Proc. Nati. Acad. Sci. USA Vol. 81, pp. 3019-3023, May 1984 Biochemistry Internal homologies in the two aspartokinase-homoserine dehydrogenases of Escherichia coli K-12 (gene duplication/evolution/bifunctional proteins) PASCUAL FERRARA, NATHALIE DUCHANGE, MARIO M. ZAKIN, AND GEORGES N. COHEN The Unite de Biochimie Cellulaire, Ddpartement de Biochimie et Gdndtique Moldculaire, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France Communicated by Norman H. Horowitz, January 27, 1984

ABSTRACT In Escherichia coli, AK I-HDH I and AK II- METHODS HDH II are two bifunctional proteins, derived from a common ancestor, that catalyze the first and third reactions of the com- Protein sequence comparisons were done by computer. mon pathway leading to threonine and methionine. An exten- Each sequence was compared with itself by a simple frame- sive amino acid sequence comparison of both molecules reveals shift method using different segment lengths. After small ho- two main features on each of them: (i) two segments, each of mologous fragments were found, comparison was extended about 130 amino acids, covering the first one-third of the poly- in both directions, allowing gaps to maximize homologies. peptide chain, are similar to each other and (ii) two segments, Finally, the fragments were aligned with a computer pro- each of about 250 amino acids and covering the COOH-termi- gram, provided by T. F. Smith and W. M. Fitch, using the nal 500 amino acids also present a significant homology. These matrix algorithm of Sellers (8), modified by Smith et al. (9). findings suggest that these two regions may have evolved inde- A deletion weight of 0.7 for each gap plus 1.5 times the pendently of each other by a process of gene duplication and number of residues in each gap was initially used. Some of fusion previous to the appearance of an ancestral aspartoki- the alignments were also checked using a deletion weight nase-homoserine dehydrogenase molecule. of 1.5 for each gap and 1.5 times the number of residues in each gap. Unmatched terminal sequences were always In Escherichia coli, aspartokinase I-homoserine dehydroge- weighted. nase I and aspartokinase TI-homoserine dehydrogenase II The number of introduced gaps in the compared se- are two bifunctional proteins that catalyze the first and third quences is between 0.22 and 0.38 times the number of identi- reaction of the common pathway leading to threonine and ties plus homologies produced by their introduction. If we methionine (1). Recently, the sequence of the corresponding restrict ourselves to identities, this ratio varies between 0.42 genes, thrA and metL has been established in our laboratory and 0.61. (2, 3). The comparison of the two amino acid sequences es- In one case (see Fig. 5), in which segments of 51 residues tablishes beyond doubt that the two proteins derive from a were compared, no gaps needed to be introduced, and we common ancestor (3). took advantage of this fact to compare these sequences to It has been known for more than 10 years that the homote- the National Biomedical Research Foundation data base, us- trameric aspartokinase I-homoserine dehydrogenase I (820 ing the program PROBE/EXPLOR, devised by Claverie residues) is composed of at least two functional domains: (i) (10). An NH2-terminal domain defined by a tetrameric fragment To determine the significance of the final alignment ob- extracted from a nonsense mutant possessing only the aspar- tained, two tests were applied. First, 10-20 random se- tokinase activity (4). This fragment, as extracted, extends quences, with identical amino acid composition to one of the from the NH2 terminus to approximately residue 495 (5). (ii) segments compared, were generated and subjected to the A COOH-terminal domain, defined by a proteolytic dimeric same alignment program with the other segment, to deter- fragment, endowed with homoserine dehydrogenase activity mine a set of random Sellers values. The second test was the only (4). This fragment starts, depending on the protease comparison between unrelated proteins or fragments of simi- used to generate it, between residues 293 and 300 (6, 7) and lar size. For example, with the gap penalty chosen, we were extends to the COOH-terminal residue 820. Thus, the two not able to detect any homology between the 190-residue- fragments share a common sequence between residues 293- long equine growth hormone (11) and the first 190 amino ac- 300 and ca. 495. A recent more detailed study of limited pro- ids of rabbit erythrocyte carbonic anhydrase C (12), or be- teolysis has defined three structural and functional domains tween the same length of carbonic anhydrase and a segment (7): an NH2-terminal aspartokinase I domain (Mr, -27,000; of comparable length of aspartokinase IT-homoserine dehy- 250 residues), a central domain ID (Mr, -25,000; 232 resi- drogenase II. dues) involved in subunit association and devoid of any cata- In addition to identities, we also considered the two types lytic activity; and a COOH-terminal homoserine dehydroge- of homologies normally accepted (13): those concerning ami- nase domain (Mr, 35,000; 324 residues). These fragments no acids that could be considered to derive from one another do not overlap and the sum of their molecular weights is by a single base change in the corresponding codon, and equal or nearly equal to that of the entire polypeptide chain. those concerning accepted replacements. For the first cate- To ascertain whether more information could be obtained gory, because we know the nucleotide sequence of the corre- about the origin of this long complex molecule, we decided sponding genes (2, 3), we considered only the cases in which to search for internal homologies in its sequence and to ex- a single base change was actually observed and not the other tend this study to its isofunctional counterpart, aspartoki- theoretically possible cases. The only accepted amino acid nase II-homoserine dehydrogenase 11 (809 residues), with replacements that we have taken into account are as follows: which it shares a common ancestor (3). isoleucine to leucine to valine, serine to threonine, phenylal- anine to tyrosine, arginine to lysine, and aspartate to gluta- The publication costs of this article were defrayed in part by page charge mate. When a given homology was generated either through payment. This article must therefore be hereby marked "advertisement" a single base change or through an accepted replacement, it in accordance with 18 U.S.C. §1734 solely to indicate this fact. was scored only once.

3019 Downloaded by guest on September 30, 2021 3020 Biochemistry: Ferrara et al. Proc. Nad Acad Sci. USA 81 (1984)

9 T S V A N AjR FLRA D I N A R Q G Q V ATV L S AP A KIT - NHL V AMI E K[I SG 14: P146T~~~LJi L-iI.j II-- AS Lii AR PADHMVLMAGLJOU L] FJAGN

58 Q - D ALP N I --S D A E R I F HEL T G LjAH A Q P G F P L A Q L K T FHD Q E F A Q I K HlI 195 E R G ELV V L G R N G S D Y S AAVjA A CLRAD - C C E I W T D V N - GLY - T C D P - R Qj 0 ~~~+ 0 * 0 0* +@0 0 + 0

105 L H G I S L L G Q C P D S I N A - A L I C R G E K 128

241P DAR L L- K SM SY QEA ME L SYF GA K 263

FIG. 1. Comparison of aspartokinase I segments 9-128 and 146-263. Positions compared, 125; gaps, 12; identities, 27 (21.6%); total identi- ties plus homologies, 55 (44%). In this and in the other figures, identities are boxed, homologies deriving from a single base change are denoted by e, and accepted replacements are denoted by +. Amino acids are designated by standard one-letter abbreviations. RESULTS 447, and from Thr-666 to Leu-686 (Fig. 3), showed 12 match- The Aspartokinase I Region is the Product of a Gene Dupli- es out of 63 base paits (19%) within the range expected on a cation. Segments 9-128 and 146-263 present 27 matches out random basis in an organism in which the four bases are ap- of 125 positions (22%). If the single base-change-derived proximately equally represented. However, if the chain resi- amino acids are considered, the similarity increases to 39%, dues 427-447 shifted by one base, the comparison showed 26 and'if we add ascepted replacements, the total homology be- matches out of the 62 positions (42%) (Fig. 4a). If the shifted comes 44% (Fig. 1).' The homology is distributed homoge- chain is translated in the + 1 frame and the amino acids are neously along the sequence (histogram not'shown). compared to sequence 666-685, the identity increases from The Intermediate Region ID and the Homoserine DMhydrog- 0% to 20%, and the total homology' increases from 0% to enase I'Region are Homologous. The alignment of segments 50% (Fig. 4 b and c). 336-569 and 572-812 shows 22% identity; that is, 55 matches We have chosen to apply the shift to residues 427-447 out of 255 positions (Fig. 2). Using the same accepted rules rather than to residues 666-686, since the latter must resem- of homology as described above, this percentage increases, ble the ancestral gene because its translated amino acids pre- respectively, to 34% and 39%. sent 82% identity with the corresponding homoserine dehy- It is interesting to note that within these two segments, drogenease II region (3) (Fig. 4 c and d). there are two sections, 427-447 and 666-686, that show no Further search of internal repeated sequences in the inter- homology at all. A close look at the DNA sequence of these mediate region of aspartokinase I-bomoserine dehydroge- two sectipons (Fig. 3) gave a striking result. The straight align- nase I revealed other surprising features. Segmients 316-366 ment, codon to codon in the open-reading frame, of the nu- and 397-447 showed 24 identities out of 51 positions (47%) cleotides corresponding to the regions from Ile-427 to Asp- without the introduction of any gaps (Fig. 5) and a total ho-

336 FA A M S R A R I S V V L I IS F C V P Q D C V R A E R A M L E E F YLE L K

572 JjH V V T P N K K A - -- N T M D YY H Q L R Y A A E KS R R K F L Y D I N V G A GL P V I

384 G L E P LAV A R - - - - A I I - V V G D G L R Tf- R I A Kj F A[A- -[ ]A N 617 ENLQ N LNNAG DE- K F S GIL G S L S Y I F G KLD EGM- -FS EAT RAE M

425 I N I V A I A Q G S S E R S I S VV VN N D D]T - V[ V T H Q M L F N QDT D I -E VFIV I G

664 G Y T E P D P R D D LS G M D V A RK L L I LAR E [E L E L A D E I E PIVL P A E - N A

+

473 VGG GALLELRQQSWLKNKHIj-LRH-CGJAHS- KALLTNJHHGLN

712 E GD VA A F MANLS Q D D L F A A R V A KA R DE G K VL R Y VG NI D E D G V C R VK - I A

+ +

518 L ]N W Q E - A Q A[E P F[ G R L I R L V K EH LHL N P I V NC T SS Q A - - D Q Y

761 - V D G N D P jF K VjN G E A - F Y S H - - Q PL P L JL R G Y G A G N DHT AAG V F L-iL-4 ~ ~

565 AD F R 569 808 LIL 812 FIG. 2. Comparison of intermediate fragment ID 336-569 with homoserine dehydrogenase I fragment 572-812. Positions compared, 255; gaps, 34; identities, 55 (21.5%); total identities plus homologies, 99 (39%). Abbreviations are as in Fig. 1. Downloaded by guest on September 30, 2021 Biochemistry: Ferrara et aL Proc. NatL Acad Sci USA 81 (1984) 3021

1279 ATT GTC GCC ATT GCT CAG GGA TCT TCT GAA CGC TCA ATC TCT GTC GTG GTA AAT AAC GAT GAT 1341 427 Ile Val Ala Ile Ala Gln Gly Ser Ser Glu Arg Ser Ile Ser Val Val Val Asn Asn Asp Asp 447

1996 ACC GAA CCG GAC CCG CGA GAT GAT CTT TCT GGT ATG GAT GTG GCG CGT AAA CTA TTG ATT CTC 2058 666 Thr Glu Pro Asp Pro Arg Asp Asp Leu Ser Gly Met Asp Val Ala Arg Lys Leu Leu Ile Leu 686 FIG. 3. Comparison of amino acid sequences 427-447 and 666-686, and of the corresponding nucleotide sequences 1279-1341 and 1996- 2058. Amino acid identities or homologies, 0; nucleotide identities, 12/63 (19%).

a) 1279 A TTG TCG TTG C GATACGMCCC AfC CAA TCT CGATF AA AC fA- 1341 1996 ACC GAA C GAC LCG C GAT GAT C TCT ATG GAT (GT C TAAT ATT ( 2058

b) Leu Ser Pro Leu Leu Arg Asp Leu Leu Asn Ala Gln Ser Leu Ser Trp Och Ile Thr Met 0

c) Thr Glu Pro Asp Pro Arg Asp Asn LSer Gly Met Asp Vai Ala Arg Lys LeuL I d) IThr Glu Pro Asp Pro Arg Asp Asp Leu Ser Gly|Lys IAsp ValI Ser|Arg Lys Vail Leu Ile

FIG. 4. Effect of a frameshift on the codons of the nucleotide sequence 1279-1341 and comparison of the generated translated sequence 427- 447 with sequences 666-685 of homoserine dehydrogenase I and 659-678 of homoserine II. (a) Same nucleotide sequences as in Fig. 3, but with a + 1 shift in sequence 1279-1341 (427-447). Identities, 42%. (b) Translation of the shifted nucleotide sequence 1279-1341. (c) Comparison of the amino acid sequence generated by this shift with amino acid sequence 666-685 in aspartokinase 1-homoserine dehydrogenase I. Identities, 20%; total homologies, 50%o. (d) Amino acid sequence 659-678 in aspartokinase II-homoserine dehydrogenase II.

316 A M F S V S G P G M K G M V G M A A R V F AA M S R A R I S V V LI T Q S S S E YS I SF CVP Q SD 366

397 SVIV DGL RTLRGIS AKFFA L ARN N AIAQGSS R VVVN DNDDN 447

FIG. 5. Comparison of segments 316-366 and 397-447, both parts of the intermediate region ID. Positions compared, 51; gaps, 0; identities, 24 (47%); total identities plus homologies, 36 (70%). Abbreviations are as in Fig. 1.

mology of 70%. It is obvious that such a high degree of ho- DISCUSSION mology is not due to change, but must reflect an ancestral The similarity in the positions of the main duplications in the local duplication. Even when the identity level of the two proteins is striking: residues 9-128 and 146-263 in aspar- PROBE/EXPLOR program was decreased to 27%, no other tokinase 1-homoserine dehydrogenase I, and residues 21- homologous sequence was detected in the whole NBRF data 146 and 150-274 in aspartokinase II-homoserine dehydroge- base. One of the two fragments (397-447) contains the only nase II (for the aspartokinase region); residues 336-569 and region of 25 residues (423-447) referred to above that does 572-812 in aspartokinase 1-homoserine dehydrogenase I, not present any homology with the corresponding residue in and residues 321-561 and 565-806 in aspartokinase II-homo- the homoserine dehydrogenase I region (662-686). It is obvi- serine dehydrogenase II (for middle and homoserine dehy- ous that when the + 1 frameshift (see above) is applied to the drogenase regions). DNA segment corresponding to sequence 427-447, the cor- From the internal homologies uncovered in each of the responding decrease of homology between segments 316- proteins under study, from the results of the limited proteol- 366 and 397-447 is observed. ysis of one of the proteins (7), and from the comparison of Aspartokinase II-Homoserine Dehydrogenase II. An exten- one of the proteins with the other (3), we can attempt to con- sive comparison of the aspartokinase II-homoserine dehy- struct a plausible hypothesis concerning the pathway drogenase II sequence with itself has revealed that this mole- through which the two iso- and bifunctional aspartokinase- cule also contains a number of internal sequence repetitions: homoserine dehydrogenases present in the same organism two segments covering the first one-third of the molecule are have evolved. similar and two segments covering the last two-thirds of the We postulate that two ancestral genes, AKo and HDHo (re- molecule also present a significant homology. The alignment spectively, -125 and -240 residues long), encoded the two of segments 21-146 and 150-274 leads to the matching of (active?) enzymes, aspartokinase and homoserine dehydrog- identical residues in 33 of the 133 positions (25%). The total enase, which were two separate entities as they are today in homology, using the criteria defined in Methods, is 42% many genera (e.g., Pseudomonas, Rhodospirillum, Saccha- (Fig. 6). romyces, Zea) outside the Enterobacteriaceae (14-17). At The alignment of segments 321-564 and 565-809 leads to some point during evolution, both genes were duplicated and an identity of 24%-that is, 63 matches out of 259 positions. fused to give AKo AKo and HDHb HDHo genes coding for If the single base-change-derived amino acids are consid- active enzymes, that later were fused to yield the bifunc- ered, the similarity increases to 33%, and if accepted re- tional AKo AKo HDHo HDHo gene, the direct common an- placements are added, the total homology is 36% (Fig. 7). cestor of the present day two bifunctional genes thrA and Downloaded by guest on September 30, 2021 3022 Biochemistry: Ferrara et al. Proc. NatL Acad Sci. USA 81 (1984)

21 L A D V K C Y ]V A G I MHE Y S P D D MM V VS A A G S TT N R L I -S W L K -LS QTD R L 150 L-DAREFL -IAfE- RLJAQPS jVDE--GL SJYP-LLQQLLVQHPGKRLVVTJGF I

69 HA H Q V Q QH LRRY Q CDL I S G LL PAE E A DSL I S A F VSDL E R L A A L LS G

194 SR N N A G ETV LLG R N G SDY S A T Q I GAL AG VSR V T- I WSDV A G V- Y S ADP R K L-iL-jL-i L-j + ~~~~~~~~+ + +

116 I N D A V Y A E VV G H G E V W - S A R L M S A V L N Q Q G _ - _ 146 242 V K D A C L L P L L R L D E A S E L A R L A A P V L H A R T L Q _ 274 + 00-- i 0 L -iL1

FIG. 6. Comparison of segments 21-146 and 150-274 of aspartokinase lI-homoserine dehydrogenase II. Positions compared, 133; gaps, 15; identities, 33 (25%); total identities plus homologies, 54 (40%). Abbreviations are as in Fig. 1.

metL coding, respectively, for aspartokinase 1-homoserine (21) has been found per protomer. Since the compared se- dehydrogenase I and aspartokinase II-homoserine dehy- quences cover the regions 1-263 and 336-820 in aspartoki- drogenase II (Fig. 8). An independent duplication of AKo nase I-homoserine dehydrogenase I, there is thus a region of AKo probably explains the origin of the lysC gene coding for -75 residues that is not accounted for in the comparison. aspartokinase III: although the complete sequence of this This region belongs to the domain defined as ID (ref. 7; see protein is not yet available, we know that it shares common above). Consequently, ID and HDHM in the present day as- antigenic determinants with aspartokinase I-homoserine partokinase I-homoserine dehydrogenase I do not coincide dehydrogenase I (18). with HDH'o and HDHo: an addition may have taken place by Sometime during the process, HDHo was inactivated as some undefined translocation mechanism. the result of accumulation of mutations, which could have It is, unfortunately, not known which moiety of AK'o AKo been point mutations, deletions or additions, frameshift mu- carries the aspartokinase activity, but it should be noted that tations, or tandem duplications affecting part of an already only one binding site for ATP (22, 23) is found in the present duplicated larger segment. This statement is based not only aspartokinase I-homoserine dehydrogenase I. on the examination of actual sequences, but on the fact that The hypothesis presented takes into account the fact that in aspartokinase 1-homoserine dehydrogenase I (7) and in three fragments in the polypeptide chain that approximately aspartokinase TI-homoserine dehydrogenase 11 (19) distal correspond in size and position to AK'o AKO (aspartokinase fragments of respective molecular weights 35,000 and 37,000 I region), HDH'o (ID region), and HDHo (homoserine dehy- possess homoserine dehydrogenase activity and that, at least drogenase region) have been characterized after limited pro- in aspartokinase I-homoserine dehydrogenase I, only one teolysis of aspartokinase 1-homoserine dehydrogenase I (7). binding site for NADPH (20) and for aspartate semialdehyde The distribution of homologies between the two aspartoki-

321 F QNP AHQ D F AH K E IDQ I LKR AV R P LA- - - VGVH- - -ND R Q L - Q F C 565 F HL- Ij- A N - G A S 5 NKY RQI H D -AF E K TGRHW L YA T V G A GLP L N

363 YT S E V AFAL KID E AL P G E L RLR Q GLALV A M VGA GVT R N PLH C H R SW 610 HIV R D L I G D TILS I SGI F S G -THS - WLFL- Q F DG- SVP FT ELV DQ A -W

412 JL K G QJV E F T W Q[D D G I L V AHL K T G P T E S LIQ G L Hj SVFRAK R I G L V 655 G L T ED P RD D LSG K D VsR K LV I L A RE A G Y N I - E P DQI-I-VE- S L V P A 0 ~~~ *~+I L~+ U Li L..J L.4 L. +

462 L F G KNIG -S R W LEF A R E Q STMSR T GF E FVLA GRV D DFS R R S LL SY 701 H C E G GS ID H FF E N G DELN E Q M V Q R LE AA R E M G LVLR Y VA R F D A N G K A R V G

509 L DHS - -ALAF F N D E A V E Q D EESL F LWMRA HPY D D L V V LID V T AIS QQ 751 V EAVRE DH PLAS L L P C DNV F A I ESR -WYnD NPL V I R G P G A G RDVTAG A I

553 L A[Q Y L D FHS H G 564 799 Q S D I NR -L A Q L L 809

FIG. 7. Comparison of segments 321-564 and 565-809 of aspartokinase TI-homoserine dehydrogenase II. Positions compared, 262; gaps, 35; identities, 63 (24%); total identities plus homologies, 93 (35.5%). Abbreviations are as in Fig. 1. Downloaded by guest on September 30, 2021 Biochemistry: Ferrara et aL Proc. NatL Acad. Sci. USA 81 (1984) 3023 AK0 HDHQ

AK' AK 0 0 HDH' 0 HDH0 I- Gene duplication and fusion

/ / AK' AK 0 HDH' HDH Gene duplication I I I Gene fusion -* Ancestral / and fusion / aspartokinase-homoserine / / dehydrogenase /

Aspartokinase III Duplications (lysC)

Aspartokinase II-homoserine Aspartokinase I-homoserine dehydrogenase II dehydrogenase I (metL) (thrA)

FIG. 8. Hypothetical pathway of evolution of E. coli aspartokinase-homoserine dehydrogenases.

nases-homoserine dehydrogenases has shown that, even 4. Veron, M., Falcoz-Kelly, F. & Cohen, G. N. (1972) Eur. J. though the identities and homologies extend throughout the Biochem. 28, 520-527. sequence (31% identity, 46% homology), maxima are found 5. Sibilli, L., Le Bras, G., Fazel, A., Bertrand, O., Dautry-Var- in the and 42% in sat, A., Cohen, G. N. & Zakin, M. M. (1982) Biochem. Int. 4, NH2-terminal (26% identity, respectively, 331-336. residues 1-140 and 150-280) and COOH-terminal (38% iden- 6. Sibilli, L., Le Bras, G., Le Bras, G. & Cohen, G. N. (1981) J. tity in the last 350 residues) regions, whereas only 20% iden- Biol. Chem. 256, 10228-10230. tity is found in the middle region (3), which is postulated to 7. Fazel, A., Muller, K., Le Bras, G., Garel, J. R., Veron, M. & be implicated in subunit contacts (7). It is interesting to note Cohen, G. N. (1983) Biochemistry 22, 158-165. that fragment 427-447, apparently derived through a frame- 8. Sellers, P. H. (1974) SIAM J. Appl. Math. 26, 787-793. shift mutation, is located in this region of the molecule. 9. Smith, T. F., Waterman, M. S. & Fitch, W. M. (1981) J. Mol. Whether this segment and its analogous counterpart in frag- Evol. 18, 38-46. ment 316-366 are directly involved in contact areas between 10. Claverie, J. M. (1984) Nucleic Acids Res. 12, 397-407. subunits must await the determination ofthe detailed tertiary 11. Zakin, M. M., Poskus, E., Langton, A. A., Ferrara, P., Della- cha, J. M., Santome, J. A. & Paladini, A. C. (1976) lnt. J. and quaternary structure of the protein. It will be interesting Pept. Protein Res. 8, 435 444. to establish whether the possible frameshift mutation that 12. Dayhoff, M. 0. (1976) Atlas of Protein Sequence and Struc- may have given rise to the sequence 427-447 as it exists in ture, Suppl. 2, (National Biomedical Research Foundation, the present day aspartokinase I-homoserine dehydrogenase Washington, DC), Vol. 5. I is related to the fact that the latter is an allosteric tetramer, 13. Dayhoff, M. 0. (1972) Atlas of Protein Sequence and Struc- whereas aspartokinase TI-homoserine dehydrogenase II is a ture, Suppl. 3, (National Biomedical Research Foundation, nonallosteric dimer. Washington, DC), Vol. 5. Further structural and biochemical studies on E. coli as- 14. Robert-Gero, M., Poiret, M. & Cohen, G. N. (1970) Biochim. as well as on isolated from other Biophys. Acta 206, 17-30. partokinase III, enzymes 15. Robert-Gero, M., Le Borgne, M. & Cohen, G. N. (1972) J. organisms, and crystallographic analysis of the two bifunc- Bacteriol. 112, 251-258. tional proteins in E. coli may provide the final clues as to the 16. de Robichon-Szulmajster, H., Surdin, Y. & Mortimer, R. K. origin of these complex molecules. (1966) Genetics 53, 609-619. 17. Bryan, J. K. (1969) Biochim. Biophys. Acta 171, 205-216. We thank Drs. J. M. Claverie, T. F. Smith, and W. M. Fitch for 18. Mouhli, H., Zakin, M. M., Richaud, C. & Cohen, G. N. (1980) kindly making their programs available; Drs. A. Fazel, K. Muller, Biochem. Int. 1, 403-409. and C. Parsot for helpful discussions; and L. Girardot for her expert 19. Dautry-Varsat, A. & Cohen, G. N. (1977) J. Biol. Chem. 252, handling of the manuscript. 7685-7689. 20. Vdron, M., Saari, J. C., Villar-Palasi, C. & Cohen, G. N. 1. Cohen, G. N. & Dautry-Varsat, A. (1980) in Multifunctional (1973) J. Biochem. 38, 325-335. Proteins, ed. Schmincke-Ott, (Wiley, New York), pp. 49-121. 21. Hirth, C. G., Veron, M., Villar-Palasi, C., Hurion, M. & Co- 2. Katinka, M., Cossart, P., Sibilli, L., Saint-Girons, I., Chalvig- hen, G. N. (1975) Eur. J. Biochem. 50, 425-430. nac, M. A., Le Bras, G., Cohen, G. N. & Yaniv, M. (1980) 22. Truffa-Bachi, P. & D'A Heck, H. (1971) Biochemistry 10, Proc. Natl. Acad. Sci. USA 77, 5730-5733. 2700-2706. 3. Zakin, M. M., Duchange, N., Ferrara, P. & Cohen, G. N. 23. Ehrlich, R. S. & Takahashi, M. (1973) Biochemistry 12, 4309- (1983) J. Biol. Chem. 258, 3028-3031. 4315. Downloaded by guest on September 30, 2021