Aspergillus nidulans swoF encodes an N-myristoyl transferase

Abstract

Polar growth is a fundamental process in filamentous fungi and is necessary for disease initiation in many pathogenic systems. Previously, swoF was identified in Aspergillus nidulans as a single-locus, temperature-sensitive (ts) mutant aberrant in both polarity establishment and polarity maintenance. The swoF gene was cloned by complementation of the ts phenotype and sequenced. The derived protein sequence had high identity with N-myristoyl transferases (NMTs) found in fungi, plants, and animals. In addition, wild-type growth at restrictive temperature was partially restored by the addition of myristic acid to the growth medium. Sequencing revealed that the mutation in swoF changes the conserved aspartic acid 369 to a tyrosine. The predicted A. nidulans SwoF protein, SwoFp, was homology modeled based on crystal structures of 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 binding sites. In wild-type NMTs, D369 appears to stabilize a structural beta-strand bend through two hydrogen bonds and an ionic interaction. These stabilizing bonds are abolished in the D369Y mutant. We hypothesize that a substrate of SwoFp must be myristoylated for proper polarity establishment and maintenance. The mutation prevents the proper function of SwoFp at restrictive temperature and thus blocks polar growth.

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.

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 β-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.

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).

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 (β strands) or cylinders (α 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.

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 β-strand βk and is shown in ball-stick representation and colored black. Helix αH 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.

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.