AspergillusnidulansswoF Encodes an N-Myristoyl Transferase

Polar growth is a fundamental process in filamentous fungi andis necessary for disease initiation in many pathogenic systems.Previously, swoF was identified in Aspergillus nidulans as asingle-locus, temperature-sensitive (ts) mutant aberrant inboth polarity establishment and polarity maintenance. The swoFgene was cloned by complementation of the ts phenotype and sequenced.The derived protein sequence had high identity with N-myristoyltransferases (NMTs) found in fungi, plants, and animals. Inaddition, wild-type growth at restrictive temperature was partiallyrestored by the addition of myristic acid to the growth medium.Sequencing revealed that the mutation in swoF changes the conservedaspartic acid 369 to a tyrosine. The predicted A. nidulans SwoFprotein, SwoFp, was homology modeled based on crystal structuresof NMTs from Saccharomyces cerevisiae and Candida albicans.The D369Y swoF mutation is on the opposite face of the protein,distal to the myristoyl coenzyme A and peptide substrate bindingsites. In wild-type NMTs, D369 appears to stabilize a structuralß-strand bend through two hydrogen bonds and an ionicinteraction. These stabilizing bonds are abolished in the D369Ymutant. We hypothesize that a substrate of SwoFp must be myristoylatedfor proper polarity establishment and maintenance. The mutationprevents the proper function of SwoFp at restrictive temperatureand thus blocks polar growth.

INTRODUCTION

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Introduction

For many filamentous fungi, two distinct growth modes are usedduring the process of spore germination and early development.When dormancy is broken, the spore first grows isotropically,adding new cell wall material uniformly in every direction.A switch to polarized growth soon follows, with new cell walldeposition occurring only at the growing tip, leading to elongatedhyphae characteristic of filamentous fungi. In Aspergillus nidulans,the switch from isotropic to polar growth occurs just afterthe first round of mitosis (34). Two distinct processes areinvolved in the switch to polar growth: polarity establishment,i.e., choosing the spot where new material will be deposited,and polarity maintenance, i.e., the continued deposition ofwall material at the growing tip.

Not surprisingly, the process of polar growth requires an intactcytoskeleton and proper vesicle transport. Pharmacological disruptionof F actin prevents the polar growth of Neurospora crassa andSaprolegnia ferax (22). The actin-associated motor protein myosinhas a high level of tip localization, and loss of myosin functionin A. nidulans leads to large apolar cells (32, 37). Mutationsin sepA, which encodes an actin-associated protein, result inabnormally large, septumless A. nidulans hyphae (20). In N.crassa, kinesin and dynein mutants grow with abnormally short,thick germ tubes (40, 41). Normal vesicle assembly is also requiredfor polar growth. The A. nidulans ${alpha}$ -cop-related gene sod^VIC,involved in coated vesicle assembly, is essential both to establishpolarity and to maintain polarity (45).

Because polar growth is coordinated with nuclear division infilamentous fungi, it is also not surprising that cell divisionsignaling pathways are involved in polarity. A cyclin-dependentkinase mutant (phoA) and a mitogen-activated protein kinasedeletion mutant (mpkA) both have abnormal germination and polaritymaintenance (10). Strains with mutations in the protein phosphatasegene pphA display abnormal hyphal growth and mitotic defects(28). Temperature-sensitive (ts) mutations in N. crassa proteinkinase A result in swollen cells with abnormal polarity maintenance(8). Also, in N. crassa the Ser/Thr protein kinase encoded bycot1 is necessary for polar growth (18, 47). In Penicilliummarneffei, the Cdc42 (Rho) homologue encoded by cflA is requiredfor both polarity establishment and polarity maintenance (7).

The A. nidulans swo (swollen cell) mutants are characterizedby either continued isotropic growth without establishment ofpolarity or the inability to maintain polar growth at restrictivetemperatures (35). Through a series of temperature shift experimentsand genetic crosses, it was determined that the events of polarityestablishment and polarity maintenance are genetically separable.swoC and swoD were found to be involved in polarity establishment,while swoA was found to be required for polarity maintenance.swoF was found to be involved in both processes.

In this study, we describe the cloning, sequencing, and furthercharacterization of swoF (35). We show that swoF encodes anN-myristoyl transferase (NMT). NMTs are highly conserved proteinsthat catalyze the transfer of myristate (C₁₄:₀) from myristoylcoenzyme A (myristoyl-CoA) to the N-terminal glycine of a subsetof cellular proteins (4-6). This modification increases theaffinity of the target protein for membranes and has been proposedto constitute a reversible mechanism that allows the targetprotein to switch between membrane-bound and cytoplasmic states(5). Known targets of NMTs include protein phosphatases, proteinkinases, kinase substrates, and G protein ${alpha}$ subunits (9).

MATERIALS AND METHODS

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Materials and Methods

Strains and media used. Strain A773 (pyrG89 pyroA4 wA3) was crossed with strain AJB11(swoF) by using standard methods (21, 25) to produce strainAXL19 (pyrG89 swoF). All experiments reported here used strainAXL19 as the swoF mutant and strain A773 as the wild type. Mediaused were as previously reported (35).

Growth of germlings and microscopic observations. Conditions for growth and preparation of germlings for observationswere as previously reported (35). Microscopic observations weremade by using an Axioplan microscope (Zeiss, Thornwood, N.Y.),and digital images were acquired by using an Optronics (Goleta,Calif.) digital imaging system. Images were prepared by usingPhotoshop 5.5 (Adobe, Mountain View, Calif.).

Complementation and plasmid recovery. A genomic library was generously provided by Greg May (Universityof Texas M. D. Anderson Cancer Center, Houston). This librarywas constructed by ligating Sau3A fragments of genomic DNA intothe BamHI site of pRG3AMA1 (36). Protoplasts of AXL19 were producedand transformation was conducted by using standard A. nidulansprotocols (48). Transformants were selected by assaying forrestoration to pyrG prototrophy. Complementation was judgedby restoration of wild-type growth at restrictive temperature(42°C). The complementing plasmid replicated autonomously(e.g., extrachromosomally) due to the AMA1 sequence (1) containedin the library vector. The complementing plasmid was recoveredby transformation of Escherichia coli XL1-Blue with DNA isolatedfrom the complemented strain and selection on ampicillin-containingmedium. The restriction patterns of three rescued plasmids werecompared, and those showing common insert patterns were usedto retransform the swoF mutant. Two of three recovered plasmidscomplemented swoF and showed identical restriction patterns.One of these, p19c2, was chosen for sequencing.

Sequencing. p19c2 was transposon tagged by using a GPS-1 kit (New EnglandBiolabs, Beverly, Mass.) and was transformed into E. coli XL1-Blue.Colonies representing individual randomly tagged plasmids werearrayed in a 96-well format. Plasmid preparation was carriedout with an R.E.A.L. 96-well kit (Promega, Madison, Wis.). Labelwas incorporated by using Big Dye 2.0 (Perkin-Elmer AppliedBiosystems, Boston, Mass.). Sequencing was done with outward-facingprimers designed on the basis of the transposon provided withthe GPS-1 kit. Unincorporated dyes were removed by using a DyeEx96 kit (Qiagen, Valencia, Calif.). Sequencing in the 96-wellformat was performed with an ABI Prism 3700 robotic sequencer(Applied Biosystems Inc., Foster City, Calif.). Subsequent analysiswas done with the programs Phred version 0.000925c and Phrapversion 0.990319 for assembly and quality determination andConsed version 11.0 for sequence viewing and recovery (all threeUnix-based programs are available from http://depts.washington.edu/ventures/collabtr/direct/ppccombo.htm).All sequences contained at least fourfold redundancy, with aquality rating of at least 20.

Identification of the swoF gene. The complete sequence for complementing genomic DNA was comparedto sequences in the National Center for Biotechnology Informationdatabase (www.ncbi.nlm.nih.gov) by using blastX. Multiple openreading frames were found within the genomic sequence. Transposon-taggedplasmids with strategically placed transposon insertions werechosen from the plasmid array to test for complementation. Transposon-taggedplasmids were transformed into AXL19. Transformants were replicaplated at permissive and restrictive temperatures. The openreading frame which, when disrupted by transposon insertion,lost the ability to restore AXL19 to wild-type growth at 42°Cwas identified as swoF.

Sequencing of the swoF mutant allele. Genomic DNA from AXL19 was isolated by using standard methods(39). This genomic DNA was used as a template in a PCR withthe Expand High Fidelity PCR system (Roche Diagnostics, Indianapolis,Ind.). The following primers were used for PCR amplification:swoF5 BamHI, 5-CGGGATCCCGATGTCAGACTCAAAAGACTC-3, and swoF3 SpeI,5-GGACTAGTCCCTAGAGCATAACAACGCCCA-3. Each primer contains a 20-merbased on the wild-type swoF sequence and a 10-mer to createa restriction enzyme recognition site. PCR products were clonedinto the pGEM-T vector system (Promega), transformed into E.coli XL1-Blue, and selected on ampicillin-containing mediumwith blue-white selection. Clones were verified by restrictionanalysis. Three clones were sequenced by using the strategydescribed above. Sequences of all three clones were comparedto the wild-type sequence to determine the mutant lesion. Allthree had the same base change.

Protein alignment. Sequences for orthologues of NMT were obtained from GenBank(http://www.ncbi.nlm.nih.gov). Protein sequence alignment wascarried out by using the program GeneDoc version 2.6.001 (www.psc.edu/biomed/genedoc)with default parameters and minimal manual adjustment to alignsequences with secondary structural features.

Homology modeling. The model for the predicted A. nidulans SwoF protein, SwoFp,was prepared by homology modeling with the program Swiss PDBViewer version 3.7b2 (http://www.expasy.ch/spdbv/). The templatesused were the atomic structures determined by X-ray crystallographyof the Saccharomyces cerevisiae NMT complexed with myrisoyl-CoAand a peptide analogue (Protein Data Bank accession number 2NMT)and the Candida albicans NMT (PDB accession number 1NMT). Thestructure of the S. cerevisiae NMT was used as the principalmodel as it represents a conformationally closed complex representativeof the substrate-bound structure. Loops were built by usingthe Swiss PDB Viewer; in some cases, program O was used (24)because of its ability to merge coordinates and slightly bettermodeling capabilities. Poor side-chain orientations were identifiedby their high energies and corrected by using the optimize functionin Swiss PDB Viewer. After all loops were built and side-chainconformations were corrected, the model was energy minimizedby using steepest descent and conjugate gradients until thechange in energy between steps was <0.05 kJ/mol and no locallypoor energies remained. The model has threading energies, conformationalenergies, and phi-psi plots similar to those of the originalcrystal structures. Thus, no attempt was made to correct geometricor energetic problem areas that appeared to carry over fromthe atomic structures. The nomenclature for ß strandsand ${alpha}$ helices follows that used by Bhatnagar and colleagues (3).The substrate myristoyl-CoA and the peptide analogue from 2NMTwere docked by using the magic-fit option. No energy minimizationwas required.

Growth restoration with myristate. To ascertain if myristic acid medium amendment allowed polargrowth of swoF cells at restrictive temperature, A773 and AXL19were grown either in liquid or on solid minimal medium containing500 µM myristate and 1% (wt/vol) Brij 58 (polyoxyetheylene20 cetyl ether) (Sigma, St. Louis, Mo.). The presence of 1%Brij 58 helps to solubilize myristate (14).

Nucleotide sequence accession number. The full genomic sequence, as well as intron locations, andthe predicted protein sequence determined here have been depositedin GenBank under accession no. AY057437.

RESULTS

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Results

The swoF mutant was identified in a screen for ts mutants withdefects in polar growth (35). Originally, swoF was thought tobe necessary for both polarity establishment and polarity maintenancebased on the failure to initiate germ tube growth at a restrictivetemperature and upon a shift from a restrictive to a permissivetemperature. However, in our current experiments, we found thatswoF cells did sometimes initiate polar growth after 16 h ofincubation at restrictive temperature (42°C), making short,thick germ tubes with swollen apices (16%; n = 500) (Fig. 1b).However, the majority of swoF cells failed to make germ tubesat the restrictive temperature, as previously reported (Fig.1d). Both swoF cells with short germ tubes and those withoutgerm tubes completed two or three mitotic divisions after 16h of incubation at the restrictive temperature. In contrast,wild-type germlings under the same conditions produced elongatedprimary and secondary germ tubes with one or more lateral branches(Fig. 1a) and underwent five or more mitotic events after 16h of incubation.

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FIG. 1. Wild-type, swoF, and complemented cells grown in complete medium for 16 h at restrictive temperature (42°C). (a) Wild-type strain A773. (b and d) Two morphologies of swoF strain AXL19. (c) AXL19 transformed with p19c2 containing wild-type swoF. Bar, 10 µm.

Complementation and sequencing of wild-type and mutant alleles. A swoF pyrG strain was transformed with a genomic library constructedin vector pRG3AMA1, which carries the pyr4 gene and the AMA1sequence for plasmid autonomous replication (1, 36). A totalof 1,092 pyr prototrophs were screened for growth at restrictivetemperature (42°C). Three transformants were judged to becomplemented based on wild-type colony morphology at the restrictivetemperature. These transformants also showed wild-type morphologywhen examined microscopically (Fig. 1c). Restriction digestionof the complementing plasmid in each of the three transformantsrevealed that two contained precisely the same genomic insert(data not shown). One of these two, plasmid p19c2, was selectedfor sequencing by transposon insertion.

The genomic insert in p19c2 is 7,385 bp long (Fig. 2a). It encodespredicted proteins with high similarity to NMTs (see below),the C terminus of a tetrahydrofolylpolyglutamate synthase, andthe N terminus of a ß-fructofuranosidase. The swoFmutant was transformed with plasmids containing transposon insertiondisruptions in the tetrahydrofolylpolyglutamate synthase region(F10 and B11), the NMT region (E8 and B7), or the large regionwithout a predicted open reading frame (C11). Only an insertionin the NMT region disrupted the ability of p19c2 to complementswoF temperature sensitivity (Fig. 3). Both tested transposoninsertions in the NMT homologue failed to complement swoF (Fig.3d and e); therefore, the complementing gene encodes an NMT.The A. nidulans NMT gene, swoF, is 1,639 bp long and containsthree introns of 55, 56, and 49 bp, making the cDNA 1,479 bplong (Fig. 2b). Intron placement was deduced on the basis ofconsensus splice site sequences (2) and protein alignment withknown NMT sequences. Transposons E8 and B7 are inserted in thethird exon of the gene at 637 and 1,358 bp, respectively.

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FIG. 2. Complementing genomic DNA and swoF. (a) Schematic representation of the 7,385-bp genomic fragment that complements swoF. Three open reading frames were revealed by a search of GenBank, the full NMT gene and portions of a tetrahydrofolylpolyglutamate synthase (TPS) gene and a ß-fructofuranosidase gene. The locations of five transposon insertions are indicated by arrowheads. (b) Schematic representation of swoF (NMT), a 1,639-bp open reading frame containing three introns represented by black boxes. The light gray arrowhead shows the location of mutant lesion D369Y.

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FIG. 3. Transposon insertion verifies that the NMT region complements swoF. Colonies of swoF cells were grown at a permissive temperature (30°C) (top) or at restrictive temperature (42°C) (bottom). (a) swoF. (b to g) swoF transformed with p19c2 containing no transposon insertion (b) or transposon insertion C11 (c), E8 (d), B7 (e), F10 (f), or B11(g).

Alignment of the SwoFp sequence with several other known NMTproteins revealed a high degree of conservation (Table 1 andFig. 4). The highest identity and similarity (80 and 88%, respectively)were to a predicted NMT from Aspergillus fumigatus found inGenBank. The highest identity and similarity to an experimentallyverified NMT were to S. cerevisiae Nmt1p (50 and 65%, respectively).The N-terminal 30 to 100 residues of these proteins were poorlyconserved; however, the remaining 350 to 400 residues were highlyconserved.

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TABLE 1. Identity and similarity of SwoFp and other NMTs

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FIG. 4. Alignment of A. nidulans SwoFp (Anid), putative Nmtp from A. fumigatus (Afum), Nmt1p from S. cerevisiae (Scer), and Nmt1p from C. albicans (Calb). Black shading shows residue identity in at least three proteins. Gray shading shows similarity in at least two residues. Secondary structures are shown above the corresponding amino acid sequence as colored arrows (ß strands) or cylinders ( ${alpha}$ helices) coded blue (N terminus) to red (C terminus). The nomenclature for the secondary structures follows that used by Bhatnager et al. (3). Residues involved in binding myristoyl-CoA are indicated by gray stippled bars below the corresponding sequence, residues involved in binding the peptide substrate are indicated by red bars, and residues involved in binding both ligands are indicated by gray and red stippled bars. Tn, sites of transposon insertion disruption at K175 (E8) and at Q415 (B7). Solid black triangles below the sequence indicate residues interacting with D369. S. cerevisiae ts mutations reported by Zhang et al. (49) are indicated by a mutant residue enclosed by a black circle below the wild-type residue. The A. nidulans swoF mutation is denoted by a mutant residue (Y) enclosed by a red circle below the wild-type D residue.

The swoF mutant allele was amplified from AXL19 by PCR. Sequencedetermination for three replicate PCR clones showed that theswoF mutant allele contains a change in base 1216 of the genomicsequence from G to T, resulting in a change in protein residue369 from aspartic acid to tyrosine.

Protein modeling. The crystal structures for the C. albicans and S. cerevisiaeNMTs have been determined with and without bound substrate peptideand myristoyl-CoA analogues (3, 16, 44). The Nmt1p fold containsa large ß sheet bisected by a pseudo-twofold symmetryaxis and flanked by several ${alpha}$ helices. The ligand binding sitesare related by a pseudo-twofold axis, with the myristoyl-CoAbinding to the N-terminal half of the protein and the peptidesubstrate binding to the C-terminal half. Using these structuresas templates, we generated a homology model for A. nidulansSwoFp (Fig. 5). The N-terminal 76 amino acids are missing fromthe crystal structures and so are not included in our model.Most of the residues found to be important in myristoyl-CoAbinding, peptide binding, or transferase activity are conservedin SwoFp (Fig. 4). Amino acids critical for structural stabilityare also conserved among all NMTs. The swoF D369Y mutation changesone of these structurally important residues, an aspartic acidthat participates in stabilizing a turn in ß-strandßk via hydrogen bonds and an ionic interaction (Fig.5b).

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FIG. 5. Ribbon representation of the A. nidulans SwoFp homology model. (a) The SwoFp homology model lacking the first 76 residues is portrayed in a ribbon representation with secondary structure features colored blue (N terminus) through red (C terminus). The substrate molecules are shown in ball-stick representation with myristoyl-CoA colored gray and a peptide analogue colored red. The side chain of D369 projects from ß-strand ßk and is shown in ball-stick representation and colored black. Helix ${alpha}$ H is at the lower right of this view and is colored yellow. (b) Enlargement of the region near D369. The view is rotated about 90° relative to that in panel a, looking down the arrow toward D369. The side chains of T368, D369, T399, and R413 are shown in ball-stick representation. Broken lines represent hydrogen bonds. Bond distances are indicated.

Partial growth restoration with myristate. In S. cerevisiae, the addition of myristate restores wild-typegrowth to most ts nmt mutants (14). Strain AXL19 containingthe swoF mutation did not produce a mycelial colony at 42°C(Fig. 6c). However, with the addition of 500 µM myristate,AXL19 produced a filamentous colony (Fig. 6d), although it wassmaller than the colony produced by swoF⁺ strain A773 (Fig.6b). Microscopically, A773 establishes characteristic long filamentousgerm tubes in minimal medium with or without 500 µM myristate(Fig. 6e and h). swoF cells grown in minimal medium at restrictivetemperature either failed to establish polarity (Fig. 1d and6g) or established but could not maintain polarity (Fig. 1b).Viewed microscopically, swoF cells grown in minimal medium amendedwith 500 µM myristate established and maintained polaritybut showed abnormal swelling within spores and hyphae. Germtubes were also thicker and shorter than those of the wild typein the presence of myristate (Fig. 6f).

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FIG. 6. Medium supplementation with myristate partially restores the growth of swoF cells. (a and c) Wild-type and swoF colonies, respectively, after 48 h of incubation in minimal medium at restrictive temperature. (b and d) Wild-type and swoF colonies, respectively, after 48 h of incubation in minimal medium supplemented with 500 µM myristate and 1% Brij 58 at restrictive temperature. (e and g) Wild-type and swoF germlings, respectively, after 16 h of incubation in minimal medium at restrictive temperature. (h and f) Wild-type and swoF germlings, respectively, after 16 h of incubation in minimal medium supplemented with 500 µM myristate and 1% Brij 58 at restrictive temperature. Bars: d (for a to d), 1 cm; f (for e to h), 10 µm.

DISCUSSION

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Discussion

SwoFp is an NMT. The swoF mutant was restored to wild-type growth by introductionof an A. nidulans gene predicted to encode an NMT. Given thehigh level of sequence conservation among NMTs, the identificationof the complementing gene as an NMT gene is unambiguous. Thepresence of the D369Y mutation in the swoF gene in the mutantargues that the NMT is not a suppressor of swoF. Myristate amendmentof medium restores wild-type growth to nmt mutants in S. cerevisiae,Cryptococcus neoformans, and other organisms (23, 30). Indeed,the first S. cerevisiae nmt mutant was discovered as a myristateauxotroph (33). Thus, the ability of myristate to partiallyrestore wild-type growth to the swoF mutant further argues thatthe mutant lesion is within the NMT gene. Myristate remediationis thought to increase the pools of myristoyl-CoA enough toovercome any myristoyl-CoA binding defect found in mutant Nmtproteins. It is also possible that myristate stabilizes thefolding of the mutant protein. In this light, it is interestingthat myristic acid allows only limited growth of the swoF mutant.Perhaps lower levels of NMT activity are sufficient for thevery limited polar growth of yeasts such as S. cerevisiae andC. neoformans but are not sufficient for the highly polar growthof filamentous fungi such as A. nidulans.

Many NMT orthologues have been reported in the literature, includingrepresentatives from S. cerevisiae (15), C. albicans (46), Ajellomycescapsulatus (31), C. neoformans (30), and Homo sapiens (17).In addition, NMT orthologues from Schizosaccharomyces pombeand A. fumigatus are present in GenBank. NMT genes are single-copygenes in all fungi in which they have been characterized (30).Disruption of this single copy is lethal in S. cerevisiae (15),C. albicans (43), and C. neoformans (30). NMTs from C. albicans,C. neoformans, and H. sapiens functionally complement the lethalS. cerevisiae null allele (31). A preliminary search of theA. nidulans expressed sequence tag database (http://www.genome.ou.edu/asperblast.html)indicates only one NMT in A. nidulans. Additionally, in Southernanalysis of A. nidulans genomic DNA, a swoF probe hybridizesto only one band (B. D. Shaw and M. Momany, unpublished data).

SwoFp homology model. Using the X-ray crystal structures of NMTs from S. cerevisiaeand C. albicans as templates, we homology modeled A. nidulansSwoFp (Fig. 5). Because most residues involved in substratebinding, catalysis, and structural stability among the NMTsare absolutely conserved, our model is likely very accurate.As can be seen in the template structures, the predicted SwoFpconsists of two ß sheets related by a pseudo-twofoldsymmetry axis and flanked by ${alpha}$ helices. Most of the ts mutationspreviously reported for NMTs are either in the central coreor in residues very near the substrate binding sites and resultin higher K_m and/or lower V_max values relative to those forthe wild type (Fig. 4) (3, 49). We expected that the D369Y lesionin the swoF mutant would also fall within the core or near thepockets where myristoyl-CoA and peptide ligands bind. However,the mutated residue is at the amino-terminal end of ß-strandßk, near the surface of the protein and on the faceopposite that of the substrate binding sites. The carboxy-terminalend of ß-strand ßk interacts with the peptidesubstrate. Therefore, one explanation for the temperature sensitivityis that the D369Y mutation distorts the orientation of strandßk and thus distorts peptide substrate binding. Itseems more likely, however, that the D369Y mutation destabilizesthe tertiary structure in the local region around strand ßkand thus has a more global effect. Figure 5b illustrates thelocal environment around D369. In the wild-type protein, D369stabilizes a bend at the amino-terminal end of ßkby hydrogen bonding to T368 and bridges the side chains of neighboringsecondary structures via an ionic interaction with R413 (helix ${alpha}$ H) and hydrogen bonds with T368 and T399 (ß-strandßi). T368 and D369 are absolutely conserved acrossspecies (49), suggesting that these residues play a criticalrole in NMTs. Alhough R413 and T399 are not conserved in highereukaryotes, conservative replacements, such as K for R413 andS for T399, could maintain these interactions and substitutefunctionally. While modeling suggests that the bulky side chainof Y369 in the swoF mutant could be accommodated with some adjustmentof the surrounding residues, the stabilizing effects of theionic and hydrogen bonds would be completely lost. Destabilizationof strand ßk in the mutant could result in lower SwoFpactivity levels through conformational changes affecting substratebinding and catalytic function. Alternatively, destabilizationmight lead to lower steady-state levels of SwoFp either fromimproper folding or from increased proteolytic sensitivity.If improper folding is the main defect in SwoFp, it raises theintriguing possibility that myristate remediation acts by furnishinga scaffold around which the myrisotyl-CoA binding pocket canbe folded or stabilized.

Conclusion. Polar growth is essential for the pathogenicity of A. fumigatusand many other fungal pathogens of animals and plants. Polargrowth is a two-step process, and SwoFp plays a role in thefirst step (polarity establishment) and is absolutely requiredfor the second step (polarity maintenance). Our current modelfor the involvement of SwoFp in polarity is that a target protein(s)required for polar growth must be myristoylated to be properlylocalized and/or fully active. At restrictive temperature, mutantSwoFp is unable to properly myristoylate its target. In thislight, a recent report that an sgdD germination mutant is defectivein a malonyl-CoA synthetase (36) is intriguing. This proteinis involved in the formation of acetyl-CoA, a necessary precursorin the production of myristoyl-CoA. It is possible that sgdDis needed for the production of the myristoyl-CoA necessaryfor the myristoylation of an as-yet-unknown target responsiblefor the initiation of germination and polar growth.

NMTs are a highly conserved class of eukaryotic proteins; however,their substrate specificities have diverged, with fungal NMTsgenerally having targets different from those of mammalian NMTs(12, 13, 27, 30, 38). This target divergence, coupled with thefact that deletion of the single copy of the NMT gene in fungiis lethal (15, 30, 43), has led to considerable interest inNMTs as possible targets for a new class of antifungal drugs.This approach has met with success in C. albicans and C. neoformans,for which compounds that specifically inhibit myristoylationare being tested (11, 12, 26, 29, 42). To our knowledge, thisoption has not yet been explored for A. fumigatus, now the mostcommon fungal pathogen found in autopsies of immunocompromisedpatients in European hospitals (19). This antifungal potential,coupled with the key role that myristoylation plays in polargrowth (a critical aspect of fungal pathogenesis), is a fruitfularea for further investigation.

ACKNOWLEDGMENTS

This work was supported by DOE biosciences grant DE-FG02-97ER20275to M.M.

We thank Greg May (University of Texas M. D. Anderson CancerCenter) for providing the genomic library used in this study,Xiaorong Lin (University of Georgia) for assistance in sequencingand crosses to create the swoF strain used in this study, andGreg Derda (University of Georgia) for computer assistance.

FOOTNOTES

* Corresponding author. Mailing address: Department of Botany, University of Georgia, Athens, GA 30602. Phone: (706) 542-2014. Fax: (706) 542-1805. E-mail: momany{at}botany.uga.edu.

REFERENCES

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