Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7714-7718, August 1995 Biochemistry

Eukaryotic methionyl : Two classes of cobalt-dependent (cotranslational processing/initiation of protein synthesis/evolution) STUART M. ARFIN*, RICHARD L. KENDALL*t, LINDA HALL*, LARRY H. WEAVERt, ALBERT E. STEWART*II BRLAN W. MATrHEWSt, AND RALPH A. BRADSHAW* *Department of Biological Chemistry, College of Medicine, University of California, Irvine, CA 92717-1700; and *Institute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, University of Oregon, Eugene, OR 97403 Contributed by Brian W Matthews, May 24, 1995

ABSTRACT Using partial sequence data de- bacteria (11, 12), yeast (13), and porcine liver (14) display rived from porcine methionyl (MetAP; me- similar substrate specificities that are largely consistent with thionine aminopeptidase, peptidase M; EC 3.4.11.18), a full- that predicted from these analyses. In all cases, the penultimate length clone of the homologous human has been amino acid is apparently the primary determinant of specific- obtained. The cDNA sequence contains 2569 nt with a single ity; cleavage occurs when the penultimate residue is small open reading frame corresponding to a protein of 478 amino (G,A,S,C,T,P,V) and not when it is large (D,E,N,Q,K,H,R, acids. The C-terminal portion representing the catalytic do- L,I,M,F,Y,W). main shows limited identity with MetAP sequences from The genes encoding the Escherichia coli (11), Salmonella various prokaryotes and yeast, while the N terminus is rich in typhimurium (12), Bacillus subtilis (15), and Saccharomyces charged amino acids, including extended strings of basic and cerevisiae (16) MetAPs have been cloned and sequenced. The acidic residues. These highly polar stretches likely result in the prokaryotic enzymes are similar in size ("30 kDa), whereas spuriously high observed molecular mass (67 kDa). This the yeast enzyme contains a 125-amino acid N-terminal ex- cDNA sequence is highly similar to a rat protein, termed p67, tension with sequences resembling zinc-finger domains. The which was identified as an inhibitor of phosphorylation of remainder of the sequence shares "40% amino acid identity initiation factor eIF2a and was previously predicted to be a with the bacterial enzymes. All MetAPs studied to date are metallopeptidase based on limited sequence homology. Model activated by Co2+ and appear to require this ion for activity. building established that human MetAP (p67) could be The x-ray structure of E. coli MetAP has been determined to readily accommodated into the Escherichia coli MetAP struc- 2.4-A resolution (17). The enzyme contains a distinctive fold ture and that the Co2+ ligands were fully preserved. However, that displays internal two-fold symmetry ofthe first and second human MetAP was found to be much more similar to a yeast halves of the sequence and appears to represent another class open reading frame that differed markedly from the previ- of proteolytic enzyme. Protein data base searches using both ously reported yeast MetAP. A similar partial sequence from sequence- and structure-based profiles identified several other Methanothermusfervidus suggests that this p67-like sequence proteins likely to share the same fold (18). These proteins is also found in prokaryotes. These findings suggest that there included creatinase (19), aminopeptidase P (20, 21), proline are two cobalt-dependent MetAP families, presently composed (22, 23), and p67 (24, 25), a eukaryotic initiation ofthe prokaryote and yeast sequences (and represented by the factor 2 (eIF2)-associated protein proposed to regulate protein E. coli structure) (type I), on the one hand, and by human synthesis by protecting the eIF2 a subunit from phosphoryla- MetAP, the yeast open reading frame, and the partial pro- tion by eIF2 kinases. karyotic sequence (type II), on the other. With a panel of substrates that reflect the precise specificity associated with cotranslational processing, a porcine liver Protein synthesis is generally initiated with (in MetAP was isolated and characterized (14). The enzyme has eukaryotes) or N-formylmethionine (in prokaryotes). How- an apparent Mr of 67,000, is monomeric, and requires Co2+ ever, the products of this process are subject to one or more ions for activity. Using partial sequence data obtained from N-terminal modification reactions, occurring co- or posttrans- endoprotease Lys-C fragments, we report here the sequence of lationally, that often result in mature proteins with a variety of a full-length clone of the homologous human protein. The free (or N"-acylated) N-terminal amino acid residues other deduced amino acid sequence of the human protein is highly than methionine (1-3). Although the physiological importance similar to the rat p67 protein (eIF2-associated protein). Model of these modifications is incompletely understood, they likely building suggests that substantial segments of the C-terminal operate in functional regulation, intracellular targeting, and domain are fully compatible with the three-dimensional struc- protein turnover. ture of E. coli MetAP, confirming the prediction (18) that p67 Removal of the initiator methionine of nascent polypeptide may function as a metal-dependent aminopeptidase. The chains (when it occurs) is catalyzed by a methionyl aminopep- subsequent identification of a yeast open reading frame (ORF) tidase (MetAP; methionine aminopeptidase, peptidase M; EC coding for a putative protein 55% identical to human MetAP 3.4.11.18). In bacteria, the N-formyl group of the initiator (p67) (although lacking most ofthe N-terminal extension) (26) N-formylmethionine is first removed by an appropriate de- formylase. Analyses of the protein data base (2, 4-6) and Abbreviations: MetAP, methionine aminopeptidase; RACE, rapid studies of the processing of mutant proteins (6-8) or proteins amplification of 5' cDNA ends; ORF, open reading frame. with systematically modified N-terminal sequences (9, 10) tPresent address: Department of Biochemistry, Merck Research Lab- suggest that MetAPs from diverse sources have quite similar oratories, Rahway, NJ 07065-0900. substrate specificities. In addition, purified enzymes from §Present address: Imperial Cancer Research Fund, Structural Molec- ular Biology Unit, Department of Crystallography, Birkbeck College, Malet Street, London WCIE 7HX, United Kingdom. The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in liThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U29607).

Downloaded by guest on September 29, 2021 7714 Biochemistry: Arfin et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7715 supports the view that this protein (and the yeast homolog) can be associated with specific conformations of the polypep- represent a second class of MetAPs that are distantly related tide backbone. to the previously described prokaryote/yeast enzymes and share specificity, metal requirements, and general backbone fold (in the catalytic domain). RESULTS Human MetAP cDNA and Predicted Protein Sequence. Using the highly selective, nonprocessive activity attributed to MATERIALS AND METHODS the N-terminal processing of the initiator methionine residue Determination of Partial Amino Acid Sequences. For pep- on nascent chains during eukaryotic protein synthesis, a 67- tide analyses, 200 jig of pure porcine MetAP was incubated kDa protein was isolated and characterized from porcine liver with endoprotease Lys-C (Boehringer Mannheim) for 6 hr at (14). The enzyme is monomeric and is strongly stimulated by 37°C. The resulting peptides were separated by HPLC on an cobalt ions, similar to other MetAPs that have been identified Aquapore C8 column. Peptides were eluted using a gradient (11-13). Treatment of 200 jig of porcine MetAP with endo- from 10 to 70% (vol/vol) acetonitrile in 0.1% trifluoroacetic Lys-C, followed by fractionation of the digest by acid (Applied Biosystems model 140A). The column effluent HPLC and automated peptide sequencing, yielded four pep- was monitored at 212 nm with an ISCO model CV4 capillary tide sequences (see Materials and Methods and Fig. 1). These electrophoresis detector. Selected peaks were analyzed by data were used to generate oligonucleotides for PCR, yielding automated liquid pulse sequencing on an Applied Biosystems a fragment that was suitable for the identification of a partial model 477A sequencer equipped with an online phenylthio- porcine clone. This sequence was used to identify a nearly hydantoin analyzer. The N-terminal sequences of four peptides complete human cDNA sequence (pMB).The 5' sequence was were determined: peptide 1, TVPIV; peptide 2, GGEATRM- generated by the RACE procedure (27). EEGEVYAIETFG; peptide 3, NLXDLGIVDPYPPL; and Fig. 1 shows the nucleotide sequence of the putative full- peptide 4, KALDQASEEIWNDFREAAEAXXQV (see also length cDNA of human MetAP, reconstituted from the RACE Fig. 1). and pMB sequences, and the predicted amino acid sequence. cDNA Cloning ofPorcine and Human MetAP. Pairs of sense The cDNA sequence contains a total of 2569 nt and a single and antisense degenerate oligonucleotides corresponding to ORF extending from nt 1 to nt 1439. There is no poly(A) tail, regions of peptides 2 and 4 were synthesized by using phos- although several possible polyadenylylation signals can be phoramidite chemistry on an Applied Biosystems model 391A found in the 3' untranslated region. The 5' end of the cDNA instrument and used in all combinations for PCR amplification lies within a (GC)-rich region, suggesting that it is a GpC island of oligo(dT)-primed first-strand cDNA synthesized from tem- and probably the start of transcription. Although there is no plate porcine liver RNA. The combination of the sense oligo- in-frame stop codon preceding the first ATG, the nucleotide nucleotide from 5'-ATG GAR GGN GAR sequence surrounding it agrees well with the consensus se- peptide 2, GAR quence proposed for translation initiation in higher eukaryotes GTN TT-3' and the antisense oligonucleotide 5'-GG RTA (29). NGG RTC NCA DAT NCC-3' (where D is T, G, orA) from pep- The ORF encodes a protein of 478 amino acids with a tide 4 yielded a 273-bp product that was purified and ligated calculated molecular mass of 52,832 Da. The N-terminal into the pCRII vector (TA cloning kit, Invitrogen). The portion of MetAP is rich in charged amino acids, 53 of the first translated nucleotide sequence contained the C-terminal se- 110 residues of and quence of peptide 2 and the N-terminal sequence of peptide consisting arginine, lysine, glutamic, 4. The PCR was aspartic acids. These residues include runs of 9 (residues fragment 32P-labeled by the random-primer 36-44) and 10 (residues 98-107) basic amino acids and a run method and used to probe a porcine liver AgtlO cDNA library of 11 (residues 77-87) charged amino acids, of which 9 are (Clontech). Forty positive clones were obtained from screen- acidic and 2 are basic. Comparison of the deduced amino acid ing 1.5 x 106 recombinant phage. A 2.1-kb clone, p71A1, was sequence to that of the MetAPs from E. coli (11), S. typhi- subsequently used to screen a cDNA library prepared from murium (12), B. subtilis (15), and S. cerevisiae (13) shows that human fetal brain poly(A)-selected RNA in the AZAP II the yeast and human enzymes have N-terminal extensions of vector (Stratagene). Sequencing of the human cDNA clone 125 and 160 amino acids, respectively, relative to the prokary- (pMB) with the largest insert (-2.4 kb) revealed that this clone otic forms (Fig. 2). There is no obvious sequence similarity did not contain an in-frame stop codon upstream of the first between the two extensions. In the portion of the protein AUG. An internal primer and a library-specific primer were shared by human MetAP and the yeast and prokaryotic then used to amplify the 5' ends of all positive clones. None of enzymes, there is 20-24% sequence identity. In contrast, the these clones proved to be longer at their 5' ends than clone yeast enzyme is 40% identical to the bacterial enzymes in this pMB. Therefore, rapid amplification of 5' cDNA ends same region. (RACE) (27) was done with first-strand cDNA prepared by The substantial sequence differences, as well as the overall reverse transcription of human HepG cell RNA as template to organizational differences distinguishing the human MetAP determine the 5' sequences. The product was cloned into enzyme and the characterized MetAPs, raised the question of pBluescript II. Five independent clones were isolated and whether the human MetAP was truly homologous to this sequenced. group, particularly because the extent of sequence similarity was marginal. Accordingly, the human MetAP sequence was MODEL BUILDING fitted to the E. coli structure to ascertain the extent to which it could (or could not) be accommodated. Using the known crystal structure of E. coli MetAP (17) as a Significantly, none of the deletions or insertions occur reference, the model-building program FRODO (28) was used within a-helices or (3-sheets. Rather, they are located near to determine whether the sequence of the human enzyme, residues 58, 84, 117, 163, 169, 176, and 220 of the E. coli aligned as in Fig. 2, was compatible with a similar three- sequence, all of which are within irregular or loop regions on dimensional fold. The backbone of the protein was kept the surface of the E. coli MetAP molecule, where insertions constant, and the side chains corresponding to the human and deletions might be anticipated. Model building also sug- MetAP sequence were built to have standard geometry and, so gests that the changes in sequence that occur within a-helices far as possible, to avoid steric interference with neighboring and }3-sheets can be accommodated with only minor pertur- parts of the structure. Particular attention was given to sub- bations of the packing of these secondary-structure elements. stitutions involving glycine and proline because these residues This hypothesis is illustrated by the region surrounding resi- Downloaded by guest on September 29, 2021 7716 Biochemistry: Arfin et al. Proc. Natl. Acad. Sci. USA 92 (1995)

-22 M A G V E E V A A S G S H L N G D L D P D D R E E G A A S T A E E 33 A S S F G R T S 99 _ _S ~~~~~~~~~CTGAiGGACGAAGATAAAAATCAGGACCAGGTAAGTAGCAGCGCGAAAG AA K K K RR K K K K S K G P S A A G E Q E P D K E S G A S V D E V A R Q L E R 73 G A V S Q L T K 219 AT _G; ATGGCGATGGAGAT GGY3C CAAAC S A L E D K E R D E DD E D G D G D G D G A T G K K K K K K K K K R G P K V Q T 113 Q E K D A R 339 AGAT G ATGrGACT TGGAGGA D PP S V P I C D L Y P N G V F P K G Q E C E Y P P T Q D G R T AA W R T T S E 153 459 PC TTAGATCAGGCAAGrCAGAGATTTGGAATGATTTAGCCAGGGATCACAAT E K K A L D Q A S E E I W N D F R E AA E A H R Q V R K Y V M S W I K P G M T M 193 579 GATACIAAAT I E I C E K L E D C S R K L I K E N G L N A G L A F P T G C S L N N C AA H Y T 233 699 T PAATP N A G DTD T_ T V L Q Y DD GCCGGKII C K I D F G T H I S F G R THACAGICAACATTAVTCFNPATYAI I DC A F T V T F N P K Y 2-73

939 _____ IGCATTGAAAGG E I D G K TY Q V K P I RN L N G H S I G Q Y R I H A GK T V P I V K GG E A T 353 P 1059 A RGMI _ CA7GAACTTGrGTr-CGAAAGTGQT_ rrAGATGAATAWTGGATTTCCArATTACATGAAAATmATTTTGAT&GACATGrGCC R M E E G E V Y A I E T F G S T G K G V V H D D M E C S H Y M K N F D V G H V P 393 1179 AAA AGGCTTCMAGATAAC'G'AAGTCCCATCCIY; CCiACCCTGCCTTGGCC,APTGCGATCGCTTCCiGAAAGrAAAW GATGGCrCTGAA I R L P R T K H LL N V I N E N F G T L A F C R R W LD R L G E S K Y L M A L K 433 1299 GAATCTGrGTATTG TCCATATCCACCATTATGTGTATACAGCGA CATACCATCCTGTTGCGTCCAATGrGMALGCAG N L C DL G I V D P Y PP L C D I KG S Y T A Q F E H T I L LR P T C K E VV S 473 C A Q P V K K L S 1419 TCGGCGAGTTTGTTGGAAAACATGATA(XAT;AATTAATTTGCCACATG rTGTarGTTTTTAAGTGGAC R G D D Y * 478 A EE M T I K T 1539 CCATGTAATACTTTTT 1659 C AATCTAAGAG_TGGKTATTT GAATGCTTT 1779 _GECTGC CCT GGAATCATTTTCTAAAGATCT ACTATTC_C 1899 A CATATGAAGrAAAGCr 2019 A 2139 TACCAGAATCCTA 2259 A CTTCAATGM 2379 2499 cAAA.r AA FIG. 1. Nucleotide sequence and deduced amino acid sequence of human MetAP (p67). Differences in the predicted rat liver p67 amino acid sequence are indicated below the human MetAP sequence. Sequences corresponding to the endoprotease Lys C-derived peptides are underlined. dues 240-250 (E. coli MetAP numbering) (Fig. 3). In this the surface, so that the arginine at this position can extend into stereo projection, the model-built residues are shown with the solvent. open bonds, and the side chains of the E. coli MetAP model Additional confirmation that human MetAP (p67) and the are drawn in black circles. The backbone is retained exactly as E. coli enzyme adopt similar conformations is provided by the in the E. coli structure. Clearly, the backbone conformation at retention of the Co2+ ligands (defined in the E. coli structure) Asp-242 is perfectly set up to accept the proline at this position (17). As shown in Fig. 2, these motifs are fully conserved for in the human sequence. Gly-244 of the E. coli sequence can be the human and E. coli MetAPs and, with the exception of a replaced with the larger cysteine in the human molecule single Gln -- Glu replacement, for the yeast MetAP as well. without introducing serious steric clashes. Cys-245 in the E. coli The requirement of the human enzyme for Co2+ for activity enzyme is largely buried, but it appears that a lysine at this has been established (14). position, as in the human, could extend toward the surface, so Comparison to Other Sequences. A search of the GenBank that the e-amino group might reach the solvent. Thr-241 is on data base revealed that human MetAP shared 92% sequence

S.c.I 1 M S T T T T V T T S D Q A S H P t K I Y CSGLQCGR H.. 1 H A G V EE V AA S G S H L N GD L D P D D R E E G AA S T A EE A A K KK R R KKKK S K G P S A A G E Q E P D K E S S.c.I 30 ET SQHMCPVCLkQGiVSIFC TSCYe NNYKAHK LHNA OGLEGAYD FPKFKYSGKVK H.s. 61 G A S V D E VA R Q L E R S A L K IE RD E D D E D G E G 0 G 0 G A T G KKK K K K K K K R G P K V Q T D PP S V P I E.C. 1 M i S I K t P D I E KM V GkLAAE L S.c.I 90 A YPLTPRRYVPEdIPK DWAANGLPVSEQRNdRLNNiPIYKKdQIKKI K CMLG E L H.S. 121 C D L Y P N G V F P KG Q E C EY P P T Q D G R T A A W K TT S E E K K A L D Q A S E E I W N D F R E AA E A H R Q V R S.c.I 1 H H X Q d f N L Q R T T V E E S R Y L K R d L E r E H W N D V R K G A e I H R RV R RA I K D M.f. 1 MEK kK GKI A S K E.c. 26 E M i E PY :: TG 1 D R I C N Y I V N E Q H A V S A C L G Y Y P K S V C I i E VV C I D- S. c. I 150 D I AAA Hr T -D IvHNEt I R G A Y - P S P L N YY N F P K S LC T v E V I C G V H. s. 181 K Y V HS W I P GX T M HIk - I CEKLEDCSRKLIKEN - - - GLNAGLAFPTGCS L NNC AA H Y T P N A S.c.II 51 IVPGHK1MDIAD N TT R Y T G A E N L AM D- P K S . L .H f M.f. 16 K A K A v iGE K i 1 d -l A F i N E I E - m G IPK C,I '-:IT 5 PC

E.c. 84- A1 G VN V T V I KD F H G T S K HF I V G K - PT I G e KR C r I T Q e S L Y L A 1 r M V K S.c.I 206 K EG V V S L Y Y Q Y HA L N E YY V G E2 I S K E A L N T T ET S reCLKLA M C K H. s. 237 D DTYLQICXIDIF1THISGRIIIICA FTVT F N - - - - P K Y D I LLKAVKDAT.NTGIK X C A G S.C.I 100oe K v y . QN N. S s - - -- N A.E Y M.f. 66 N D R K iL P G 1 V I V vO1):F G T T L V E ----G Y E K N Y N D E L EKNKKMI E A

E.c. 142 P Gi N R.e'.l::: A: KF v AEGF-SVVrE- C G G -RF EE P Q L H Y D S.c.I 265 P.G.T.T FQe d:H E.K H A T ENKKC-SVVr- C b v E f - F C S P N i H Y A H.s. 293 I D CVLCD V.O E A I Q Vv M E Y IE V E.I D 0 K T X Q V - - - - K P I R N L N G H S I G Q Y R I H A GK TV P I V K S.c.II 164 :' : . N ----...... CC. A PS a M. f. 123 A e s A E N A I N T }R dO v 'I G K i G E V I E N T I N K F G F S T _ t D R WV 1 S L s i N

E.c. 188 S R E T NV V 1 Tf P H V N A K E i R T MK d - - S.c.I 311 K N R T P G : HY.. TDMTP P H IN- TWK DMTWPd M.S. 349 G --FGEASRMI-.OT ..Y.YA I KT.F'G.S.TKV.VHS D.DI.ME C S H Y M K N F D VG H V P IRLPRT K H L L N V I N S.c220 N ELd .r:TY A r S A e DHQ H PT D S A N K T D H. f. 183 - Q N S H k 1... E.c. 220- GW T v KT K D R LS y v v T D N S.c.I 343-- D T S T T Q D K L s .1 v TSE H.s. 408 EINFFT L A. F C KR WN L D R L o EIS K Y L: M.-AL .K N L C:D L G I OVD P YPP L C D I K G S Y T A Q F EH T I LL R P T SoeII 279K QI~E RH 100 N P HANH E.c. 244 G C i 1 t L R K D T I P A I I S H D E * S.c.I 367 G V i 1 t A K N K K S P G G P RQ R I K * H. s. 468 CKEV..R0DDX: S. .II 33933 K ..:....--...-..:..::...:.-...... :-..:k.-..:::..X ... - *..*

FIG. 2. Primary sequence alignment of E. coli MetAP (E.c.), S. cerevisiae MetAP-1 (S.c. I), human MetAP (H.s.), S. cerevisiae hypothetical protein YBLO91c (S.c. II), and a partial ORF from Methanothermusfervidus (M.f.). Blank spaces indicate that the amino acid residues are identical to those in the human sequence; dashes indicate deletions introduced to maximize alignments. The three dots at the end of the M. fervidus sequence indicate that it is derived from a partial clone and that the sequence extends beyond this point for an unknown distance. Lowercase letters denote conservative substitutions. Residues that are cobalt ligands in the structure of E. coli MetAP and are conserved in the other sequences are boxed. Regions of substantial homology between human MetAP and one or more of the other sequences are indicated by shading. Downloaded by guest on September 29, 2021 Biochemistry: Arfin et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7717

FIG. 3. Stereo drawing illustrating the compatibility of the amino acid sequence of human MetAP with the three-dimensional structure of E. coli MetAP (17). The flgure shows the results of model building in which residues Thr-461-Ser-473 of the human sequence (open bonds) were model-built onto the backbone of the E. coli structure (solid bonds). [This is the region in which the alignment of the human vs. E. coli MetAP sequence has been revised relative to that previously proposed for the rat P67 sequence (18) (see text).] Numbering corresponds to the E. coli sequence. D242P, for example, indicates that Asp-242 in the E. coli sequence is replaced by a proline in the human enzyme. 0, Carbon atoms; 0, oxygen atoms. To show the overall context within the three-dimensional E. coli MetAP structure, neighboring residues are shown in solid black and are unlabeled. identity at the amino acid level with that previously reported ably in conjunction with Na-acetylation (3). Perhaps this for a rat liver eIF2-associated protein designated p67 (Fig. 1). apparent association with the initiation complex represents the p67 copurifies with eIF2 and is postulated to regulate protein mechanism by which MetAP in higher eukaryotes is recruited synthesis by protecting the eIF2 a subunit against phosphor- to the ribosome to insure stoichiometric interactions that lead ylation. The amino acid differences occur almost exclusively in to the highly selective (and apparently quantitative) removal of the N-terminal region and at the C-terminal end. The observed methionine from susceptible sequences (9). N-terminal differences are probably due to species variations Why the human enzyme has a significantly different orga- (rat vs. human). However, at the C terminus the p67 sequence nization than that of the yeast MetAP, including two major can be transformed into the MetAP sequence by a + 1 frame- inserts (one of 63 amino acids) in the catalytic domain (as shifting over 18 codons. The insertion of an extra nucleotide at indicated by the prokaryotic forms) and an entirely different position 1412 in the MetAP sequence is compensated for by a N-terminal extension, is also unclear. The N-terminal se- single-base deletion 50 nt further downstream. Interestingly, quence of the yeast MetAP has two putative zinc-finger this revised sequence markedly improved alignment with domains that may allow interactions with nucleic acid (13). The prokaryotic MetAPs in this region (Fig. 2). Identity of the excision of these domains has little effect on the catalytic human MetAP and rat liver p67 proteins (excepting species properties of the enzyme, but the construct expressing this variations) was, in fact, presaged by the recent suggestion that truncated form is significantly less effective in rescuing the p67 belongs to a small family of structurally related proteins slow-growth phenotype of a MetAP mutant as compared with that includes the MetAPs ofE. coli, S. typhimurium, B. subtilis, the wild-type MetAP (34). In contrast, the human enzyme and S. cerevisiae and the prediction that it would function as a lacks the consensus zinc-finger sequences and instead contains metal-dependent aminopeptidase (18). the extended runs of basic and acidic residues. The function of these sequences is unknown. DISCUSSION These differences are sufficiently great to suggest that Determination of the human MetAP sequence based on human MetAP may be a member of a distinct subfamily of peptides derived from the homogeneous enzyme (14) estab- MetAPs, distantly related to but distinct from the prokaryotic lished the predicted link between eIF2-associated protein and yeast MetAPs. Strong support for this view is provided by (p67) and MetAP (18). The identity of these proteins is further the recent report of a yeast ORF that is distinctly more closely established by their unusual behavior on SDS gels. Both related to human MetAP than is either the yeast or prokaryotic proteins show apparent Mr values of 67,000 but have calculated MetAPs (26). This protein (designated S.c. II in Fig. 2) con- molecular weights in the low 50,000 range. Datta et al. (30) attributed this difference to intracellular 0-linked glycosyla- CATALYTIC (Co+2) DOMAIN tion (31), but because these modifications introduce only a kDa I------I single monosaccharide per site, a rather substantial number of ,,E. coli 30F :I -B. subtilis sites would be required to account for the difference. A more L S. typhimuriumn likely explanation is the observation that elongated tracts of I43L acidic and basic residues can produce anomalously high Mr Yeast values on SDS gels (32, 33). Yeast Fpr3 (also called yFKBP-47) is a protein of Mr 46,800 (calculated) but shows an apparent mass of 65 kDa on SDS gels; this yeast protein is characterized II 53 Porcine, by similar stretches of basic and acidic residues (32). Human Although it is not unusual for a protein to display more than M. fervidus one activity, the association of MetAP with the initiation complex rather than the ribosome was unexpected. Cotrans- lational excision of the initiator methionine occurs when the FIG. 4. Schematic representation of the two proposed subfamilies nascent chain is still quite short (20-40 residues) and presum- of MetAP. Downloaded by guest on September 29, 2021 7718 Biochemistry: Arfin et aL Proc. Natl. Acad. Sci. USA 92 (1995) Table 1. Amino acid identities in the catalytic domains of MetAPs 3. Arfin, S. M. & Bradshaw, R. A. (1988) Biochemistry 27, 7979- 7984. Type I Type II 4. Persson, B., Flinta, C., von Heijne, G. & Jornvall, H. (1985) Eur. E.c. S.c. I H.s. S.c. II M.f J. Biochem. 152, 524-528. 5. Boissel, J.-P., Kasper, T. J., Shah, S. C., Malone, J. I. & Bunn, Type I E.c. - 104/244 50/234 48/234 41/189 H. F. (1985) Proc. Natl. Acad. Sci. USA 82, 8448-8452. T S.c. I 43 - 49/231 40/231 34/189 6. Sherman, F., Stewart, J. W. & Tsunasawa, S. (1985) BioEssays 3, H.s. 21 21 185/314 52/189 27-31. Type II S.c. II 20 17 59 43/189 7. Tsunasawa, S., Stewart, J. W. & Sherman, F. (1985)J. Biol. Chem. WM. 21 18 28 23 - 260, 5382-5391. Entries above the diagonal refer to the number of identical residues 8. Moerschell, R. P., Hosokawa, Y., Tsunasawa, S. & Sherman, F. per total number of residues compared based on the alignments in Fig. (1990) J. Biol. Chem. 265, 19638-19643. 2. Entries below the diagonal refer to percentage identities. The 9. Huang, S., Elliott, R. C., Liu, P. S., Koduri, R. K., Weickmann, remainder of the M. fervidus' sequence is unavailable. Ec., E. coli J. L., Lee, J. H., Blair, L. C., Ghosh-Dastidar, P., Bradshaw, MetAP; S.c. I, S. cerevisiae MetAP; H.s., human MetAP; S.c. II, S. R. A., Bryan, K. M., Einarson, B., Kendall, R. L., Kolacz, K. H. cerevisiae hypothetical protein YBLO91c; Mf., partial ORF from M. & Saito, K. (1987) Biochemistry 26, 8242-8246. fervidus. 10. Boissel, J.-P., Kasper, T. J. & Bunn, H. F. (1988) J. Biol. Chem. 263, 8443-8449. tains the same organization as human MetAP with respect to 11. Ben-Bassat, A., Bauer, K., Chang, S.-Y., Myambo, K., Boosman, the inserts in the catalytic domains but lacks a long N-tertninal A. & Chang, S. (1987) J. Bacteriol. 169, 751-757. extension [it possesses an N-terminal segment of 25 amino 12. Miller, C. G., Strauch, K. L., Kukral, A. M., Miller, J.-L., Wing- field, P. T., Mazzei, G. J., Werlen, R. C., Garber, P. & Mowa, acids beyond the catalytic domain that is unrelated to either N. R. (1987) Proc. Natl. Acad. Sci. USA 84, 2718-2722. human MetAP or the initially identified yeast MetAP (desig- 13. Chang, Y.-H., Teichert, U. & Smith, J. A. (1990) J. Biol. Chem. nated S.c. I in Fig. 2)]. This yeast protein is "80% identical to 265, 19892-19897. the human MetAP in the catalytic domain, including complete 14. Kendall, R. L. & Bradshaw, R. A. (1992) J. Biol. Chem. 267, conservation of the putative Co2+ ligands. Thus, yeast appar- 20667-20673. ently expresses both a prokaryotic-type and a human-type 15. Nakamura, K., Nakamura, A., Takamatsu, H., Yoshikawa, H. & MetAP. We propose to designate the former as MetAP-I and Yamane, K. (1990) J. Biochem. (Tokyo) 107, 603-607. to designate the latter as MetAP-II. In addition, a partial ORF 16. Chang, Y.-H., Teichert, U. & Smith, J. A. (1992) J. Biol. Chem. from M. fervidus, previously identified as a potential homolog 267, 8007-8011. of rat p67 (18) (and, hence, MetAP-1I type) (see Fig. 2), shows 17. Roderick, S. L. & Matthews, B. W. (1993) Biochemistry 32, significant similarity to the human and yeast MetAP-II pro- 3907-3912. teins (40% and 34% similarity, respectively, over the known 18. Bazan, J. F., Weaver, L. H., Roderick, S. L., Huber, R. & Mat- that across thews, B. W. (1994) Proc. Natl. Acad. Sci. USA 91, 2473-2477. segment), suggesting the MetAP-II family extends 19. Hoeffken, H. W., Knof, S. H., Bartlett, P. A., Huber, R., Moel- the eukaryotic-prokaryotic boundary. It is, therefore, possible lering, H. & Schumacher, G. (1988) J. Mol. Biol. 204, 417-433. that a human MetAP-I-type enzyme also exists. 20. Henrich, B., Monnerjahn, U. & Plapp, R. (1990)J. Bacteriol. 172, The general structure of the two MetAP families is shown 4641-4651. in Fig. 4, and their relationships in size and similarity are 21. Butler, M. J., Bergeron, A., Soostmeyer, G., Zimny, T. & Malek, summarized in Table 1. Although all are likely Co2+ requiring L. T. (1993) Gene 123, 115-119. (this remains to be shown for the S.c. II and M. fervidus 22. Yoshimoto, T., Tone, H., Honda, T., Osatomi, K., Kobayashi, R. sequences), the MetAP-I family differs from the MetAP-II & Tsuru, D. (1989) J. Biochem. (Tokyo) 105, 412-416. family by significant structural features in the catalytic domain. 23. Endo, F., Tanoue, A., Nakai, H., Hata, A., Indo, Y., Titani, K. & However, more striking are the various N-terminal structures. Matsuda, I. (1989) J. Biol. Chem. 264, 4476-4481. Prokaryotic MetAPs of both families lack an N-terminal 24. Wu, S., Gupta, S., Chatterjee, N., Hileman, R. E., Kinzy, T. G., domain, as does yeast MetAP-I1. Yeast MetAP-I and human Danslow, N. D., Merrick, W. C., Chakrabarti, D., Osterman, J. C. MetAP-II have such extensions and are distinct in this & Gupta, N. K. (1993) J. Biol. Chem. 268, 10796-10801. clearly 25. Chakraborty, A., Saha, D., Bose, A., Chatterjee, M. & Gupta, regard. Because the substrate specificity of both families is N. K. (1994) Biochemistry 33, 6700-6706. similar (and in keeping with that expected for cotranslational 26. Feldmann, H., Aigle, M., Aljinovic, G., Andre, B., Baclet, M. C., processing), it raises the interesting question whether one or et al. (1994) EMBO J. 13, 5795-5809. both function in this regard and why two such families are 27. Frohmann, M. A., Dush, M. K. & Martin, G. R. (1988) Proc. Natl. necessary, at least in the case of yeast. At the least, these Acad. Sci. USA 85, 8998-9002. observations suggest that cotranslational processing is a more 28. Jones, T. A. (1982) in Crystallographic Computing, ed. Sayre, D. complex process than was originally thought. (Oxford Univ. Press, Oxford), pp. 307-317. 29. Kozak, M. (1991) J. Biol. Chem. 266, 19867-19870. We thank Mrs. Gale D. Trudeau for her expert assistance in the 30. Datta, B., Ray, M. K., Chakrabarti, D., Wylie, D. E. & Gupta, preparation of this manuscript. This work was supported by U.S. Public N. K. (1989) J. Biol. Chem. 264, 20620-20624. Health Service Grants DK32465 (S.M.A. and R.A.B.) and GM20066 31. Hart, G. W. (1992) Curr. Opin. Cell Biol. 4, 1017-1023. (B.W.M.). 32. Fuller, R. S., Brake, A. & Thorner, J. (1989) Proc. Natl. Acad. Sci. USA 86, 1434-1438. 1. Brown, J. L. & Roberts, K. W. (1976)J. Biol. Chem. 251, 1009- 33. Benton, B. M., Zang, J.-H. & Thorner, J. (1994) J. Cell Biol. 127, 1014. 623-639. 2. Driessen, H. P. C., deJong, W. W., Tesser, G. I. & Bloemendahl, 34. Zuo, S., Guo, Q., Ling, C. & Chang, Y.-H. (1995) Mol. Gen. H. (1985) CRC Crit. Rev. 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