INTRODUCTION

Myristoyl-CoA:protein N-myristoyltransferase (Nmt¹, EC 2.1.3.97) catalyzes the co-translational, covalent attachment of myristate (C14:0) to the NH₂-terminal glycine of eukaryotic cellular and viral proteins (1). Nmt is a potential target for antiviral (2, 3, 4), antifungal (5, 6, 7), and antineoplastic therapy (8, 9, 10).

Saccharomyces cerevisiae Nmt1p has been used as a model for examining the enzyme's kinetic mechanism, substrate specificities, and biological functions. The NMT1 gene is essential for vegetative growth (11, 12). It encodes a 455-residue cytoplasmic protein (13). Nmt1p has no known co-factor requirements (14). The enzyme's mechanism is ordered Bi Bi (15). Apoenzyme binds myristoyl-CoA, forming a high affinity myristoyl-CoA·Nmt1p complex (K_d = 15 nM; Ref. 16). There is heterotropic cooperativity between the enzyme's acyl-CoA and peptide binding sites; formation of the binary complex allows synthesis of a peptide binding site (16). After assembly of a ternary myristoyl-CoA·Nmt1p·peptide complex, C14:0 is transferred from CoA to the peptide substrate, and the products are released (CoA followed by myristoylpeptide). Nmt1p's acyl-CoA and peptide specificities have been defined in vitro using purified enzyme, >300 fatty acid analogs with systematic alterations in chain length, polarity, conformation, and steric bulk, plus >100 octapeptides representing variations in the NH₂-terminal sequences of known N-myristoylproteins (reviewed in Ref. 1).

The primary structures of six orthologous Nmts have been determined (11, 17, 18, 19). The 450-529-residue proteins contain 105 absolutely conserved amino acids. Searches of current protein data bases with these primary structures has failed to disclose any discernible homology to other sequence entries.

Only a modest amount of information exists about Nmt1p's structure/activity relationships. Deletion analyses suggest that the minimal domain required for myristoyltransferase activity spans Ile⁵⁹-Phe⁹⁶ through Gly⁴⁵¹-Leu⁴⁵⁵ (20). Two distinct genetic selections have identified two residues important for function. An allele with a single amino acid substitution, Gly⁴⁵¹  Asp (nmt1-451D) was recovered during a screen for mutations that cause temperature-sensitive (ts) fatty acid auxotrophy (12, 21). nmt1-451D produces growth arrest at various stages of the cell cycle 1 h after shifting from 24 to 37 °C and produces lethality after an 8-12-h incubation at nonpermissive temperatures (12, 22). The growth arrest and lethality can be rescued at 37 °C by adding myristate but not shorter or longer chain saturated fatty acids. Another allele with a single Leu⁹⁹  Pro substitution (nmt1-99P) is associated with undermyristoylation of Gpa1p, thereby reducing this  subunit's affinity for the polypeptides of a heterotrimeric G protein involved in the mating response (23, 24). The resulting free subunits produce constitutive activation of the mating pathway and growth arrest. Leu⁹⁹  Pro results in less global alterations in protein N-myristoylation than does Gly⁴⁵¹  Asp (24). Gly⁴⁵¹ and Leu⁹⁹ are conserved in all six known Nmts. Introduction of the Gly  Asp substitution in Cryptococcus neoformans and Candida albicans Nmts also produces temperature-sensitive growth arrest and myristic acid auxotrophy in these fungal pathogens (5, 6).

Site-directed mutagenesis has been used to replace each of human Nmt's four conserved His residues with Asn and each of its two conserved Cys residues with Ser (25). Site-directed mutagenesis of absolutely conserved amino acids can be very useful for identifying residues critical for substrate recognition, binding, and/or catalysis. In the case of Nmt, designing such experiments is hindered by the high percentage of conserved amino acids, by the lack of homology to other proteins, and by the lack of a rapid screening assay for disabling mutations. In this report, we describe how random PCR mutagenesis and an in vivo screen can be used to recognize mutations of conserved residues in Nmt1p that affect the functional properties of its myristoyl-CoA and/or peptide binding sites.

EXPERIMENTAL PROCEDURES

Yeast Strains and Media

pBB110 is a 2 µ YEp plasmid containing NMT1 (11). The isogenic strains YB332 (MATa NMT1 ura3-52 his3200 ade2-101 lys2-801 leu2-3,112) and YB336 (MATa nmt1-181 ura3-52 his3200 ade2-101 lys2-801 leu2-3,112) were described by Johnson et al. (24). YB510 (MAT nmt1::HIS3 ura3-52 his3200 ade2-101 ade3 lys2-801 leu2-3,112 trp1901, pBB110) was obtained from a cross between YB133 (MATa nmt1::HIS3 ura3-52 his3200 ade2-101 lys2-801 trp1901 can1 leu2::pRY181, pBB110) and YB428 (MAT nmt1-181 ura3-52 his3200 ade2-101 ade3 leu2 trp1901). YB523 was obtained from YB510 by replacing pBB110 with pBB290, a high copy 2 µ NMT1 ADE3 URA3 plasmid derived from pTSV31A (kindly supplied by Alan Bender, Indiana University).

Strains were incubated at 24-40 °C on YPD (1% yeast extract, 2% peptone, 2% dextrose)/agar plates with or without myristate (500 µM; NuChek-Prep). Brij 58 (1% (w/v); Sigma) was included in media containing myristate. Synthetic complete medium (SC) minus leucine and/or uracil (BIO 101) was used for selection of plasmids with LEU2 and URA3 markers. Episomes with URA3 were removed from cells by plating them on SC supplemented with 0.1% 5-fluoro-orotic acid (5-FOA; PCR, Inc.).

Random PCR Mutagenesis and Colony Color Sectoring Assay

The entire open reading frame (ORF) of NMT1 plus three overlapping subdomains of the ORF were amplified by PCR (Fig. 1A). PCR was performed using the conditions described in Fig. 1B plus the following thermocycling protocol: 94 °C for 1 min (denaturation), 55 °C for 2 min (annealing), and 72 °C for 3 min (extension) for a total of 30 cycles.

Each PCR fragment was used to replace a segment of the NMT1 ORF by in vivo recombination with a gapped plasmid (26). Replacement involved the following steps: (i) the PCR product was purified by agarose gel electrophoresis and the GeneClean kit (BIO 101); (ii) pBB361 (NMT1 LEU2) was digested with either NcoI and BalI, NcoI and AvrII, SphI and MluI, or BalI alone (Fig. 1A), and the linearized, gapped plasmids were purified as in the first step; (iii) S. cerevisiae strain YB523 (nmt1, ura3-52, ade2, ade3, leu2, pBB290) was co-transformed with the PCR fragment and gapped plasmids (27); (iv) cells were plated on SC minus leucine and incubated for 3 days at 30 °C.

A colony color sectoring assay was performed as outlined in Fig. 2. Leucine prototrophs were replated on YPD and incubated at 30 °C for 5 days. Individual red colonies were restreaked on YPD and incubated for 5 days at 30 °C. Colonies that continued to exhibit a nonsectoring (red) phenotype due to retention of the 2 µ pBB290 episome (NMT1 ADE3 URA3), were replica plated onto SC/5-FOA minus leucine and incubated at 24 °C and 35-40 °C to identify nmt1 mutations that produced a ts growth phenotype.

Total cellular nucleic acids were extracted (28) from ts isolates and used to transform Escherichia coli strain JS5. Plasmids were purified from bacterial colonies that grew on Luria broth (LB) supplemented with kanamycin (50 µg/ml). The nucleotide sequence of each plasmid's nmt1 insert was determined using a panel of 14 oligonucleotide primers (11, 12), a DyeDeoxy terminator cycle sequencing kit, and a model 373 Automated DNA Sequencer (Applied Biosystems). The primers cover both strands of NMT1's ORF at intervals of 200 base pairs. 400-500 base pairs of sequence were obtained per primer, allowing 200-300-base pair overlaps between each primer-driven reaction.

Site-directed Mutagenesis

Twenty-three of the ts nmt1 alleles contained more than one amino acid substitution. These multiply mutated protein sequences were aligned with one another and with the sequences of orthologous Nmts using the algorithm included in GeneWorks (version 2.4). The alignments identified eight residues that were conserved in five or six of the six known Nmts and mutated in the ts nmt1 alleles. Site-directed mutagenesis was used to introduce each of the eight mutations (individually) into NMT1's ORF: Asn¹⁰²  Thr (AAC  ACC); Leu¹⁷¹  Ser (TTG  TCG); Lys³⁸⁹  Ile (AAA  ATA); Val³⁹⁵  Asp (GTT  GAT); Leu⁴⁰⁸  Ser (TTG  TCG); Phe⁴¹³  Ser (TTC  TCC); Asp⁴¹⁷  Val (GAC  GTC); and Leu⁴²⁰  Ser (TTG  TCG). The entire ORF of each site-directed mutant nmt1 allele was sequenced to confirm that only the desired nucleotide substitution was present.

Measurement of Steady-state Levels of Wild Type and Mutant Nmts in S. cerevisiae

pBB290 (see above) was removed from strain YB523 (nmt1) and replaced (29) with low copy centromeric pRS315-derived plasmids (30) that contained a LEU2-selectable marker, kanamycin and ampicillin resistance genes, and a mutant nmt1 ORF under the control of the NMT1 promoter (31). These plasmids include pBB391 (nmt1-102T); pBB387 (nmt1-202T); pBB386 (nmt1-217R); pBB380 (nmt1-328P); pBB392 (nmt1-395D); pBB381 (nmt1-404Y); pBB393 (nmt1-420S); pBB385 (nmt1-426I); pBB382 (nmt1-451D); and an NMT1 control (pBB361).

One hundred-milliliter cultures of the various transformants were grown at 24 °C in YPD to an A₆₀₀ = 0.8. Twenty-five-milliliter aliquots were removed and incubated at 24 or 37 °C for 2 h. Cells were subsequently harvested by centrifugation at 1,600 × g for 10 min at 4 °C and washed twice with 15 ml of phosphate-buffered saline. The cell pellet was resuspended in 0.5 ml of lysis buffer (2% SDS, 80 mM Tris, pH 6.8, 200 µM Pefabloc SC, 2 µM leupeptin, 2 µM pepstatin; all protease inhibitors from Boehringer Mannheim). Cells were disrupted by vortexing with 0.5 ml of 425-600-µm glass beads (Sigma; vortexing was in four cycles, 1 min/cycle). The mixture was boiled for 10 min. Cellular debris were removed by centrifugation at 10,000 × g for 5 min. The protein concentration in the cleared lysates was determined using the BCA assay kit (Pierce). Equal masses of protein from each sample (100 µg) were reduced, denatured, fractionated by SDS-PAGE (32), and transferred to polyvinylidene difluoride membranes (Amersham Corp.). Western blots were probed with a previously characterized rabbit anti-C. albicans Nmt sera that recognizes the orthologous S. cerevisiae acyltransferase (Ref. 19; diluted 1:5000 in Blotto). Antigen-antibody complexes were visualized using an enhanced chemiluminescence (ECL) kit and the protocol recommended by its manufacturer (Amersham).

Purification of Nmts from E. coli

pBB376 (kindly supplied by Jennifer Lodge, Washington University) was derived from pMON22310 (origin of replication, pBR322; selectable marker, streptomycin resistance gene). pBB376 contains the NMT1 ORF with an NH₂-terminal tag of six histidine residues (6XHis). Expression of 6XHis-Nmt1p is controlled by the isopropyl--D-thiogalactopyranoside-inducible Ptac promoter. Appropriate restriction enzymes were used to place a fragment of an nmt1 ORF containing an amino acid substitution into similarly digested pBB376, yielding the following plasmids: pBB390 (6XHis-nmt202Tp); pBB389 (6XHis-nmt217Rp); pBB378 (6XHis-nmt328Pp); pBB379 (6XHis-nmt404Yp); pBB388 (6Xhis-nmt426Ip); and pBB377 (6XHis-nmt451Dp). The ORFs contained in each 6XHis-nmt1p expression vector were sequenced on both strands to verify that no additional nucleotide changes had been introduced during vector construction.

1.5-liter cultures of E. coli strain JM101, containing a 6XHis-nmt1p expression plasmid, were grown in LB/streptomycin (30 µg/ml) and 100 µM isopropyl--D-thiogalactopyranoside at 24 °C for ~12 h until they reached an A₆₀₀ = 1. Bacteria were harvested by centrifugation at 5,000 × g for 20 min at 4 °C and resuspended in 50 ml of buffer A (0.3 M NaCl, 5 mM -mercaptoethanol, 1 mM Pefabloc SC, 2 µM leupeptin, 2 µM pepstatin, 25 mM sodium phosphate buffer, pH 7.0). Cells were lysed with French press at 3,200 p.s.i. The lysates were centrifuged at 20,000 × g for 30 min at 4 °C. Each supernatant was collected and mixed with 5 ml of Ni²⁺-NTA-agarose (Qiagen; the resin was washed three times with buffer A before use). The resulting suspension was incubated on ice for 2 h with intermittent shaking and then subjected to centrifugation at 3,000 × g for 5 min at 4 °C. The supernatant was removed and discarded. The Ni²⁺-NTA-agarose was washed four times with ice-cold buffer A (30 ml/wash) and loaded into an empty 10-ml Poly-Prep column (Bio-Rad). The column was washed with 10 ml of buffer A containing 40 mM imidazole. Nmt activity was eluted by sequentially washing the column with 3 ml of buffer A containing 60 mM, 80 mM, 100 mM, 120 mM, and finally 150 mM imidazole. Wild type and mutant enzymes eluted with buffer A, 120 mM imidazole and were used on the day of their purification for kinetic studies (see below). Wild type 6XHis-Nmt1p and each mutant 6XHis-nmt1p were purified on at least three separate occasions. The purity of each preparation was verified by SDS-PAGE followed by Coomassie staining.

Nmt1p without an NH₂-terminal 6XHis tag was also expressed in E. coli and purified to apparent homogeneity using a protocol described by Rudnick et al. (33).

Kinetic Studies of Wild Type and Mutant Nmts

A coupled in vitro Nmt assay system was employed (33, 34). [³H]Myristoyl-CoA was generated first using [³H]myristate, CoA, and Pseudomonas acyl-CoA synthetase (Boehringer Mannheim). A purified wild type or mutant Nmt was then added together with a peptide substrate. Following a 10-min incubation at 24 or 37 °C, [³H]myristoylpeptide was purified from the reaction mixture by reverse phase high pressure liquid chromatography using a 4.6 µm (internal diameter) × 150-mm C4 column (Vydac) and a linear gradient from H₂O containing 0.1% trifluoroacetic acid and 0.05% triethylamine to acetonitrile, 0.1% trifluoroacetic acid. The amount of [³H]myristoylpeptide produced was quantitated with an in-line scintillation counter (Packard).

The peptide substrate specificities of Nmt1p, 6XHis-Nmt1p, and each mutant 6XHis-nmt1p were compared at 24 and 37 °C using a panel of five previously defined octapeptide substrates of Nmt1p. Each octapeptide was assayed at a final concentration of 90 µM in the presence of 230 nM [³H]myristoyl-CoA and 0.1-250 µg/ml acyltransferase. The panel was composed of GNSSSKSS-NH₂ (NH₂-terminal sequence of S. cerevisiae Ppz1p), GNSGSKQH-NH₂ (Ppz2p), GAAPSKIV-NH₂ (Cnb1p), GNAAAARR-NH₂ (bovine C subunit of protein kinase A), and GARASVLS-NH₂ (human immunodeficiency virus I Pr55^gag).

Peptide kinetics were determined at 24 and 37 °C using 100 ng/ml of Nmt1p, 1-250 µg/ml of 6XHis-Nmt1p or a mutant 6XHis-nmt1p, 230 nM [³H]myristoyl-CoA, and GNSGSKQH-NH₂ (0.1-20 µM) or GAAPSKIV-NH₂ (0.01-4 µM). Peptide K_m and V_max were calculated using double reciprocal plots.

A series of K_m values were defined for the Ppz2p octapeptide substrate (10-20 µM) at 24 and 37 °C using 230 nM [³H]myristoyl-CoA, 10-1000 nM of the peptidomimetic inhibitor SC-58272 (7), and 0.1-250 µg/ml of Nmt1p, 6XHis-Nmt1p, or a 6XHis-nmt1p. The K_m at each inhibitor concentration and the type of inhibition were determined using double reciprocal plots. A K_i was obtained from a plot of the K_m values versus the inhibitor concentration.

The acyl chain length selectivities of Nmt1p, 6XHis-Nmt1p, and each 6XHis-nmt1p were compared at 24 and 37 °C using C12:0-CoA, C14:0-CoA, and C16:0-CoA (final concentration = 18 µM) and GAR[³H]ASVLS-NH₂ (Ref 34; specific activity = 373 mCi/mmol, final concentration = 25 µM).

A series of myristoyl-CoA K_m values were determined at 24 and 37 °C using [³H]myristoyl-CoA (0.2-10 µM), the Ppz2p octapeptide (10-20 µM), varying amounts of the competitive inhibitor S-(2-oxo)pentadecyl-CoA (0.005-1 µM), and purified Nmt1p, 6XHis-Nmt1p, or a 6XHis-nmt1p. The K_m at each inhibitor concentration and type of inhibition were determined using double reciprocal plots. K_i values were calculated from a plot of K_m versus the inhibitor concentration.

Each 6XHis-tagged enzyme preparation was studied at each temperature using purified Nmt1p without a 6XHis tag as a reference control. Analysis of myristoylpeptide production by the Nmts at 24 and 37 °C indicated that the enzymes were stable over the course of the 10-min incubation period. The kinetic studies involved over 3,000 datapoints, each obtained either in duplicate or triplicate. The mean percentage difference between duplicate datapoints was 9.9 ± 9.4 (S.D.). The mean percentage difference between triplicate datapoints was 12.8 ± 11.6.

Gel Mobility Shift Assay for Assessing the Degree of N-Myristoylation of Arf1p in Strains Containing Wild Type or Mutant Nmts

pBB290 in YB523 (nmt1) was replaced with low copy centromeric pRS315-based plasmids containing NMT1 or one of the ts nmt1 alleles. One hundred-milliliter cultures of the various strains were grown at 24 °C in YPD to an A₆₀₀ = 0.8. Twenty-five-milliliter aliquots were removed and incubated at 24 or 37 °C for 2 h. Cell lysates were prepared using the same protocol employed for measurement of steady-state Nmt levels. Western blots were probed with rabbit antiserum R23 raised against a peptide (SNSLKNST) encompassing the C-terminal eight residues of Arf1p (kindly supplied by Richard Kahn, Emory University; final dilution = 1:2000 in Blotto. Note that R23 does not cross-react with Arf2p, whose C-terminal sequence is SNNLKNQS).² Antigen-antibody complexes were visualized as described above.

RESULTS AND DISCUSSION

Random PCR Mutagenesis and a Colony Color Sectoring Assay Yields a Series of nmt1 Alleles Encoding Temperature-sensitive Mutants

Specific regions of NMT1 were targeted for random mutagenesis using in vivo recombination to repair a gapped plasmid with PCR fragments (Figs. 1 and 2). A colony color sectoring assay (35, 36) was used to identify nmt1 alleles encoding mutant enzymes with reduced activity. This assay (Fig. 2) takes advantage of the fact that two mutations within the S. cerevisiae purine nucleotide biosynthetic pathway, when present in different combinations, give rise to colonies with different colors. Mutations in ADE2 cause accumulation of the chromophore phosphoribosylaminoimidazole, resulting in red colonies. Strains with ADE3 mutations do not accumulate the chromophore and grow as white colonies. ADE3 mutations also affect an enzymatic step upstream of Ade2p. Therefore, ade2 ade3 strains grow as white colonies. Introduction of a low copy centromeric plasmid containing NMT1 and ADE3 into an ade2 ade3 nmt1 strain produces cells that form red colonies (Fig. 2). Since NMT1 is essential, if these cells lose this plasmid they die. Viable colonies containing the episomal copy of NMT1 and ADE3 remain red. If a second episome is introduced containing products from the PCR mutagenesis, one of two color phenotypes will be observed. If the mutation does not reduce Nmt1p's activity below the point required to acylate essential cellular N-myristoylproteins at levels compatible with viability, then there will be no selective pressure to retain the plasmid; i.e. during the course of cell division, 5-20% of newly formed daughter cells will lose the NMT1 ADE3 plasmid, and colonies will appear as red circles containing white sectors (sectoring phenotype). If a mutation inactivates NMT1, then the colony will be forced to retain the NMT1 ADE3 plasmid and will remain red (nonsectoring phenotype). Replica plating nonsectoring red colonies onto 5-FOA-containing plates will force the nmt1 ade2 ade3 leu2 host strain to remove the URA3-containing NMT1 ADE3 episome, thereby revealing the phenotype produced by the remaining LEU2 episome with its mutant nmt1 allele (Fig. 2).

Three overlapping regions of NMT1's ORF (Met¹  Leu³⁴⁵, Ala²⁰²  Asn³⁹⁷, and Lys³⁸⁹  Leu⁴⁵⁵) or the entire ORF were replaced by DNA fragments generated using standard conditions for the polymerase chain reaction or conditions designed to increase the error rate of Taq polymerase. A total of 52,000 transformants were screened; 2122 transformants (4%) had a nonsectoring phenotype, 1093 of the nonsectoring colonies (51%) had a lethal phenotype when incubated at 24 °C on synthetic complete media containing 5-FOA but lacking leucine, and 35 nonsectoring colonies had a ts phenotype based on differences in their growth in this media at 24 and 35 or 40 °C (Fig. 1B).

Polymerase chain reactions 1-3, performed under standard conditions, produced 47 nonsectoring transformants with a lethal phenotype and two with a ts phenotype (Fig. 1, A and B). Plasmids were recovered from 38 of the transformants with a lethal phenotype: 25 of the 38 did not contain any NMT1 sequences and presumably arose from self-ligation of the gapped plasmids. Sequence analysis of the remaining 13 plasmids revealed that they encoded truncated enzymes. Nine contained insertions or deletions that produced frameshifts, resulting in the introduction of three to nine amino acid substitutions followed by a stop at Val²¹⁴, Val³¹⁰, Leu³⁹⁸, Lys³⁹⁹, Ser⁴⁰⁰, Leu⁴¹¹, or Leu⁴²⁰; four contained single base changes that produced a stop codon at Glu³¹⁷, Ser³³¹, Gln⁴⁰², and Leu⁴⁰⁶. The two plasmids that produced a ts phenotype contained nmt1 alleles with single amino acid substitutions affecting conserved residues: Ser³²⁸  Pro (nmt1-328P) and Asn⁴⁰⁴  Tyr (nmt1-404Y ) (Fig. 3, A and B).

The number of nonsectoring ts colonies obtained with a given set of primers was affected by the PCR conditions employed and by the size of the region of NMT1 targeted for replacement by in vivo repair of the gapped plasmid. Manipulating these two parameters (reactions 4-8 in Fig. 1B) yielded 33 additional plasmids that conferred a ts phenotype. Three plasmids had nmt1 alleles with single amino acid substitutions affecting absolutely conserved residues: Ala²⁰²  Thr (nmt1-202T ), Cys²¹⁷  Arg (nmt1-217R), and Asn⁴²⁶  Ile (nmt1-426I) (Fig. 3, A and B). Nucleotide sequence analysis of the remaining plasmids revealed 24 unique nmt1 alleles, all of which contained multiple amino acid substitutions (range = 2-6 substitutions). A total of 64 amino acids were altered in these 24 alleles. Of these 64 residues, 6 were mutated in at least two different alleles: Ala²⁰², Lys³⁸⁹, Val³⁹⁵, Leu⁴⁰⁸, Asp⁴¹⁷, and Asn⁴²⁶. All of these amino acids are conserved (Fig. 3B). Two of these substitutions, Ala²⁰²  Thr and Asn⁴²⁶  Ile, were also represented in other ts nmt1 alleles with single amino acid changes (see above). Moreover, mutations that affected four of the six residues did not always result in the same amino acid substitution: Ala²⁰² was replaced with either Thr or Gly, Lys³⁸⁹ with Asn or Ile, Val³⁹⁵ with Asp, Ala, or Gly, and Asp⁴¹⁷ with Asn or Val. Together, these findings suggested that the PCR mutagenesis of NMT1 was comprehensive.

Alignments of the 24 multiply substituted protein sequences with the six orthologous Nmts revealed a total of 11 substitutions that affected eight conserved residues. Site-directed mutagenesis was used to generate eight nmt1 alleles, each containing a single substitution: Asn¹⁰²  Thr, Leu¹⁷¹  Ser, Lys³⁸⁹  Ile, Val³⁹⁵  Asp, Leu⁴⁰⁸  Ser, Phe⁴¹³  Ser, Asp⁴¹⁷  Val, and Leu⁴²⁰  Ser (Fig. 3B). (In the three instances where a conserved residue was replaced by more than one amino acid (Lys³⁸⁹, Val³⁹⁵, and Asp⁴¹⁷), we selected the replacement that was more frequently encountered in our library of ts nmt1 alleles.)

The eight nmt1 alleles engineered by site-directed mutagenesis were tested to determine whether they conferred a ts phenotype. An nmt1 strain, with a low copy centromeric plasmid containing NMT1 (wild type control), nmt1-451D (ts mutant control, see the Introduction), or one of the eight nmt1 alleles, was incubated at 24, 30, 33, 35, 37, and 40 °C on YPD/agar. The NMT1 episome supported similar rates of growth over this broad temperature range. An episome with the previously characterized nmt1-451D allele resulted in growth arrest at 40 °C. Asn¹⁰²  Thr (nmt1-102T), Val³⁹⁵  Asp (nmt1-395D), and Leu⁴²⁰  Ser (nmt1-420S) also produced complete growth arrest at 40 °C (Fig. 4A). In contrast, plasmids containing nmt1 alleles with Leu¹⁷¹  Ser, Lys³⁸⁹  Ile, Leu⁴⁰⁸  Ser, Phe⁴¹³  Ser, or Asp⁴¹⁷  Val substitutions had no detectable effect on growth, even at 40 °C (data not shown).

nmt1

Fig. 4A also shows the phenotypes produced by the five ts nmt1 alleles obtained directly from the colony color sectoring screen and found to contain single amino acid substitutions. Growth arrest occurred at 30 °C (Ala²⁰²  Thr, nmt1-202T), 35 °C (Ser³²⁸  Pro, nmt1-328P, and Asn⁴²⁶  Ile, nmt1-426I), 37 °C (Asn⁴⁰⁴  Tyr, nmt1-404Y) or 39 °C (Cys²¹⁷  Arg, nmt1-217R).

The remaining 1046 nonsectoring colonies with lethal phenotypes were generated by using modified PCR conditions designed to increase the error rate of Taq polymerase or by targeting the entire NMT1 ORF. There was no obvious difference in the frequency of nonsectoring colonies with lethal phenotypes when the modified PCR conditions were used to target Met¹  Leu³⁴⁵, Ala²⁰²  Asn³⁹⁷, or Lys³⁸⁹  Leu⁴⁵⁵ (reactions 4-6 in Fig. 1B). Our analyses of the 35 nonsectoring colonies with a ts phenotype and the 47 nonsectoring colonies with a lethal phenotype suggested that the majority of the nmt1 alleles that would be recovered from these 1046 colonies would contain multiple amino acid substitutions and/or stop codons. Therefore, they were not analyzed further.

Steady-state Levels of Mutant Nmts at Permissive and Nonpermissive Temperatures

nmt1 cells, containing episomal copies of one of the eight ts nmt1 alleles with single amino acid substitutions, were incubated in YPD broth at the permissive temperature (24 °C) until they reached mid-log phase. The temperature was then raised to 37-40 °C for 2 h. Western blots of total cellular proteins were prepared and probed with a previously characterized rabbit anti-Nmt sera (19). The steady-state levels of six of the mutant acyltransferases were equivalent to each other and to Nmt1p (Fig. 4B). The concentration of nmt420Sp at 24 °C was equal to Nmt1p but was 50% that of the wild type enzyme at 37-40 °C. nmt328Pp was ~3-fold less abundant than Nmt1p at permissive and nonpermissive temperatures (Fig. 4B). Thus, with the exception of nmt1-328P and nmt1-420S, differences in temperature sensitivity produced by the mutant nmt1 alleles could not be simply ascribed to differences in the steady-state levels of their protein products.

In Vitro Kinetic Studies of Purified Wild Type and Mutant Nmts

As noted above, five of the eight ts nmt1 alleles produced growth arrest on YPD at 39 °C. To define the effects of their Ala²⁰²  Thr, Cys²¹⁷  Arg, Ser³²⁸  Pro, Asn⁴⁰⁴  Tyr, and Asn⁴²⁶  Ile substitutions on substrate recognition and/or catalysis, each mutant was expressed in E. coli, a bacterium with no endogenous Nmt activity (37). Nmt1p and nmt451Dp were used as reference controls. A genetically engineered NH₂-terminal tag of six histidine residues (6XHis) allowed rapid purification of wild type and mutant enzymes by Ni²⁺-NTA-agarose affinity chromatography. The purification protocol produced a 100-1000-fold increase in specific activity over what was observed in unfractionated bacterial lysates and yielded apparently homogeneous preparations of each enzyme based on Coomassie staining of SDS-polyacrylamide gels (data not shown).

The acyl chain length selectivity of each mutant was examined in an in vitro assay system that used purified enzyme, unlabeled C12:0-CoA, C14:0-CoA, or C16:0-CoA, and a fixed concentration of an extensively characterized radiolabeled octapeptide substrate of Nmt1p (GAR-[³H]ASVLS-NH₂; Refs. 34, 38, and 39). Neither the addition of the 6XHis tag to Nmt1p nor any of the amino acid substitutions had statistically significant effects on the specificity of the acyltransferase reaction at 24 or 37 °C (range of lauroyltransferase activity = 2-15% of myristoyltransferase activity; palmitoyltransferase activity = 0-8%; n = 2 independent preparations of each mutant, each assayed on two separate occasions in triplicate).

Having established that all of the mutants "retained" their character as myristoyltransferases, we examined the effects of the amino acid substitutions on the enzyme's affinity for S-(2-oxo)pentadecyl-CoA (40). The rationale was as follows. None of the mutants could be purified in sufficient quantity or were sufficiently stable at 24 or 37 °C to permit thermodynamic analysis of myristoyl-CoA binding by isothermal titration calorimetry (ITC), as has been done with wild type apo-Nmt1p (16). For an ordered reaction such as the one catalyzed by Nmt1p, a competitive inhibitor for the first of the two substrates, obtained at any concentration of the second substrate, is a dissociation constant for E + I. S-(2-Oxo)pentadecyl-CoA is a competitive Nmt inhibitor containing a single methylene insertion between the CoA sulfur and the fatty acid carbonyl carbon of myristoyl-CoA. Determination of its K_i required less enzyme and could be performed more rapidly than ITC analysis of myristoyl-CoA binding. Moreover, ITC has established that purified apo-Nmt1p perceives S-(2-oxo)pentadecyl-CoA as a close approximation of myristoyl-CoA: the enthalpy of binding of the nonhydrolyzable inhibitor (25.8 kcal/mol) is equivalent to that of myristoyl-CoA (24.8 kcal/mol) (Ref. 16).³

The K_i for S-(2-oxo)pentadecyl-CoA was defined at 24 and 37°C using the purified mutant Nmts, varying amounts of [³H]myristoyl-CoA, and an Nmt1p substrate (GNSGSKQH-NH₂; Ref. 22) derived from the NH₂-terminal sequence of Ppz2p, a protein phosphatase that suppresses the cell lysis defect produced by a protein kinase C null allele (41, 42). The concentration of peptide was 2-100-fold over its K_m for each mutant Nmt (see below).

For Nmt1p, the K_i is 5 nM (Table I), a value identical to the K_d obtained from ITC (16). The addition of an NH₂-terminal 6XHis tag to Nmt1p has no appreciable effect on its affinity for S-(2-oxo)pentadecyl-CoA (K_i = 4-7 nM). The Cys²¹⁷  Arg substitution in nmt217Rp does not produce any detectable alteration in affinity for the inhibitor (5 nM at 24 and 37 °C). In contrast, Asn⁴²⁶  Ile results in the greatest reduction in affinity, with K_i increases of 12- and 20-fold at 24 and 37 °C (compared with 6XHis-Nmt1p). Asn⁴⁰⁴  Tyr produces no change in affinity at 24 °C but a 5-fold reduction at 37 °C (K_i = 21 nM). The other substitutions result in ~3-fold increases in K_i at 24 °C and 6-8-fold increases at 37 °C (Table I).

Table I. Inhibition of wild type and mutant Nmts with S-(2-oxo)pentadecyl-CoA Ki values were determined using purified enzyme, S-(2-oxo)pentadecyl-CoA, varying amounts of [3H]myristoyl-CoA, and an octapeptide substrate derived from the NH2-terminal sequence of Ppz2p. The averages of duplicate determinations are shown. Duplicate values reported in Tables I, II, III varied by <30% (see "Experimental Procedures"). Ki 24 °C 37 °C nM Nmtlp 5 5 6XHis-Nmtlp 7 4 6XHis-nmt202Tp 23 24 6XHis-nmt217Rp 5 5 6XHis-nmt328Pp 17 32 6XHis-nmt404Yp 4 21 6XHis-nmt426Ip 85 79 6XHis-nmt451Dp 25 30

As noted in the Introduction, once a binary myristoyl-CoA·Nmt1p complex forms, an allosteric transition occurs that allows formation of a functional peptide binding site. Previous in vitro and in vivo studies using myristic acid analogs have emphasized that changes in interactions between apo-Nmt1p and its acyl-CoA substrates can produce changes in the functional characteristics of the enzyme's peptide binding site (16, 43, 44). Therefore, we surveyed myristoylpeptide production at 24 and 37 °C using Nmt1p, 6XHis-Nmt1p, each of the five mutants, saturating amounts of [³H]myristoyl-CoA, and a panel of five octapeptide substrates. Two of the peptides had been used for defining each enzyme's acyl chain length selectivity and its affinity for S-(2-oxo)pentadecyl-CoA (GARASVLS-NH₂ from human immunodeficiency virus type I Pr55^gag and GNSGSKQH-NH₂ from Ppz2p, respectively).

The addition of a 6XHis tag to Nmt1p has no demonstrable effect on peptide specificity as judged by myristoylpeptide production at 24 and 37 °C. When compared with 6XHis-Nmt1p, the specific activity of each 6XHis-nmt1p mutant is reduced at 24 and 37 °C for all peptides surveyed. For example, in the case of the Ppz2p peptide, the decrease ranges from 10- to 50-fold at 24 °C and from 15- to 300-fold at 37 °C. The extent of the reduction in specific activity varies between the mutants and as a function of the peptide substrate.

Two peptides were selected for more detailed kinetic analysis: GNSGSKQHP-NH₂ (Ppz2p) and GAAPSKIV-NH₂ (representing the NH₂-terminal sequence of Cnb1p, the N-myristoylated regulatory subunit of yeast Ca²⁺/calmodulin-dependent phosphoprotein phosphatase (45, 46)). They were chosen because all of the mutants were able to generate readily detectable amounts of these myristoylpeptides at 24 and 37 °C and because Ppz2p had been used when computing K_i values for S-(2-oxo)pentadecyl-CoA.

Asn⁴²⁶  Ile, which results in the greatest reduction in S-(2-oxo)pentadecyl-CoA affinity (12-20-fold), produces minimal (2-fold) changes in K_m for either peptide at either temperature when compared with 6XHis-Nmt1p (Table II).

Table II. Summary of peptide kinetics Peptide 6XHis-Nmtlp 6XHis-nmt202Tp 6XHis-nmt217Rp 6XHis-nmt328Pp 6XHis-nmt404Yp 6XHis-nmt426Ip Km (nM) GNSGSKQH-NH2 (Ppz2p)   24 °C 200a 10,000 11,100 800 600 300   37 °C 400 12,500 9500 400 3200 400 Vmaxb   24 °C 1450 1790 72 3 26 170   37 °C 4600 1100 82 9 37 147 Km (nM) GAAPSKIV-NH2 (Cnb1p)   24 °C 50 300 400 30 400 100   37 °C 60 800 1800 60 1000 100 Vmaxb   24 °C 1000 37 4 3 59 36   37 °C 2570 42 17 7 70 25 a  All kinetic parameters represent the average of duplicate determinations (see legend to Table I). b  pmol/min/mg enzyme.

Cys²¹⁷  Arg, which has no detectable effect on S-(2-oxo)pentadecyl-CoA affinity, has a marked effect on peptide K_m: 24-56-fold increases for Ppz2p and 8- and 30-fold increases for Cnb1p (Table II).

Asn⁴⁰⁴  Tyr, which produces the greatest temperature-dependent change in K_i for S-(2-oxo)pentadecyl-CoA among the mutants (Table I), is associated with perturbations in peptide K_m that also worsen with increasing temperature (Table II).

Ala²⁰²  Thr, which produces 3-6-fold decreases in S-(2-oxo)pentadecyl-CoA affinity at 24 and 37 °C, results in increases in the K_m for both peptides. The increase is more pronounced for the Ppz2p peptide (30-50-fold) than for the Cnb1p peptide (6-13-fold; Table II). Ser³²⁸  Pro, which results in a similar 3-6-fold decrease in affinity for the nonhydrolyzable analog as Ala²⁰²  Thr, has more modest effects on peptide K_m; the K_m of the Ppz2p octapeptide is only increased 2-4-fold, and there is no significant change for the Cnb1p peptide (Table II).

All of the amino acid substitutions result in decreases in peptide V_max. Ser³²⁸  Pro produces the greatest reductions: ~350- and 500-fold for the Cnb1p and Ppz2p peptides, respectively (Table II).

We selected the two mutants with the greatest increases in peptide K_m (nmt217Rp (Cys  Arg) and nmt202Tp (Ala  Thr)) for additional analysis. Increases in peptide K_m cannot be used to conclude that there are decreases in the mutant enzymes' affinities for these substrates. For an inhibitor of the second substrate of an ordered reaction, the inhibition constant is a dissociation constant under conditions where the first substrate is at a saturating level. Therefore, the effects of these two amino acid substitutions on the functioning of Nmt's peptide binding site were explored by determining the K_i for a peptidomimetic inhibitor. SC-58272 was derived from the NH₂-terminal eight residues of a yeast N-myristoylprotein, ADP-ribosylation factor-2 (Arf2p; Ref 7). Amino acids 1-4 were replaced with a para-(2-methylimidazole-N-butyl) phenylacetic acid derivative, amino acids 7 and 8 (Leu-Ser-NH₂) were deleted, and the 2-cyclohexylethyl amide derivative of lysine was used as the C-terminal residue (see Fig. 5). SC-58272 is competitive for binding of peptide substrates but not for myristoyl-CoA (7). ITC has been used to determine that the K_d of SC-58272 for myristoyl-CoA·Nmt1p binary complexes is 29 nM.³

Table III presents K_i values for SC-58272 defined in the presence of saturating amounts of [³H]myristoyl-CoA and varying amounts of the Ppz2p peptide. With Nmt1p, the K_i at 24 °C was very similar to the K_d defined calorimetrically (43 versus 29 nM). The addition of a 6XHis tag to Nmt1p produces <2-fold changes in the K_i at 24 and 37 °C. Ala²⁰²  Thr results in K_i increases of 6- and 9-fold at 24 and 37 °C compared with 6XHis-Nmt1p. Cys²¹⁷  Arg produces 3- and 6-fold increases at these two temperatures.

Table III. Inhibition of wild type and mutant Nmts by the peptidomimetic SC-58272 The Ki for SC58272 was defined using purified Nmts, saturating amounts of [3H]myristoyl-CoA, and varying amounts of the Ppz2p octapeptide substrate (see "Experimental Procedures"). The averages of duplicate determinations are presented (see legend to Table I). Ki 24 °C 37 °C nM Nmtlp 43 14 6XHis-Nmtlp 24 9 6XHis-nmt202Tp 138 82 6XHis-nmt217Rp 74 55

The finding that Cys²¹⁷  Arg produces an increase in the K_i for SC-58272 but no change in the K_i for S-(2-oxo)pentadecyl-CoA indicates that this substitution of a conserved Cys has selective effects on the functional properties of the enzyme's peptide binding site. In contrast, substitution of the conserved Asn⁴²⁶ with Ile appears to selectively affect the functional properties of the enzyme's myristoyl-CoA binding site. Ala²⁰²  Thr, which produces the most severe ts phenotype, provides an example of a substitution that affects the functional properties of both sites, resulting in increases the K_i for S-(2-oxo)pentadecyl-CoA and SC-58272. Such a substitution may perturb specific contacts between the enzyme and the myristoyl or CoA moieties, which, in turn, could disturb the subsequent allosteric transition required for creation of a fully functional peptide binding site. Alternatively, Ala²⁰²  Thr may produce more global changes in the conformation of Nmt1p.

Defining Levels of Acylation of an N-Myristoylprotein in NMT1 and nmt1 Cells

At 37 °C, adding 500 µM myristate to YPD media rescues growth of all but one of the ts nmt1 mutants (data not shown). (nmt1-328P is the exception. Recall that at 37 °C nmt328Pp has the lowest steady-state cellular concentration among the mutants (Fig. 4B), the greatest reduction in peptide V_max in vitro (Table II), and a 6-fold decrease in affinity for the myristoyl-CoA analog.)

At 39-40 °C, adding 500 µM myristate to YPD only partially rescues nmt1 cells with nmt1-426I (lowest affinity for S-(2-oxo)pentadecyl-CoA among the mutants), nmt1-202T (lowest affinity for SC-58272 plus moderate reductions in affinity for S-(2-oxo)pentadecyl-CoA), or nmt1-404Y (most marked change in S-(2-oxo)pentadecyl-CoA affinity between 24 and 37 °C).

Levels of cellular protein N-myristoylation produced by the wild type and mutant nmt1 alleles were defined under these various growth conditions using an Arf gel mobility shift assay. Arf1p and Arf2p are two functionally interchangeable but essential N-myristoylproteins involved in vesicular trafficking (47, 48). They depend upon N-myristoylation for expression of their biological functions (49). Arf1p represents ~90% of cellular Arfs (47). N-Myristoylation produces a change in the electrophoretic mobility of Arf1p during SDS-PAGE; the acylated species migrates more rapidly than the nonmyristoylated species (19). We therefore reasoned that Arf1p could be used to report the extent of cellular protein N-myristoylation by noting the ratio of its distinctively migrating N-myristoylated and nonmyristoylated forms.

nmt1 cells containing plasmids with NMT1 or ts nmt1 inserts were grown in YPD at 24 °C to mid-log phase. Aliquots of the cultures were then incubated for an additional 2 h at 24 °C or 33-40 °C in YPD alone or in YPD plus 500 µM myristate. Western blots of total cellular proteins were prepared and probed with rabbit antibodies that react with Arf1p but not Arf2p,² and the ratio of N-myristoylated to nonmyristoylated Arf1p was determined. Fig. 6 shows representative results obtained with NMT1, nmt1-404Y, and nmt328P. Only myristoyl-Arf1p is detectable in NMT1 cells, whether they are cultured in YPD at 24, 37, or 40 °C. Defects in protein N-myristoylation are evident in nmt1-404Y cells when cultured in YPD alone. Even at the permissive temperature of 24 °C, only ~60% of Arf1p is acylated; at 37 °C, the value falls below 50%. In nmt1-328P cells, >95% of Arf1p is N-myristoylated when grown at 24 °C on YPD; however, between 33 and 37 °C, myristoyl-Arf1p decreases to 40% of total Arf1p. The ability of myristate to increase levels of myristoyl-Arf1p to 50% of total cellular Arf1p can be directly correlated with its ability to rescue the growth of a given nmt1 strain. At 37 °C, myristoyl-Arf1p levels increase in nmt1-404Y cells from <50% to >95% 2 h after exposure to myristate (Fig. 6). These cells are able to sustain growth in this media at this temperature. In contrast, myristate has no detectable beneficial effects on the efficiency of Arf1p acylation in nmt1-328P cells at 37 °C (Fig. 6), a temperature where they are unable to survive on YPD/myristate. The gel shift assay was used to establish that a reduction in levels of myristoyl-Arf1p to 50% is also associated with a failure of cells containing the other ts nmt1 alleles to survive (data not shown).

Gel shift assay for determining the extent of Arf1p N-myristoylation in nmt1 cells containing NMT1, nmt1-328P, and nmt1-404Y episomes.

Prospectus

A panel of mutant Nmts, with defined defects in the functioning of their myristoyl-CoA and/or peptide binding sites has been generated using an error-prone PCR strategy. A full understanding of the structural significance of their amino acid substitutions must await determination of the atomic structure of Nmt1p with and without bound substrates or substrate analogs. Nonetheless, the temperature-sensitive growth arrest produced by various members of this panel offers an opportunity to conduct genetic screens for factors that affect Nmt activity, to examine the adaptive responses of cells to undermyristoylation of cellular proteins, and to identify the functions of cellular N-myristoylproteins in dividing and nondividing yeast cells.