Escherichia coli H+-ATPase: Loss of the carboxyl terminal region of the γ subunit causes defective assembly of the F1 portion

ARCHIVESOFBIOCHEMISTRYAND BIOPHYSICS Vol. 251, No. 2, December, pp. 458-464,1986

Escherichia co/i H+-ATPase: Loss of the Carboxyl Terminal Region of the y Subunit Causes Defective Assembly of the F, Portion JUNJI MIKI, MICHIYASU TAKEYAMA, TAKATO NOUMI, HIROSHI KANAZAWA: MASATOMO MAEDA, AND MASAMITSU FUTA12 Department of Organic Chemistry and Biochemistry, The Institute of S&&i& and Industrial Research, Osaka Unive-rsity, Ibaraki, Osaka 567, Japan Received May 27,1986, and in revised form August 81986

Mutant genes for the y subunit of H+-translocating ATPase (H+-ATPase) were cloned from eight different strains of Eschmichiu coli isolated in this laboratory. Determination of their nucleotide sequences revealed that they are amber nonsense mutations: a Gln codon at position 15,158,227,262, and 270, respectively, was replaced by a termination codon in these strains. As terminal Met is missing in the y subunit, these results indicate that these strains are capable of synthesizing fragments of y subunits of 13,156, 225, 260, and 268 amino acid residues, respectively. Studies on the properties of membranes of these strains suggested the importance of the region between Gln 269 and the carboxyl terminus (residue 286) for forming a stable F1 complex with ATPase activity and the region between Gln 226 and Gln 261 for normal interaction of F1 with FO.The sequence from Gln 261 to Gln 269 also seemed to be important for stability of F1 assembly on the membranes. The high frequency of the nonsense mutations suggested that the number of essential residues is limited in this subunit. Comparison of the homologies of the amino acid sequences of the y subunits from four different sources confirmed this notion: 19% of amino acid residues are identically conserved in these four strains, and the conserved regions are the amino terminal and carboxyl terminal regions. o 19% Academic Press. Inc.

The H+-ATPase (FOF1)of Escherichia coli catalyzes the synthesis of ATP utilizing energy derived from an electrochemical gradient of H+ across the cytoplasmic membranes (l-4). The nucleotide sequences of the genes (uric operon) for the F,-,Fl subunits and their encoded amino acid sequences have been determined. The catalytic portion F1 consists of five different subunits (a, B, y, 6, and E). The y subunit plays crucial roles in assembly and function of the entire FoFl. This subunit is essential for the formation of a catalytic complex in i Permanent address: National Cancer Center Research Institute, Tokyo 164, Japan. a To whom correspondence should be addressed at ISIR-Sanken, Osaka University, Suita Campus, 8-l Mihogaoka, Ibaraki, Osaka 567, Japan. 0003-9861/86 $3.00 Copyright All rights

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

the complex with ATPase activity could be reconstituted by recombination of isolated (Y,/3, and y subunits from E. coli, but not from a! and /3 subunits only (5, 6). The F1 could be reconstituted from the c@y complex and 6 and c subunits (6) possibly through a variety of subunit-subunit interactions, including that of y and E subunits (7). Studies with chloroplasts (8) and a thermophilic F1 (9) suggested that the y subunit acts as a gate for the H+-pathway, playing a key role in H+-translocation coupled with ATP synthesis or hydrolysis. The genetic approach using E. coli may be very useful for understanding the roles of the y subunit in assembly and catalysis, because a specific amino acid replacement can be introduced by mutation, and functional alterations can be analyzed both in

vitro:

458

ROLE

OF C-TERMINAL

REGION

viva and in vitro (1). We previously analyzed five mutants with defective y subunit and located mutations in defined DNA segments (10). One of these strains, NR70, had a deletion of seven amino acid residues (residues 21 to 27), resulting in loss of assembly of F1 in wivo (11). The deleted residues are in the highly conserved region of the subunit of four different species (1216). In this study we cloned mutant alleles from four previous strains and also from four newly isolated strains. Determination of their nucleoticle sequences revealed that all these strains were amber mutants capable of forming only fragments of the subunit lacking carboxyl terminal regions. The F1 portions of these strains did not have ATPase activity and were not properly assembled, suggesting the essential roles of the carboxyl terminal region of the subunit for F1 assembly. EXPERIMENTAL

PROCEDURES

Bacterial strains and growth conditions. E. wli wildtype strain KY7230 (thy-, thi-, asn-, recA+) and its y subunit mutants (uncG-, thy-, thi-, recA+) KFl, KFlO, KF12, and KF13 were as described previously (lo), and similar strains KF21, KF68, KF84, and KF88 were isolated in this study by the method described previously (10). Minimal medium supplemented with thymine (100 pg/ml), thiamine (2 &ml), and a carbon source (0.5% either glucose or succinate) and rich me-

OF H+-ATPase

SUBUNIT

Y

459

dium (L-broth) were used for genetic analysis. The same minimal medium with 0.5% glycerol was used for the preparation of membranes (10). For isolation of strains carrying antibiotic resistance, 20 Kg/ml of ampicillin or tetracycline was added to L-broth agar plate. Plasmids and genetic analysis Hybrid plasmids pFT1501, pFT302, pTN1606, and pTN1607 carrying various portions of the gene cluster for ATPase were constructed previously (10) (Fig. 1). Plasmid DNA and competent cells were prepared by a published method (17), and complementation and recombination tests were carried out as described previously (10).

Cloning the mutant aUelea coding for the y subunit. Total chromosomal DNA of the mutant (KF12, KF13, KF21, or KF84) mapped in the carboxyl terminal region was digested with Sal1 and EwRI (Fig. 1) and subjected to electrophoresis on polyacrylamide gel (11). The fraction of DNA fragments corresponding to 696 bp (SC&-EwRIz) was eluted from the gel matrix and ligated with pBR322 digested with the corresponding endonucleases. The ligated plasmid was introduced into strain KFl (a mutant mapped in the middle of the y subunit). Cells with plasmids carrying mutant genes showed ampicillin resistant and tetracycline sensitive phenotypes and became able to grow on succinate (Uric+ phenotype) utilizing oxidative phosphorylation after recombination of chromosomal and plasmid DNA. Mutant DNA cloned was further digested with Hind111 and EcoRI and the 440 bp DNA fragment was ligated into pUC18 for determination of the DNA sequence (18). pFT302 was digested with appropriate endonucleases and fragments were used as standards for molecular weight estimation. For cloning the mutant gene of KFlO, total chromosomal

FIG. 1. Portions of the gene for the y subunit carried by recombinant plasmids. DNA segments carried by recombinant plasmids are shown with reading frames for a, y, and @ subunits. (A) Reading frames of the a, 7, and B subunits. As amino terminal Met is missing in the isolated y subunit (7), amino acid residues are numbered from the second codon. (B) Sites of E. cdi chromosomal DNA cleaved by restriction endonucleases. Numbers indicate base pairs starting from the BgZII cleavage site. (C) DNA segments carried by each hybrid plasmid. See text for further details.

460

MIKI

DNA was digested with B&II and HindIII, and 1036bp-long DNA was isolated and ligated with pBR322 digested with BamHI and HindIII. The cloned DNA fragment was cut out with Sal1 and a 412-bp DNA fragment was recloned into pUC18. For KFl, KF68, and KF88, essentially the same procedure was used except that cloned DNA was cut out with Sal1 and HindIII. Other procedures. Membranes were prepared from cells (late logarithmic phase) that had been passed through a French press (10) and washed with dilute buffer containing EDTA to remove F1 (19). The amounts of (Y and fl subunits in mutant membranes were determined quantitatively by an immunochemical method (20). Membranes (unwashed, 150 ng protein) from each strain were incubated in 1.0% SDSx containing 2.0% 6-mercaptoethanol for 5 min in a boiling water bath and applied to 12.5% polyacrylamide gel containing 0.1% SDS. After electrophoresis, the protein bands on the polyacrylamide gel were transferred electrophoretically to nitrocellulose paper. The paper was incubated with FITC (fluorescein isothiocyanate)-labeled anti-F1 IgG (immunoglobulin G) obtained previously (20). The paper was washed extensively and dried at room temperature. The fluorescence on the paper was scanned with a Shimadzu dual wave length TLC Scanner CS930 and the peak areas corresponding to the position of the (Yor fi subunit from the wild-type and mutant membranes were compared. The ATPase activity (21), formation of an electrochemical gradient of protons (lo), and amount of protein (22) were assayed by reported procedures. F1 was prepared from E. wli ML 308-225 (21). Materials. Restriction endonucleases and T4 DNA ligase were purchased from Takara Shuzo Company, Kyoto, Japan. cyaaP-dCTP (400 Ci/mmol) was purchased from Amersham Corp. Other reagents used were of the highest grade available commercially. RESULTS

Identi)!cation of Mutants Defective in the y Subunit Mutants defective in the y subunit were identified using plasmid pFT302 carrying the entire cistron for the y subunit and roughly mapped using pTN1606, pTN1607, and pFT1501 carrying amino terminal (from the amino terminus to residue 81), central (residue 82 to 166), and carboxyl terminal (residue 167 to the carboxyl terminus) portions of the cistron, respectively (Fig. 1). The results of mapping showed a Abbreviations used: DCCD, dicyclohexylcarbodiimide; SDS, sodium dodecyl sulfate; FITC, fluorescein isothiocyanate.

ET AL.

that KFlO had a defect in the amino terminal portion of the y subunit cistron, KFl, in the central portion, and KF12 and KF13, in the carboxyl terminal portion (10). Similarly, the strains isolated in this study, KF68 and KF88, and KF21 and KF84, were found to have defects in the central and carboxyl terminal portions, respectively. The DNA fragment corresponding to the defective region was cloned from each strain and its nucleotide sequence was determined. It is interesting that all eight strains were amber mutants in which Gln codons (CAG) were replaced by the termination codons (TAG) (Table I). Of all the Gln residues replaced in the mutants, only Gln 269 is conserved in amino acid sequences of the y subunit from E. wli (12, 13), Rhodopseudomonas blustica (14), Rhodospirillum rubrum (15), and bovine heart mitochondria (16). Absence of ATPase Activity and Normal Assemblies of Fl in Mutant Membranes The specific activity of membrane ATPase of all the mutants was less than 2% of that of the wild type, and their cytoplasmic fractions did not show significant activity (Table II). These results suggest

TABLE

I

DETERMINATION OF MUTATION SITES IN STRAINS DEFECTIVE IN THE y SUBUNIT

Strain

Gln residue replaced”

Conservation of Gln residues”

KFlO KFl, KF68, KF88 KF21 KF84 KF12, KF13

14 157 226 261 269

-e -d

+

a In all cases Gln (CAG) codons were replaced by the termination codon (TAG). As indicated previously, the isolated y subunit does not have a Met residue at the amino terminus and so residues are numbered from Ala residue (second codon) (7). *Residues conserved (+) or not conserved (-) in E. wli, R. blastica, R. r&rum, and the bovine subunits are indicated. ‘This Gln is conserved except in they subunit from E. coli d This Gln is conserved except in the y subunit from bovine heart mitochondria.

ROLE OF C-TERMINAL TABLE II ATPASE AGTIVITIESOF MEMBRANESAND

REGION OF H+-ATPase SUBUNIT y

461

(10). In this study the amounts of the (Yand ,!?subunits in unwashed membranes were CYTOPLASMOFMUTANTS determined immunochemically. KFl, KFlO, KF21, and KF84 had about 10% as ATPase activity (units/mg) much fi subunit as the wild type, and no Strain Membranes Cytoplasm detectable (Y subunit, suggesting that in these strains assembly of F1 was incomKY7230 (wild) 1.5 0.24 plete, or assembled F1 was unstable and KFlO 0.010 0.05 degraded during preparation of memKFl 0.016 0.06 branes. On the other hand, the amounts of KF21 0.025 0.04 KF84 (Yand fi subunits in KF12 were about half 0.018 0.04 KF12 0.016 0.05 those in the wild type. It was of interest to know whether other Note. Membrane vesicles (Membranes) and the cyminor subunits (7, 6, and C)were released toplasmic fraction (Cytoplasm) were prepared as defrom membranes by washing or whether scribed previously (10). The amounts of protein recovered in the two fractions were similar for all the they remained on the membranes. The strains tested. ATPase activity was assayed by a re- washed membranes of the wild type, KF12 and KF84, could bind purified normal F1 ported method (21). and showed about the same specific activthat the F1’s of the mutant membranes ities as unwashed wild-type membranes were defective in ATPase activity or were (Table III). On the other hand, washed not properly assembled into the mem- membranes of KFl, KFlO and KF21 branes. For further characterization of the showed 40-50s of the specific activities of mutant membranes, they were washed unwashed wild-type membranes after rewith dilute buffer containing EDTA under binding of F1. These results suggest that conditions that released normal F1 from all the F. portions in the membranes of wild-type membranes and the extracts strains KF12 and KF84 were exposed after (EDTA extracts) were subjected to elec- washing with EDTA, whereas about half trophoresis on polyacrylamide gel. The the F0 portions in washed membranes of EDTA extract of KF12 contained protein KFl, KFlO, and KF21 remained possibly bands corresponding to the CYand /3 sub- occupied by defective complexes of F1. units, and their amounts were about half These defective complexes may consist of those in the wild-type extract. These pro- the p subunit and subunits other than the tein bands could not be detected in extracts (Ysubunit, because the latter was not deof the other strains. As expected, only neg- tectable as described above. However, most ligible ATPase activities were detectable of the defective complexes may be formed from 6, t, and y subunits, because memin the mutant extracts: the ATPase activity branes of KFl, KFlO, and KF21 had only in wild-type EDTA extract was 9.0 units/ 10% as much /3 subunit as the wild type. mg protein, whereas those in mutant exAn alternative interpretation of the above tracts were less than 0.1 unit/mg protein. The fact that only traces of the (Yand /3 results is that about half of F1 binding sites subunits were present in EDTA extracts of in KFl, KFlO, and KF21 may be degraded most of the mutants raised the question of during preparation of membranes. This whether these subunits were present in seems to be unlikely because similar treatmembranes. Previously we showed by the ment of membranes of wild type, KF12 and two-dimensional gel electrophoresis that KF84, did not degrade F1 binding sites in the amounts of both subunits were much FO. However, the degradation of sites in less in unwashed membranes of KFl and viva could not be excluded. KFlO than in those of the wild type, whereas the amounts in membranes of Formation of a Respi~atcny or ATPDependent Proton Gradient in KF12 were about half those in the wild type Membranes from Mutants (10). Moreover immunochemical analysis indicated that the (Y, /3, y, and c subunits The formation of a proton gradient in were present in the membranes of KF12 membranes was determined semiquanti-

462

MIKI TABLE III

ET AL.

not attached to a defective F1 complex. When F1 was added to the mutant membranes, respiration-dependent quenching ATPase activity reached essentially the maximal level, of membranes while ATP-dependent quenching was about (units/mg protein) half of the maximal level of respirationdependent quenching, suggesting that the Strain +FI -FI amount of active FoFl in these membranes KY7230 (wild) 1.4 (1OO)Q 0.086 was much less than that in wild-type KFlO (GlniJ 0.54 (39) 0.010 membranes. 0.59 (42) 0.006 KF1 Glnd When membranes of the wild type had KF21 (GIna) 0.71 (51) 0.007 been washed to remove Fi, only a low level KF84 (Gln& 1.3 (93) 0.021 of quenching was observed with ATP or KF12 (Gln& 1.4 (100) 0.011 lactate, indicating that F,, portions were Note Membrane vesicles (about 1.5mg protein) from exposed and the membranes became almost completely permeable to protons as each strain were suspended in 8.0 ml of 1.0 mM TrisHCl (pH 8.0) containing 0.5 mM EDTA and 10% glycreported previously (10). The respiratory erol, and the suspension was centrifuged at 100,OOOg or ATP-dependent quenching was reconfor 120 min. The resulting pellet (washed membranes) stituted by rebinding F1 to the washed was suspended in 10 rnreTris-HCl (pH 8.0) containing membranes (Fig. 2A). This was also essen5 mM MgSO,, 140 mM KCl, 2 rnrd fl-mercaptoethanol, tially the case with KF12: the respiratory and 10% glycerol. The washed membranes (2 mg proquenching was greatly reduced by washing tein) were incubated with 1.3 mg of purified F1 (from ML308225) in 2 ml of the same buffer for 15 min at and completely restored by adding F1, and 20°C. The mixture was centrifuged at 100,OOOg for 120 the ATP-dependent quenching after remin after addition of 23 ml of buffer. The pellet was constitution was significantly greater than washed with 25 ml of the same buffer and finally sus- that of the unwashed membranes (Fig. 2B). pended at 1.0 mg/ml. ATPase activity of washed However, the washed and unwashed memmembranes with (left column) or without (right col- branes of KFl showed essentially the same umn) F1 were assayed. The strains are shown together magnitude of respiratory quenching. Furwith the positions of mutations (in parentheses). thermore the respiratory or ATP-depen’ Percentage ATPase activities is shown in parendent quenching after F1 binding was simtheses. ilar to that of unwashed membranes (Fig. 2C). These results suggest that the defectatively by measuring quenching of quin- tive F1 complex of KF12 could be released acrine fluorescence (Fig. 2). As expected, from the membranes by extensive ,washing, whereas that of KFl could not. Membranes no ATP-dependent quenching of quinacrine fluorescence was observed with membranes of KF84 were similar to those of KF12, but from any of the mutants (Figs. 2B, C). slightly more leaky. Membranes of KFlO When lactate was added to membranes of and KF21 showed the same properties as mutants to drive respiration, quenching those of KFl. These results are consistent was observed, indicating that a proton with the above finding that washed memgradient was established. However, mutant branes from KFl, KFlO, and KF21 could membranes showed less quenching than rebind much less F1 than those of wild type, those of the wild type, indicating that they KF12 and KF84 (Table III). were partially permeable to H+. DCCD, DISCUSSION which is known to block the H+-pathway All the mutants isolated were found to of Fo, only slightly affected quenching of the wild type (Fig. 2A). On the other hand, be nonsense (amber) y subunit mutants. We isolated these strains by mutagenesis the extent of quenching in KF12 and KFl membranes increased on addition of DCCD using a transducing phage Pl. The phages to a similar maximal level to that with were mutagenized with hydroxylamine, wild-type membranes (Figs. 2B, C). These which changes GC to AT but does not specifically induce nonsense mutations. Acresults suggest that part of the population of F,, complexes in mutant membranes was cording to a simple calculation based on BINDING

OF F1 TO WASHED

MEMBRANES

OF MUTANTS

ROLE A

KY7230

OF C-TERMINAL (wild

type)

REGION B KF12

OF H+-ATPase

(Gln269)

end 1

ATP

F, +Lac.

-

1

ATP

-

v

F, +ATP

SUBUNIT C KFl

v

q

(Gln157)

463

y end)

ATP

Ft +Lac.

DCCD

ATP

F, +Lac.

c DCCD

FIG. 2. Formation of a proton gradient in membrane vesicles of mutants measured by quenching of quinacrine fluorescence. Membrane vesicles (200 pg) from the wild-type (A) and mutants KF12 (B) and KFl (C) in 2.0 ml of 10 mM Tricine-choline buffer (pH 8.0), containing 140 mM choline chloride and 1 PM quinacrine, were mixed with 20 pl of 1 M MgClc and then fluorescence (emission, 500 nm; excitation, 420 nm) was monitored in a Hitachi fluorescence spectrophotometer F3000. Washed membrane vesicles were prepared as described in the legend of Table III. At the indicated or 2 ~1 of 20 mM DCCD (ethanol solution) was times, 10 ~1 of 0.20 M ATP, 10 ~1 of 1.0 M D-kictate, added. For Fi + ATP or F1 + lac, 20 pg of purified wild-type F1 was added to reaction mixtures containing membranes and then the mixtures were incubated for 10 min before adding ATP or Dlactate.

the DNA sequence of the gene and the mechanism of action of hydroxylamine, the possibility of obtaining amino acid replacements is about ll-fold higher than that of nonsense mutations. Therefore, our results suggest that amino acid replacements in the y subunit, if any in our mutagenesis experiments, did not result in a serious defect(s) in function or assembly of the entire complex and that the numbers of essential residues are limited in the y subunit. Studies on the homologies of amino acid sequences of the y subunits from different sources confirmed this notion: comparison of the sequences of the y subunits from E. coli (12, 13), R. bhtim (14), R. r&rum (15), and bovine heart (16) indicated that 19% of the amino acid residues are identically conserved (Fig. 3). The conservations of the (Yand @subunits are much higher than that of the y subunits: 45 and 63% ,respectively, of the amino acid

residues in the (Y and p subunits of the above four species are conserved. It is noteworthy that the amino and carboxyl terminal regions of the y subunit are highly conserved (Fig. 3). Thus loss of the conserved carboxyl terminal region by nonsense mutation resulted in a serious defect(s) in the entire Fi, as shown above. Similarly, deletion in the conserved region near the amino terminus resulted in an unassembled F1 complex, as shown previously (11). From the results of this study it seems possible to discuss the role(s) of the carboxy1 terminal region of the y subunit. Immunochemical studies have suggested that KF12 (lacking from Gln 269 to the carboxyl terminus) has defective F1 containing LY,p, y, and c subunits, although it was easily dissociated during purification (10). Thus the region between Gln 269 and the carboxy1 terminus (residue 286) may be es-

464

MIKI

ET AL.

and Technology from the Science and Technology Agency of the Japanese Government, and a grant from the Naito Memorial Foundation. A N

C

REFERENCES

: 8

I 14

IA

non- conserved

I I

157

226

251259;

A

A

AA

FIG. 3. Schematic illustration of conserved amino acid residues in y subunits from four different species. (A) Amino acid sequences of y subunits from E. wli (286 amino acid residues), R. blastica (286 residues), R ?ubrum (299 residues), and beef heart mitochondria (272 residues) aligned to obtain maximal homology. The regions deleted in the E. wli sequence were omitted and residues are numbered according to the E. coli sequence. The positions of identical residues in the four species are indicated by upward vertical bars on the schematic y subunit and those having four different residues by downward vertical bars. Conserved regions are near the amino (N) and carboxyl (C) termini. (B) Positions of nonsense mutations shown by triangles.

sential for formation of a stable F1 complex with ATPase activity. Strain KF84 (lacking Gln 261 to the carboxyl terminus) had defective complexes of F1 subunits and it could be released by washing the membranes with dilute buffer, suggesting that the region between Gln 261 and the carboxy1 terminus, or possibly between Gln 261 and Gln 269, may be important for stable assembly of F1 on the membranes. Other strains KFlO (lacking residues from Gln 14 to the end), KFl (Gln 157 to the end), and KF21 (Gln 226 to the end) had defective complexes of F1 subunits that could not be released from the membranes. Thus the region between Gln 226 and Gln 261 may be important for forming an F1 assembly that interacts normally with F,,. It is of interest to know whether any single amino acid residues in these regions are essential for assembly of Fr. ACKNOWLEDGMENTS We are grateful to Ms. H. Hama and Ms. Y. Sakai for technical assistance in the early stage of this study. This study was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan, a Special Coordination Fund for Promotion of Science

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2. WALKER,J. E., SARASTE,M., ANDGAY, N. J. (1984) Biochim Biophys. Acta 768, X4-200. 3. SENIOR,A. E. (1985) Cum: Top. Membr. Transp. 23,135-151. 4. FILLINGAME,R. H. (1980) Annu. Rev. B&hem 49, 1079-1113. 5. FUTAI, M. (1977) Biochem Biophys. Res. Cwmmun 79,1231-1237. 6. DUNN, S. D., AND FUTAI, M. (1980) J. Bid C&m 255,113-118. 7. DUNN, S. D. (1982) J. Biol Chem. 257,7354-7359. 8. MCCARTY,R. E. (1982) Membrane and Transport (Martonosi, A. N., ed.), Vol. 2, pp. 599-603. 9. YOSHIDA,M., OKAMOTO,H., SONE,N., HIRATA, H., AND KAGAWA, Y. (1977) Proc NatL Acud Sci. USA 74.936-940.

10. KANAZAWA,H., NOUMI,T., FUTAI, M., ANDNITTA, T. (1983) Arch Biochem. Biophya 223,521-532. 11. KANAZAWA,H., HAMA, H., ROSEN,B. P., ANDFUTAI, M. (1985) Arch B&hem Biophys. 241,364-370. 12. SARASTE,M., GAY, N. J., EBERLE,A., R~NSWICK, M. J., AND WALKER, R. E. (1981) Nucleic Acid Res. 9,5287-5296.

13. KANAZAWA, H., KAYANO, T., MABUCHI, K., AND FUTAI, M. (1981) B&hem Biophys. Res. Cornmun 103.604-612. 14. TYBULEWICZ,V. L. J., FALK, G., ANDWALKER,J. E. (1984) J. Md Biol. 179.185-214. 15. FALK, G., HAMPE, A., AND WALKER, J. E. (1985) Biochem. J. 228.391-407.

16. WALKER,J. E., FEARNLEY,I. M., GAY, N. J., GIBSON, B. W., NORTHROP,F. D., POWELL,S. J., RUNSWICK, M. J., SARASTE,M., AND TYBULEWICZ, V. L. J. (1985) J. Mel Bid 184,677-701. 17. NOUMI, T., MOSHER,M. E., NATORI, S., FUTAI, M., AND KANAZAWA, H. (1984) J. BioL Chem. 259, 10071-10075. 18. SANGER,F., COULSON, A. R., BARRELL,B. G., SMITH, A. J. H., ANDROE,B. A. (1981) J. Mol. Bid 143, 161-178. 19. KANAZAWA,H., HORIUCHI,Y., TAKAGI, M., ISHINO, Y., AND FUTAI, M. (1980) J. Biochem. 88, 695703. 20. NOUMI,T., OKA, N., KANAZAWA,H., ANDFUTAI, M. (1986) J. Bid Chem. 261.7070-7075. 21. FUTAI, M., STERNWEIS,P. C., AND HEPPEL, L. A. (1974) Proc Nat1 AC&. Sci. USA 71,2725-2729. 22. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem 193, 265-275.