N-Myristoylation is an irreversible modification that affects the membrane 
binding properties of crucial cytoplasmic proteins from signal transduction 
cascades. We characterized the two putative N-myristoyltransferases of 
Arabidopsis thaliana as a means of investigating the entire N-myristoylation 
proteome (N-myristoylome) in a higher eukaryote. AtNMT1 compensated for the nmt1 
defect in yeast, whereas AtNMT2 and chimeras of the two genes did not. Only 
AtNMT1 modified known N-myristoylated proteins in vitro. AtNMT1 is therefore 
responsible for the A. thaliana N-myristoylome, whereas AtNMT2 does not seem to 
have usual myristoylation activity. We began with the whole set of 
N-myristoylated G proteins in the A. thaliana proteome. We then used a 
reiterative approach, based on the in vitro N-myristoylation of more than 60 
different polypeptides, to determine the substrate specificity of AtNMT1. We 
found that the positive charge on residue 7 of the substrate was particularly 
important in substrate recognition. The A. thaliana N-myristoylome consists of 
437 proteins, accounting for 1.7% of the complete proteome. We demonstrated the 
N-myristoylation of several unexpected protein families, including innate 
immunity proteins, thioredoxins, components of the protein degradation pathway, 
transcription factors, and a crucial regulatory enzyme of glycolysis. The role 
of N-myristoylation is discussed in each case; in particular, this process may 
underlie the "guard" hypothesis of innate immunity.