Abstract
Myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) catalyzes the co-translational addition of myristic acid to the amino-terminal glycine residue of a number of important proteins of diverse functions. We have isolated a full-length Arabidopsis thaliana cDNA encoding NMT (AtNMT1), the first described from a higher plant. This AtNMT1 cDNA clone has an open reading frame of 434 amino acids and a predicted molecular mass of 48,706 Da. The primary structure is 50% identical to the mammalian NMTs. Analyses of Southern blots, genomic clones, and database sequences suggested that the A. thaliana genome contains two copies of NMT gene, which are present on different chromosomes and have distinct genomic organizations. The recombinant AtNMT1 expressed in Escherichia coli exhibited a high catalytic efficiency for the peptides derived from putative plant myristoylated proteins AtCDPK6 and Fen kinase. The AtNMT was similar to the mammalian NMTs with respect to a relative specificity for myristoyl CoA among the acyl CoA donors and also inhibition by the bovine brain NMT inhibitor NIP(71). The AtNMT1 expression profile indicated ubiquity in roots, stem, leaves, flowers, and siliques (approximately 1.7 kb transcript and approximately 50 kDa immunoreactive polypeptide) but a greater level in the younger tissue, which are developmentally very active. NMT activity was also evident in all these tissues. Subcellular distribution studies indicated that, in leaf extracts, approximately 60% of AtNMT activity was associated with the ribosomal fractions, whereas approximately 30% of the activity was observed in the cytosolic fractions. The NMT is biologically important to plants, as noted from the stunted development when the AtNMT1 was down-regulated in transgenic Arabidopsis under the control of an enhanced CaMV 35S promoter. The results presented in this study provide the first direct molecular evidence for plant protein N-myristoylation and a mechanistic basis for understanding the role of this protein modification in plants.
Highlights
Myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) catalyzes the co-translational addition of myristic acid to the amino-terminal glycine residue of a number of important proteins of diverse functions
The NMT is biologically important to plants, as noted from the stunted development when the AtNMT1 was down-regulated in transgenic Arabidopsis under the control of an enhanced CaMV 35S promoter
Cloning of an Arabidopsis cDNA Encoding Myristoyl-CoA: Protein N-Myristoyltransferase (AtNMT1) and Its Predicted Structural Features—To identify NMT homologues in Arabidopsis, we first demonstrated the presence of NMT in Arabidopsis by a series of biochemical experiments
Summary
Materials—gt cDNA and EMBL3 genomic libraries of Arabidopsis were obtained from CLONTECH Laboratories Inc. Similar results were obtained when either the purified recombinant AtNMT1 or the peptide conjugate was adsorbed onto an enzyme immunoassay plate (data not shown). These results confirm the specificity of the antipeptide antibody for AtNMT1. The microsomal supernatant was further centrifuged at 100,000 ϫ g for 70 min, yielding the ribosomal pellet and the cytosolic fraction with supernatant Each of these pellets was washed once using resuspension buffer (100 mM Tris-HCl, pH 7.6, containing 4 mM EDTA, 2 mM EGTA, 10 mM 2-mercaptoethanol, 1 mM benzamidine, 0.2 mM PMSF, and 0.7% g/ml leupeptin), centrifuged again, and redissolved in resuspension buffer. For the standard enzyme assays, the reaction mixture contained 0.4 M [3H]myristoyl-CoA, 50 mM Tris-HCl, pH 7.8, 0.5 mM EGTA, 0.1% Triton X-100, synthetic peptide and AtNMT1 enzyme in a total volume of 25 l. The enrichment of ribonucleoprotein in the ribosomal fraction was assessed by A260/A280 and A235/A280 absorption ratios [51]
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