamidation and Methylationi n Tsr Stewart, R. C., and Dahlquist, F. W. (1987) Chem. Rev. 87,997-1025 Stewart. R. C.. and Dahlauist. F. W. (1988) J. Bucteriol. 1 7 0 , 57285738 Stock. J. B.. and Koshland, D. E., Jr. (1978) Proc. Nutl. A c d . Sci. U. S. A . 75,'3659-3663 Stock. J. B., and Koshland, D. E., Jr. (1981) J. Biol. Chem. 2 5 6 , 10826-10833 .

I

Tenvilliger, T. C., Wang, J. Y., and Koshland, D. E., Jr. (1986) J. Biol. Chem. 2 6 1 , 10814-10820 VanDerWerf, P., and Koshland, D. E., Jr. (1977) J. Biol. Chm. 252,2793-2795 Weis, R. M., and Koshland, D.E., Jr. (1988) Proc. Nutl. Acud. Sci. U. S. A. 85,83-87

A List of Tar Plasnida pcs20 pMSR131

pHSRl4l pMSF202 pMSRZO5 pMSR206 pnsP.201 plSR208 pUSR210 pMSR212 pMSR422

TABU 1

E.

063 D90 091 Dl11

0227 0228 0260 0269 0283 0290 0311

D349 D347 D405 Dl14 0413

wild

type

3.3. Parkinaon, -487 5 . 5 . Parkinaon, ~ 9 3 8 4 1

M. simon, ~ ~ 5 8 8 4 Parkinaon, -2896 J . S . Parkinson. R94968 J.S. Parkinaon, -4972 J . S . Parkinson, W 3 0 9 8 C. Sprecher, th&e lab by P1 tramduction of D31 to 0228. J S . Parkinaon, Rp5114 R . C . Stewart, thze 1aD. G.L. wazelbruer, C P 3 6 z J.S.

96.

9753