Abstract
N-terminal myristoylation is a cotranslational lipid modification common to many signaling proteins that often serves an integral role in the targeting and/or function of these proteins. Myristoylation is catalyzed by an enzyme activity, N-myristoyltransferase (NMT), which transfers myristic acid from myristoyl coenzyme A to the amino group of a protein's N-terminal glycine residue. While a single human NMT cDNA has been isolated and characterized (hNMT-1), biochemical evidence has indicated the presence of several distinct NMTs in vivo, often varying in either apparent molecular weight and/or subcellular distribution. We now report the cloning and characterization of a second, genetically distinct human NMT (hNMT-2), as well as the isolation of the respective mouse NMT homologue for each human enzyme. The mouse and human versions of each NMT are highly homologous, displaying greater than 95% amino acid sequence identity. Comparisons between the NMT-1 and NMT-2 proteins revealed reduced levels of sequence identity (76-77%), indicating that NMT-1 and NMT-2 comprise two distinct families of N-myristoyltransferases. Transient transfection of either the hNMT-1 or hNMT-2 cDNA into COS-7 cells resulted in the expression of high levels of NMT enzyme activity. Both hNMT-1 and hNMT-2 were found to myristoylate several commonly studied peptide substrates with similar, but distinguishable, relative selectivities. Western analysis revealed that while hNMT-2 appeared as a single 65-kDa protein in transfected COS-7 cells, hNMT-1 was processed to provide four distinct protein isoforms ranging from 49 to 68 kDa in size. Collectively, these studies demonstrate a heretofore unappreciated level of genetic complexity underlying the enzymology of N-terminal myristoylation and suggest that the specific inhibition or regulation of either NMT in vivo may in turn allow for the selective control of particular myristoylation-dependent cellular functions.
Highlights
Genetic studies have shown that the NMT gene is essential for the viability of the yeast, Saccharomyces cerevisiae [10], and the pathogenic fungi, Candida albicans [11] and Cryptococcus neoformans [12]
We report the isolation and characterization of a second distinct NMT cDNA from a human liver library, as well as the cloning of the respective mouse homologue for each of the two human NMTs
Cloning of Human and Mouse NMT cDNAs—PCR primers based on the sequence of expressed sequence tag (EST) AA036845 were designed for the amplification of a 550-base pair portion of the hNMT-2 cDNA from a human liver 5ЈStretch Plus cDNA library (CLONTECH): Primer 1, 5Ј-GCGAATTCAACATCCACACAGAGACGCCC-3Ј; Primer 2, 5Ј-GCGAATTCTCTGTAACTTCTACTTAGTCC-3Ј
Summary
(Received for publication, December 23, 1997, and in revised form, January 21, 1998). Transient transfection of either the hNMT-1 or hNMT-2 cDNA into COS-7 cells resulted in the expression of high levels of NMT enzyme activity Both hNMT-1 and hNMT-2 were found to myristoylate several commonly studied peptide substrates with similar, but distinguishable, relative selectivities. Western analysis revealed that while hNMT-2 appeared as a single 65-kDa protein in transfected COS-7 cells, hNMT-1 was processed to provide four distinct protein isoforms ranging from 49 to 68 kDa in size These studies demonstrate a heretofore unappreciated level of genetic complexity underlying the enzymology of N-terminal myristoylation and suggest that the specific inhibition or regulation of either NMT in vivo may in turn allow for the selective control of particular myristoylation-dependent cellular functions. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF043324 (hNMT-1), AF043325 (hNMT-2), AF043326 (mNMT-1), and AF043327 (mNMT-2)
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