Proc. Nati. Acad. Sci. USAVol. 89, pp. 4129-4133, May 1992Biochemistry

Mutations of human myristoyl-CoA:protein N-myristoyltransferasecause temperature-sensitive myristic acid auxotrophy inSaccharomyces cerevisiae

(protein N-myristoylation/complementation cloning/enzyme structure-function/fatty acid metabolism/drug design)

ROBERT J. DURONIO*, STEVEN 1. REEDt, AND JEFFREY 1. GORDON**Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110; and tDepartment of MolecularBiology, Research Institute of Scripps Clinic, La Jolla, CA 92037

Communicated by Carl Frieden, January 13, 1992 (received for review November 18, 1991)

ABSTRACT We have isolated cDNAs encoding humanmyristoyl-CoA:protein N-myristoyltransferase (NMT, EC2.3.1.97) by complementing the nmtl-181 mutation of Saccha-romyces cerevisiae, which causes temperature-sensitive myristicacid auxotrophy. Human NMT is derived from a single-copygene, contains 416 amino acids, is 44% identical to S. cerevisiaeNMT (yeast NMT), and can complement the lethal phenotype ofan nmtl null mutation. Human and yeast NMTs have overlap-ping yet distinct protein substrate specificities as judged by acoexpression system that reconstitutes protein N-myristoylationin Escherchia coli. Both enzymes contain a glycine five residuesfrom the C terminus. Gly -* Asp or Lys mutagenesis in theseorthologous NMTs produces marked reductions in their activ-ities in E. coli as well as temperature-sensitive myristic acidauxotrophy in S. cerevisiae. These results indicate highly con-served structure-function relationships in vivo and underscorethe usefulness of these functional assays for identifying factorsthat regulate protein N-myristoylation in mammalian systems.

Myristate, a rare 14-carbon saturated fatty acid, is cotransla-tionally attached by an amide linkage to the N-terminal glycineresidue ofcellular and viral proteins with diverse functions (1).This covalent modification appears to be irreversible and isrequired for full expression of the biological activities ofseveral known N-myristoylated proteins. For example,N-myristoylation of the a subunit of the signal-transducingguanine nucleotide binding protein (G protein) Go (termed GO,)increases its affinity for Py subunits and thereby promotesformation of the heterotrimeric complex (2). The tyrosinekinase p60v-src requires a myristoyl group to bind to its 32-kDaplasma membrane receptor and to transform cells (3, 4).N-myristoylation of the gag polyprotein precursors of severalmammalian retroviruses, including the Pr55m of human im-munodeficiency virus 1, is necessary for viral particle forma-tion (5). Myristoyl-CoA:protein N-myristoyltransferase(NMT; EC 2.3.1.97) catalyzes the transfer of myristate fromCoA to these proteins. Insertional mutagenesis of the Sac-charomyces cerevisiae NMTJ gene causes recessive lethality(6), indicating that N-myristoylation provides an essentialfunction for one or more of the 12 proteins of this yeast thatare substrates for NMT.These observations indicated that modulation of NMT

activity in vivo may be a useful antiviral and/or antifungaltherapeutic strategy. S. cerevisiae NMT has an ordered bi-bikinetic mechanism, with myristoyl-CoA binding taking placeprior to peptide binding and CoA release occurring beforeacylated peptide release (7). Cooperative interactions be-tween the acyl-CoA and peptide binding sites of NMTcontribute to its extraordinary chain-length specificity (8).

Substitution of a methylene group with an oxygen or sulfuratom yields fatty acid analogs with chain lengths and bondgeometries similar to those of myristate but with markedreductions in hydrophobicity (8). When incorporated intoproteins in vivo, these compounds can cause selective analog-dependent and specific alterations in function (9, 10). Forexample, 13-oxatetradecanoic acid is incorporated intoPr555ag and nef of human immunodeficiency virus 1, reducesproteolytic processing of the gag polyprotein precursor, andinhibits viral replication in both acutely and chronicallyinfected H9 cells without associated cellular toxicity (11).Other analogs are incorporated into N-myristoylated proteinsproduced by Candida albicans and Cryptococcus neofor-mans and either inhibit the growth of these pathogens inculture or are fungicidal (C. Langner, G. Kobayashi, G.Gokel, and J.I.G., unpublished observations). Therapeuticstrategies employing alternative NMT substrates or species-specific inhibitors require information about similarities anddifferences in the structure-activity relationships of humanand fungal NMTs. cDNAs encoding a number of humanenzymes have recently been cloned by genetic complemen-tation of a mutation in the corresponding gene of S. cerevisiae(12, 13). Using this approach, we have now isolated andcharacterized cDNAs encoding human (h) NMTA

MATERIALS AND METHODSComplementation Cloning. nmtl-181 strain YB207 (14) was

grown at 24°C in YPD [1% yeast extract/2% (wt/vol) pep-tone/2% (wt/vol) dextrose] to 5 x 107 cells per ml. Sphero-plasts were subsequently prepared and transformed (15) with2.5 ,ug of library (13) DNA plus 40 jig of sonicated herringsperm carrier DNA per i-10- cells per plate. The Ura-plates were incubated for 6-7 days at 38°C. Colonies thatformed under these growth conditions were replica-plated to5-fluoroorotic acid (5-FOA) medium (16) and incubated at 24or 36°C for 2-3 days. Isolates that grew on 5-FOA at 36°Cwere discarded. Plasmid DNA was recovered in Escherichiacoli (14) from 14 isolates that failed to grow at 36°C but couldgrow at 24°C. The nucleotide sequences of two of theseplasmids, designated pBB201 and pBB203, were determinedby the dideoxynucleotide chain-termination method. Thesequence at the 5' end of pBB203 and pBB201 begins atnucleotides 1 and 141 in Fig. 1A, respectively.Complementation of an nmtl Deletion Allele. Diploid strain

YB152 (NMTJ/NMTIA2.5::HIS3 ura3-52/ura3-52; ref. 14)was transformed to Ura+ with plasmids expressing hNMT

Abbreviations: NMT, N-myristoyltransferase; ts, temperature sen-sitive; h, human; y, yeast; G protein, guanine nucleotide bindingprotein; Ga, G protein a subunit; 5-FOA, 5-fluoroorotic acid; Metint,initiator methionine; 06, 6-oxatetradecanoate.tThe sequence reported in this paper has been deposited in theGenBank data base (accession no. M86707).

4129

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Mar

ch 6

, 202

1

4130 Biochemistry: Duronio et al.

ACCGCCGOCACCTCCGCTGCCGCAGATGATOGAAGGGAACOGGAACGGCCATGAGCACTGC

AGCGATTOCGAGAATGAGGAGGACAACAGCTACAACCGOGGTI5GTTTGAGTCCAGCCAAT

GACACTGGAOCCAAAAAG^AGAAAAAGAAACAAAAAAAGAAGAAAGAAA;AGGCAGTGAGACAGATTCAOCCCAGGATCACCCTGTGAAGATGAACTCTTTGCCAGCAGAGAGGATCCAG

MotAsnSerLeuProAlaGluArgl loGln 10

GAAATACAGAAGGCCATTGAGCTGTTCTCAGTGGGTCAGGGACCTGCCA;AACCATGGAG11 Glul leGlnLysAllIIeGluLouPhoSerValGly31nGIyProAlaLysThrMotGlu 30

GAGGCTA0CAAGCGAA,.CTACCAGTTCTGGGATACGCAGCCCGTCCCCA;ACTGGGCGA;31 Gl uAlaSerLysArgSerTyrGI nPheTrpAspThrGI nProVa IProLysLeuGl yGl u 50

GTGGTGAACACCCATGGCCCCGTGGAGCCTGACAAGGACAATATCCGCCAGGAGCCCTAC51 VaIValAsnThrHi sGlyProValGluProAspLysAspAsn IleArgGInGluProTyr 70

ACCCTGCCCCAGGGCTTCACCTGGGATGCTTTGGACCTGGGCGATCGTGGTGTGCTAAA;71 ThrLouProGInGl yPheThrTrpAspAIaLouAspLeuGlyAspArgGlyValLouLys 90

GAACTGTACACCCTCCTGAATGAGAACTATGTGGAAGATGATGACAACATGTTCCGATTT91 GluLouTyrThrLeuLouAsnGluAsnTyrValGluAspAspAspAsnMetPheArgPhe 110

GATTATTCCCCGGAGTTTCTTTTGTGGGCTCTCCGGCCACCCGGCTGGCTCCCCCAGTG;111 AspTyrSerProGluPheLeuLouTrpAlaLeuArgProProGlyTrpLouProGlnTrp 130

CACTGTGGGGTTCGAGTGGTCTCAAGTCGGAAATTGGTTGGGTTCATTAGCGCCATCCCA131 HisCysGlyValArgValValSerSerArgLysLeuValGlyPhoI leSerAlal lePro 150

GCAAACATCCATATCTATGACACAGAGAAGAAGATGGTAGAGATCAACTTCCTGTGTGTC151 AlaAsnIleHisIleTyrAspThrGluLysLysMetValGlulleAsnPheLeuCysVaI 170

CACAAGAAGCTGCGTTCCAAGAGGGTTGCTCCAGTTCTGATCCGAGAGATCACCAGGCGG171 Hsi LysLysLeuArgSerLyoArgValAlaProVaI Lou I IeArgGI u IeThrArgArg 190

GTTCACCTOGAGGGCATCTTCCAAGCAGTTTACACTOCCOGGOGTGGTACTACCAAAGCCT191 VaIHisLeuGI uGlyl lePhoGi nAlaVaITyrThrAlaGlyValValLeuProLysPro 210

GTTGGCACCTGCAGGTATTGGCATCGGTCCCTAAACCCACGGAAGCTGATTGAAGTGAAG211 VaIGlyThrCysArgTyrTrpHisArgSorLouAsnProArgLysLeul leGIuValLys 230

TTCTCCCACCTGAGCAGAAATATOACCATOCAGCGCACCATGAAGCTCTACCGACTOCCA231 PheSerHi sLouSerArgAsnMetThrMetGl nArgThrMetLysLeuTyrArgLeuPro 250

GAGACTCCCAAGACAGCTGGGCTGCGACCAATGGAAACAAAGGACATTCCAGTAGTGCAC251 GluThrProLysThrAlaGlyLeuArgProMetGluThrLysAspl ieProValValHis 270

CAGCTCCTCACCAGGTACTTGAAGCAATTTCACCTTACGCCCGTCATGAGCCAGGAGGAG271 G1 nLeuLeuThrArgTyrLeuLysGI nPheHi sLeuThrProVa IMotSerGI nGI uGI u 290

GTGGAGCACTGGTTCTACCCCCAGGAGAATATCATCGACACTTTCGTGGTGGAGAACGCA291 VaIGIuHisTrpPheTyrProGInGIuAsnl leIeAspThrPheValValGluAsnAla 310

AACGGAGAGGTGACAGATTTCCTGAGCTTTTATACGCTGCCCTCCACCATCATGAACCAT311 AsnGi yGI uVa lThrAspPheLeuSerPheTyrThrLeuProSerThr leMetAsnHi s 330

CCAACCCACAAGAGTCTCAAAGCTGCTTATTCTTTCTACAACGTTCACACCCAGACCCCT331 ProThrHisLysSerLeuLysAlaAlaTyrSerPheTyrAsnValHisThrGlnThrPro 350

CTTCTAGACCTCATGAGCGACGCCCTTGTCCTCGCCAAAATGAAAGGGTTTGATGTGTTC351 LeuLeuAspLeuMetSerAspAlaLeuValLeuAlaLysMotLysGlyPheAspValPhe 370

AATGCACTGGATCTCATGGAGAACAAAACCTTCCTGGAGAAGCTCAAGTTTGGCATAGGG371 AsnAlaLeuAspLeuMetGluAsnLysThrPheLeuGluLysLouLysPheGlyl leGly 390

GACGGCAACCTGCAGTATTACCTTTACAATTGGAAATGCCCCAGCATGGGGGCAGAGAAG391 AspGlyAsnLeuGInTyrTyrLeuTyrAsnTrpLysCysProSerMetGlyAlaGluLys 410

GTTGGACTGGTGCTACAATAACCAGTCACCAG3TGCGATTCTAGATAAAGCCACTGAAAAT411 Va 1GI yLeuVa LeuGI nEnd

TCGAACCAGGAAATGGAACCCCACCACTGTTGGTCCAATTTTCACACACGTGAGAATCCC

TGGCAAAGGGAGCAGAACTGAACCGGCTTTACCAAACCGCCAGCGAACTTGACAATTGTA

TTGCGATGGCGTGGGCTGCGTGACGTCACCTCCGGTCGTGTCTCTGGTCTCCGTGTTTTC

CAGTTAATTACATCCTCATGCAGCCGTGATCAAGGGAATGTAACTGCTGAAAACTAGCTC

GTGATTGGCATATAATGGAGTTAACGGGTGAATAATAAAAGTATATATATATATTATATA

TATAAAAAAAAAAAAAAAAAAAAAAA

BMNSLPAERIQEIOKAIELFSVGOGPAKTMEEASKRSYOFWDTOPVPKLGEV 51.:::I :: I . .. ..:..11I III.:

MSEEDKAKKLENLLKLLOLNNDDTSKFTOEQKKAMKDHKFWRTOPVKDFDEK 52

52 VNTHGPVEPDK .. DNIRQEPYTLPOGFTWDALDLGDRGVLKELYTLLNEN 50

53 VVEEGPIDKPKTPEDISDKPLPLLSSFEWCSIDVDNKKOLEDVFVLLNEN 102

100 YVEDDDNMFRFDYSPEFLLWALRPPGWLPOWHCGVRVVSSRKLVGFISAI 149MI1 1. 111:1..11: III:-III .:11.1111 ... 111:11111

103 YVEDRDAGFRFNYTKEFFNWALKSPGWKKDWHIGVRVKETOKLVAFISAI 152

150 PANIHIYDTEKKMVEINFLCVHKKLRSKRVAPVLIREITRRVHLEGIFOA 191

153 PVTLGVRGKQVPSVEINFLCVHKOLRSKRLTPVLIKEITRRVNKCDIWHA 202

200 VYTAGVVLPKPVGTCRYWHRSLNPRKLIEVKFSHLSRNMTMORTMKLYRL 241

203 LYTAGIVLPAPVSTCRYTHRPLNWKKLYEVDFTGLPDGHTEEDMIAENAL 252

250 PETPKTAGLRPMETKDIPVVHOLLTRYLKQFHLTPVMSOEEVEHWFYPOE 291

253 PAKTKTAGLRKLKKEDIDOVFELFKRYOSRFELI0IFTKEEFEHNFIGEE 302

300 ...... NIIDTFVVENANGEVTDFLSFYTLPSTIMNHPTHKSLKAAYSFY 34'.:1.:11.:1.:1111111 1:1....1 :1 :1

303 SLPLDKOVIFSYVVEQPDGKITDFFSFYSLPFTILNNTKYKDLGIGYLYY 352

344 NVHT..... .. QTPLLDLMSDALVLAKMKGFDVFNALDLM 371

353 YATDADFQFKDRFDPKATKALKTRLCELIYDACILAKNANMDVFNALTSO 402

377 ENKTFLEKLKFGIGDGNLOYYLYNWKCPSMGAE .............KVGYL 41::1.11:.1111 11 1.:11:1::...:.: .11:

403 DNTLFLDDLKFGPGDGFLNFYLFNYRAKPITGGLNPDNSNDIKRRSNVGV 452

414 VLO

453 VML

Proc. Natl. Acad. Sci. USA 89 (1992)

cDNAs. Recovery of His' Ura' haploid segregants aftersporulation and tetrad dissection of the transformants indi-cated complementation of the deletion with the hNMT plas-mids (14).

Construction of Plasmids. S. cerevisiae NMT [yeast (y)NMT] was expressed in E. coli using pBB125, which containsthe NMTJ gene linked to the recA promoter (17). pBB125 alsohas a ColEl origin and an ampicillin-resistance gene. GO andG,.c cDNAs were expressed in E. coli with plasmids pBB143and pBB144, respectively (18). These constructs have a Ptacpromoter, a piSA origin, and a kanamycin-resistance gene.An Nde I site was engineered at the initiator methionine(Metin0) codon of the hNMT cDNA using the method ofKunkel (19). This allowed the hNMT coding region to beplaced immediately downstream of the E. coli recA promoterin pMON2670, which has a ColEl origin of replication andencodes ampicillin resistance (17). The resulting plasmid wasdesignated pBB218. To express Gpal in E. coli, the codingsequences of the GPAJ (SCGI) gene (20) were fused to thePtac promoter in plasmid pBB229, which also contains a pi5Aorigin and encodes kanamycin resistance. This was achievedafter engineering an Nco I site at the Metint ofGPAI (19). TheGGA Gly412 codon of hNMT was mutated to encode eitheraspartic acid (GAT) or lysine (AAG) (19). Each of thesemutations was subcloned into the E. coli expression plasmidpBB218 and the S. cerevisiae expression plasmid pBB201.

[3H]Myristate Metabolic Labeling of Yeast and E. coli.Strains YB288 [NMT1A2.5::HIS3 pBB201 (hNMT)] andYB172 (isogenic with YB288 but with a yNMT complement-ing plasmid; ref. 14) were grown at 30°C in YPD to an A600value of -2.0 and incubated with [3H]myristic acid (39Ci/mmol, 50 ,uCi/ml of culture; 1 Ci = 37 GBq; NEN/DuPont) for 1 h at 30°C. Yeast lysates (100 ,ug of total cellularprotein) were subjected to SDS/PAGE and fluorography(14). E. coli JM101 containing pairwise combinations of theNMT and Ga-expressing plasmids were coinduced and la-beled with [3H]myristate at 50,Ci/ml exactly as described(14), except that for experiments at 24°C E. coli were grownat 37°C until the NMT induction step, at which time they wereshifted to 24°C.

RESULTS AND DISCUSSIONCloning of hNMT. Two reagents were used as starting

points for complementation cloning of a hNMT cDNA: (i) aHepG2 cDNA library constructed in a plasmid vector con-taining the glyceraldehyde-3-phosphate dehydrogenase pro-moter, a 2-,um origin of replication, and the URA3 gene as aselectable marker (13) and (ii) a S. cerevisiae strain contain-ing a mutant NMTI allele (nmtl-181) that causes ts myristic

2 acid auxotrophy (14). nmtl-181 encodes a Gly451 -- Asp point9 mutation of the 455-residue acyltransferase. Mutant cells are

unable to grow at restrictive temperatures (36-38°C) unless9 2-500 tkM myristate is added to the medium. In contrast, they2 grow at nearly wild-type rates at 24°C. This phenotype9 appears to arise from a temperature-dependent 10-fold in-~2 crease in the apparent Km of nmtl-181 for myristoyl-CoA

relative to wild-type NMT (14). After spheroplast transfor-mation of a strain carrying the nmtl-181 mutation with library

12 DNA, individual colonies were selected that could grow at38°C on medium lacking uracil. These isolates were tested fortheir ability to grow at 36°C on medium containing 5-FOA, a

k3 pyrimidine analog that kills cells with a wild-type URA3 genei2 (16). Cells carrying recombinant plasmids responsible for the

FIG. 1. Nucleotide sequence of hNMT cDNA and comparison ofits primary translation product to that of S. cerevisiae NMT1. (A)Complete sequence of hNMT cDNA and its 416-residue proteinproduct. (B) hNMT (upper sequence) and yNMT (lower sequence)were aligned using the BESTFIT program in the GCG softwarepackage (gap weight, 3.00; gap length weight, 0.10).

16

Q

3

i2

Dow

nloa

ded

by g

uest

on

Mar

ch 6

, 202

1

Proc. Natl. Acad. Sci. USA 89 (1992) 4131

suppression of temperature-sensitive (ts) growth will only beable to papillate Ura- colonies on 5-FOA-containing mediumat 240C and not at 360C. Plasmid DNA was recovered in E.coli from yeast satisfying this screen (14) and analyzed byrestriction endonuclease mapping. Plasmids containing over-lapping cDNA inserts of 1.6 and 1.8 kilobases were identified.Both recombinant plasmids abrogate ts growth after retrans-formation of the nmtl-181 strain. Furthermore, each couldrescue a strain with a complete deletion of NMTJ, indicatingthat they encode a hNMT activity.

Analysis of hNMT cDNA. Nucleotide sequence analysesindicated that both cDNAs were derived from the samemRNA. The shorter cDNA lacked most ofthe 210-nucleotide5' nontranslated domain contained in the longer cDNA,accounting for their different lengths. The open reading frameof 1248 nucleotides encodes a 416-residue polypeptide of Mr48,109 (Fig. 1A). Although there is an in-frame methioninecodon 86 nucleotides upstream of our assigned Metint, severalobservations suggest that it is unlikely to be used as a startsite for translation: (i) The designated Metint occurs in acontext that has a greater degree of similarity to the consen-sus sequence for translation initiation in eukaryotic mRNAs(21); (ii) the shorter hNMT cDNA clone contains only thedesignated Metint yet can fully rescue the lethal phenotypecaused by a S. cerevisiae nmtl deletion allele; (iii) inclusionof these additional amino acids yields a protein that is largerthan yNMT without additional sequence similarities; and (iv)the hNMT polypeptide described in Fig. 1A is catalyticallyactive when expressed in E. coli (see below).Sequence alignments revealed that the predicted primary

translation product ofhNMTmRNA has 44% identity with theproduct of S. cerevisiae NMTJ (Fig. 1B). This includes theGly412 ofhNMT, which occupies a position that is comparableto Gly451 in S. cerevisiae NMT. The overall similarity, asdefined by the alignment algorithm, is 63%. The N-terminal 40residues are the most divergent. Three gaps of 6, 17, and 13amino acids were introduced in the C-terminal half of hNMTto maximize its identity with yNMT. The primary structuresof hNMT and yNMT have no significant similarities to anyproteins in data bases searched (National Biomedical Re-search Facility/Protein Information Resource, Release 28.0;Swiss-Prot, Release 18.0; GenPept, Release 64.3).

Additional studies were performed to determine whetherthe human genome supported expression of multiple NMTsor contained related gene sequences. DNA was preparedfrom circulating nucleated cells in blood, digested with EcoRIor Spe I, and probed with the 1.6-kilobase 32P-labeled hNMTcDNA. The results (not shown) indicate that hNMT is asingle-copy gene. In addition, RNA blot hybridization anal-yses of total cellular RNA prepared from exponentiallygrowing and stationary post-confluent HepG2 cells and hu-man heart, gut, kidney, liver, and placenta revealed a singlereactive identically sized 2-kilobase mRNA (data not shown).hNMT and yNMT Have Overlapping Yet Distinct Protein

Substrate Specificities. There was no difference in growth rateor gross cellular morphology between a nmtl-deleted yeaststrain expressing only hNMT (YB288) and an isogenic strainexpressing an episomal copy ofNMTJ (YB172). Thus, hNMTmust be able to acylate all essential S. cerevisiae N-myris-toylated proteins to a degree that is sufficient for normalvegetative growth. [3H]Myristate labeling studies have re-vealed differences in the in vivo substrate specificities ofwild-type NMT and nmtl-181 at the restrictive temperature(14). When strains YB288 and YB172 were incubated with[3H]myristate and total cellular proteins were subjected tosingle-dimension SDS/PAGE and fluorography, it was ap-parent that all S. cerevisiae N-myristoylated proteins (1, 14)were substrates for hNMT and yNMT (data not shown).An in vivo assay of NMT activity was used to determine

whether there are differences in the substrate specificities ofh- and yNMTs. In this assay system, two plasmids containing

separately inducible promoters, different but compatibleorigins of replication, and distinct antibiotic resistance genesare employed to direct production of NMT and known orputative protein substrates in E. coli, a bacterium with noendogenous NMT activity (17, 18). The reconstituted eu-karyotic protein modification is specific for myristoyl-CoAand requires substrate proteins with an N-terminal glycine(17). Several homologous mammalian and yeast Ga proteinswith conserved N-terminal domains were employed for theseanalyses, since they provide an opportunity to examinesubtle variations in the protein substrate specificities oforthologous NMTs (18).NMT and Ga production were sequentially induced in E.

coli JM101 during growth in medium containing[3H]myristate. Lysates were prepared and cellular proteinswere subjected to SDS/PAGE and fluorography. Coexpres-sion ofyNMT and rat Go,, in E. coli yields a tritiated 40-kDaprotein (Fig. 2A, lane 2) that is not detected in strainsproducing yNMT alone (Fig. 2A, lane 1). Coexpression ofhNMT and Go,, results in production of similar amounts ofthis labeled 40-kDa polypeptide (Fig. 2A, lane 4). Immuno-blots indicated that the steady-state level of Go,, was com-parable in each lysate (Fig. 2C). Coexpression of S. cerevi-siae Gpal (the Ga involved in mating pheromone signaltransduction; ref. 20) and yNMT or hNMT yielded a labeled55-kDa band of equivalent intensity (Fig. 3A, lanes 4 and 5,respectively). The levels of immunoreactive Gpal in theselysates were also equivalent (Fig. 3C, lanes 4 and 5). Awell-characterized rabbit anti-yNMT serum (14) did notproduce a signal when incubated with blots of lysates pre-pared from E. coli containing a hNMT expression vector.However, Coomassie blue staining of SDS/polyacrylamide

A

66-

'0

0 45-

xU.

2 3 4B

1 2 3 4

66-

°45-_ w X

29-

c

29-

D1 2 3

66-

° 45-

29-

1 2 3

b 66-45-

2 29-

FIG. 2. Coexpression of hNMT and yNMT with Ga, in E. coli.[3H]Myristate-labeled cellular proteins recovered from E. coli coex-pressing hNMT or yNMT and a Ga substrate were subjected toSDS/PAGE and fluorography. (A) Lanes: 1, yNMT alone; 2, yNMTplus rat Go,; 3, hNMT alone; 4, hNMT plus rat Go,. The gel wasexposed for 2 days. (B) Lanes: 1, yNMT alone; 2, hNMT alone; 3,yNMT plus human Ga,; 4, hNMT plus human Ga,. The gel wasexposed for 14 days. A portion of each induced culture was nottreated with [3H]myristate, and the unlabeled proteins were used forimmunoblot analysis. (C) A 1:2000 dilution of rabbit antiserum P-960(22) was utilized to detect Goc. Lanes: 1, no Go,,; 2, yNMT plus Go,,;3, hNMT plus GO,. Antigen-antibody complexes were visualized bya chemiluminescent detection method (Amersham). (D) A 1:1000dilution of rabbit antiserum P-%1 (22) was used to detect Ga, Lanes:1, no Ga,; 2, yNMT plus Ga; 3, hNMT plus Gza. Antigen-antibodycomplexes were visualized with 1251-labeled protein A.

Biochemistry: Duronio ei al.

Dow

nloa

ded

by g

uest

on

Mar

ch 6

, 202

1

4132 Biochemistry: Duronio et al.

gels after electrophoretic separation of lysate proteins re-vealed that the steady-state levels of hNMT and yNMT wereequivalent at the end of the metabolic labeling experiments(i.e., those involving Go,, and Gpal, data not shown). Thusthese data allowed us to conclude that the open reading framedescribed in Fig. 1A yields catalytically active hNMT whenit is expressed in E. coli and that there has been someconservation of the protein substrate specificities of theseorthologous NMTs over the course of eukaryotic evolution.

Expression of yNMT alone results in the labeling of severalunidentified endogenous E. coli proteins (17, 18). These poly-peptides are not labeled by hNMT (compare Fig. 2B, lanes 1and 2), suggesting that the sequence divergence between thetwo NMTs has been accompanied by differences in theirprotein substrate specificities. Human Gzc is N-myristoylatedwhen expressed in COS cells (10) but is not labeled by yNMTin the E. coli coexpression system (Fig. 2B, lane 3). Coex-pression ofhNMT and Go, results in labeling of a 40-kDa band(Fig. 2B, lane 4) that is not detected when the G,, cDNA isabsent (Fig. 2B, lane 2). Immunoblot analyses employing aGz,-specific antiserum revealed equal amounts ofGo in eachlysate (Fig. 2D), suggesting that human G2, is a better sub-strate for hNMT than yNMT. The N-terminal 6-8 residues ofNMT substrates appear to provide the principal recognitiondeterminants for many N-myristoylated proteins (1). There arethree amino acid differences in this region of Go,, and G,(GCTLSAEE vs. GCRQSSEE, respectively). However, forGa, recent studies indicate that amino acids C-terminal tothese 6-8 residues may also play an important role in modu-lating enzyme-substrate interactions (18).

Mutation of Gly412 of hNMT Affects Catalysis. Gly45' ofyNMT plays a prominent role in myristoyl-CoA recognitionand catalysis. A Gly451 -- Asp yNMT mutant displaysreduced activity in the E. coli coexpression system, and aLys451 mutant is completely inactive (14). As noted above,the Asp451 mutation causes ts myristic acid auxotrophy in S.cerevisiae, and the Lys451 mutation of yNMT is unable to

A B

2 4 5 6 7

66 -

0

- 45-x

v: _OM

e 4 Xt

66

-- 45-

Ox L+ -

29 29

2 54 5 7 8

66-

454-:~ -O 4

r 66-

- 45-2 __V

, 4 5 t

saws

FIG. 3. Coexpression of [Asp4l2]- and [Lys412]hNMT mutantswith G0 in E. coli. [3H]Myristate-labeled proteins produced in E. colithat coexpressed wild-type yNMT, wild-type hNMT, [Asp412IhNMT,or [Lys412]hNMT and rat G,, or yeast Gpal substrates were subjectedto SDS/PAGE and fluorography. (A) Induction and labeling wereperformed at 37TC. Lanes: 1, hNMT plus Go; 2, [Asp412]hNMT plusGo; 3, [Lys412]hNMT plus Go; 4, yNMT plus Gpal; 5, hNMT plusGpal; 6, [Asp412]hNMT plus Gpal; 7, [Lys412]hNMT plus Gpal. Thearrow indicates the position of migration of Go,,, and the arrow plusasterisk indicates the location ofGpal. (B) Induction and labeling wereperformed at 240C. The lanes are as in A. (C and D) Immunoblots ofunlabeled bacterial lysates representing lanes 1-7 ofA and B, respec-tively. Lane 8 ofC represents a control lysate lacking Gpal. G. (lanes1-3) was detected with P-960, and Gpal (lanes 4-7) was detected witha 1:200 dilution of a rabbit antiserum raised against recombinantprotein (23). Antigen-antibody complexes were visualized with 1251_labeled protein A.

complement a nmtl deletion allele on YPD medium even at240C (14). Since the comparably positioned codons 451 and412 in yNMT and hNMT both encode a glycine, we examinedwhether Gly -- Asp or Lys site-directed mutations wouldhave effects on hNMT that were similar to those observed inyNMT. The activities of [Gly412]-, [Asp412]-, and[Lys412]hNMTs were compared in the E. coli coexpressionsystem at 24 and 370C by using Go, and Gpal as substrates.In contrast to wild-type hNMT (Fig. 3A, lane 1), no labelingof Go, was detected at 370C with either the [Asp412]- or[Lys412]hNMT mutants (Fig. 3A, lanes 2 and 3, respectively).At 240C, labeling of Go, was detected with each mutant (Fig.3B, lanes 2 and 3), but at a level that was lower than thatobtained with wild-type hNMT (Fig. 3B, lane 1). The[Lys412]hNMT mutant was less active than [Asp412]hNMT(compare Fig. 3B, lanes 2 and 3). Immunoblots revealed thatthese differences in incorporation of [3H]myristate were notdue to differences in the concentration of Go,, in the lysates(Fig. 3 C and D, lanes 1-3). Labeling of Gpal by the [Asp412]-and [Lys412]hNMT mutants was significantly reduced rela-tive to wild type at 37TC, with [Lys412]hNMT being less activethan [Asp412]hNMT (Fig. 3A, lanes 6 and 7). However, incontrast to the results obtained with Goa, the intensity oflabeling of Gpal with [3H]myristate was similar with all threeforms ofhNMT when the experiment was performed at 240C(Fig. 3B, lanes 5-7). Immunoblots confirmed that the amountof Gpal was equivalent in all lysates (Fig. 3 C and D, lanes4-7). The levels of yNMT, wild-type hNMT, and mutanthNMTs were equivalent in the Go,, and Gpal experiments at24 or 37°C, as judged by Coomassie blue staining of SDS/polyacrylamide gels (data not shown). Thus, it appears thatthe relative activities of the mutant hNMTs depend upontemperature and the particular protein substrate.

Gly412 Mutations ofhNMT Cause Myristic Acid Auxotrophyin Yeast. These results suggested that if the [Asp412]- or[Lys412]hNMT mutants could support vegetative growth of S.cerevisiae, they may induce a ts phenotype or even tsmyristic acid auxotrophy as caused by nmtl-181. Therefore,restriction fragments encompassing the [Asp412]- and[Lys412]hNMT mutations were used to replace the compara-ble fragment of wild-type hNMT in our original recombinantplasmid. Both [Asp412]- and [Lys412]hNMT plasmids couldcomplement the lethal phenotype of a nmtl null allele.However, the phenotype of these strains was different thanthat of isogenic strains carrying an episomal copy of eitherNMT1 or wild-type hNMT cDNA. Both the [Asp412]- and[Lys412]hNMT mutations caused ts growth on standard YPDmedium (Fig. 4). Normal growth of the [Asp412]hNMT-containing strain could be restored at the nonpermissivetemperature (36°C) by supplementing the medium with 500,uM myristate (YPD-MYR). [Lys412]hNMT caused a moredramatic ts phenotype: this strain grew well on YPD-MYR at30°C but was unable to grow on YPD-MYR at 36°C. Thisresult implies that [Asp412]hNMT is more active in vivo than[Lys412]hNMT, an observation consistent with the pattern ofactivity seen in the E. coli coexpression system (Fig. 3). Thestrain containing [Asp412]hNMT grew better under all con-ditions than a strain containing a single genomic copy ofnmtl-181 (Fig. 4). Since the hNMTs are expressed with thestrong glyceraldehyde-3-phosphate dehydrogenase pro-moter, this result may be analogous to the observation thatincreased expression of the nmtl-181 enzyme (using a GALI-nmtl-181 fusion) relieves the ts phenotype (14). Microscopicexamination of temperature-arrested [Asp412]- or [Lys412]hNMT-producing cells revealed a wide variety of bud sizes(data not shown), indicating that growth arrest did not occurat a single place in the cell cycle and suggesting that reducedN-myristoylation of Gpal, which causes G1 phase arrest and"shmoo" formation (23), was not solely responsible forgrowth arrest at 360C.

Proc. Natl. Acad. Sci. USA 89 (1992)

Dow

nloa

ded

by g

uest

on

Mar

ch 6

, 202

1

Proc. Natl. Acad. Sci. USA 89 (1992) 4133

NMTI

nmtl-181 hNMTLys412 Asp412

240C 300C 360C

YPD~

YPD-MYR

YPD M M EPAL /CER

YPD-06

FIG. 4. Phenotype of S. cerevisiae strains expressing only mutanthNMTs. Strains containing the indicated NMTs were tested forgrowth at 24, 30, and 360C on the following media: YPD, YPDsupplemented with 500 MuM myristate (YPD-MYR) or 500 AM 06(YPD-06), and YPD supplemented with 25 jiM cerulenin and 1.3 mMpalmitate (YPD-PAL/CER). All media (except YPD) also contain1% Brij 58 to help solubilize free fatty acids. The plates shown wereincubated for 2 days at the indicated temperatures. The genotype ofstrains containing [Asp4l2]- and [Lys412]hNMT is identical to YB288,except that they contain episomes carrying the mutant cDNAs [seeref. 14 for the genotype of YB218 (nmtl-181)].

Two additional phenotypic characteristics of the nmtl-181mutation were recapitulated by the [Asp412] and [Lys412]-hNMT-expressing strains. Growth of nmtl-181 strains isinhibited relative to wild type when the medium is supple-mented with cerulenin (14). Both the [Asp412]- and[Lys412]hNMT strains display growth inhibition at 24, 30, and36°C on YPD containing 25 ,M cerulenin and 1.3 mMpalmitate (Fig. 4). At this concentration, cerulenin is knownto block the de novo pathway of fatty acid synthesis (whichyields myristoyl-CoA as one of its products) by inhibiting thea436 fatty acid synthetase complex (24). Thus, the cerulenin-induced growth inhibition observed in strains producing[Asp4l2]- or [Lys412]hNMT, but not wild-type hNMT, may bedue to an increased sensitivity to reductions in intracellularmyristoyl-CoA levels-reductions that cannot be overcomeby metabolic processing of exogenous palmitate. Such sen-sitivity could arise if these mutant enzymes had reducedaffinity for myristoyl-CoA (as was found with nmtl-181; ref.14) or a perturbation in their ability to gain access to cellularpools of this substrate. Given the ordered bi-bi reactionmechanism of yNMT, any disturbance in myristoyl-CoAbinding should produce marked alterations in proteinN-myristoylation. The other phenotypic similarity shared bystrains producing [Asp451]yNMT, [Asp412]hNMT, and[Lys412]hNMT is that they cannot grow at the permissivetemperature (24°C) on YPD containing the myristic acidanalog 6-oxatetradecanoate (06, ref. 8) at 500 AM (Fig. 4).The underlying mechanism for this phenomenon is notknown. In vitro studies using purified yNMT and octapeptidesubstrates derived from known N-myristoylated proteinsindicate that 06-CoA is utilized by the enzyme (8). Theextent of accumulation of radiolabeled 06 in S. cerevisiae is1% that of C14:0 (14). Furthermore, 06 is not incorporatedinto S. cerevisiae N-myristoylated proteins (14). The simi-larities between these observations with 06 and those ob-tained with cerulenin have led us to propose that 06 maydeplete intracellular myristoyl-CoA pools, possibly throughinhibiting de novo fatty acid synthesis or import ofexogenous

Conclusions. The data presented in this report suggest thathNMT and yNMT are sensitive to the same perturbations inS. cerevisiae myristoyl-CoA metabolism. Moreover, the sim-ilar phenotypes caused by the codon 412 and 451 mutations inhNMT and yNMT, respectively, indicate that the structuraldeterminants required for myristoyl-CoA acquisition in vivomay be the same in hNMT and yNMT. In this sense, expres-sion of wild-type or mutant hNMTs in S. cerevisiae providesa powerful system for identifying and characterizing factorsthat may regulate protein N-mynistoylation in mammalian cells(e.g., systems for importing fatty acids and maintaining intra-cellular myristoyl-CoA pools). Finally, it appears that over thecourse of eukaryotic evolution these NMTs have retainedsimilarities in substrate specificity yet have developed distinctdifferences. This finding should have important therapeuticimplications and may permit development of species-specificinhibitors or alternative acyl-CoA substrates that can beselectively incorporated into target proteins.We thank Robin Johnson for excellent technical assistance. This

work was supported in part by grants from the National Institutes ofHealth (AI27179 and A130188) and Monsanto.

1. Gordon, J. I., Duronio, R. J., Rudnick, D. A., Adams, S. P. &Gokel, G. W. (1991) J. Biol. Chem. 266, 8647-8650.

2. Linder, M. E., Pang, I.-K., Duronio, R. J., Gordon, J. I.,Sternweiss, P. C. & Gilman, A. G. (1991) J. Biol. Chem. 266,4654-4659.

3. Resh, M. D. (1989) Cell 58, 281-286.4. Cross, F. R., Garber, E. A., Pellman, D. & Hanafusa, H.

(1984) Mol. Cell. Biol. 4, 1834-1842.5. Bryant, M. & Ratner, L. (1990) Proc. Natl. Acad. Sci. USA 87,

523-527.6. Duronio, R. J., Towler, D. A., Heuckeroth, R. 0. & Gordon,

J. 1. (1989) Science 243, 796-800.7. Rudnick, D. A., McWherter, C. A., Rocque, W. J., Lennon,

P. J., Getman, D. P. & Gordon, J. I. (1991) J. Biol. Chem. 266,10498-10504.

8. Kishore, N. S., Lu, T., Knoll, L. J., Katoh, A., Rudnick,D. A., Mehta, P. P., Devadas, B., Huhn, M., Atwood, J. L.,Adams, S. P., Gokel, G. W. & Gordon, J. I. (1991) J. Biol.Chem. 266, 8835-8855.

9. Johnson, D. R., Cox, A. D., Solski, P. A., Devadas, B., Adams,S. P., Leimgruber, R. M., Heuckeroth, R. O., Buss, J. E. &Gordon, J. I. (1990) Proc. Natl. Acad. Sci. USA 87, 8511-8515.

10. Mumby, S. M., Heuckeroth, R. O., Gordon, J. I. & Gilman,A. G. (1990) Proc. Natl. Acad. Sci. USA 87, 728-732.

11. Bryant, M. L., Ratner, L., Duronio, R. J., Kishore, N. S.,Devadas, B. & Gordon, J. I. (1991) Proc. Natl. Acad. Sci. USA88, 2055-2059.

12. Colicelli, J., Birchmeier, C., Michaeli, T., O'Neill, K., Riggs,M. & Wigler, M. (1989) Proc. Natl. Acad. Sci. USA 86,3599-3603.

13. Schild, D., Brake, A. J., Kiefer, M. C., Young, D. & Barr,P. J. (1990) Proc. Natl. Acad. Sci. USA 87, 2916-2920.

14. Duronio, R. J., Rudnick, D. A., Johnson, R. L., Johnson,D. R. & Gordon, J. I. (1991) J. Cell Biol. 113, 1313-1330.

15. Hinnen, A., Hicks, J. B. & Fink, G. R. (1978) Proc. Natl.Acad. Sci. USA 75, 1929-1932.

16. Boeke, J. D., LaCroute, F. & Fink, G. R. (1984) Mol. Gen.Genet. 197, 345-346.

17. Duronio, R. J., Jackson-Machelski, E., Heuckeroth, R. O.,Olins, P. O., Devine, C. S., Yonemoto, W., Slice, L. W.,Taylor, S. S. & Gordon, J. I. (1990) Proc. Natl. Acad. Sci.USA 87, 1506-1510.

18. Duronio, R. J., Rudnick, D. A., Adams, S. P., Towler, D. A.& Gordon, J. I. (1991) J. Biol. Chem. 266, 10498-10504.

19. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492.20. Dietzel, C. & Kurjan, J. (1987) Cell 50, 1001-1010.21. Kozak, M. (1986) Cell 44, 283-292.22. Casey, P. J., Fong, H. K. W., Simon, M. I. & Gilman, A. G.

(1990) J. Biol. Chem. 265, 2383-2390.23. Stone, D. E., Cole, G. M., de Barros Lopez, M., Goebl, M. &

Reed, S. I. (1991) Genes Dev. 5, 1969-1981.24. Awaya, J., Ohno, T., Ohno, H. & Omura, S. (1975) Biochim.

myristate into cells (14).

Biochemistry: Duronio et al.

Biophys. Acta 409, 267-273.

Dow

nloa

ded

by g

uest

on

Mar

ch 6

, 202

1