N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADPribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes.