N-Myristoyltransferase 1 Is Essential in Early Mouse Development

Abstract

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a ␤-galactosidase marker gene. ␤-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1؉/؊) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1؉/؊ mice yielded no viable homozygotes (Nmt1؊/؊), and heterozygotes were born at a less than predicted frequency. Nmt1؊/؊ embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1؊/؊ embryos. To explore this concept, we generated Nmt1؊/؊ embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1؊/؊ ES cells, but the total NMT activity levels were reduced by ϳ95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1؊/؊ ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.

Protein myristoylation in health and disease

“…Little is known about distinct functions of the NMT1 isoforms, but recent work investigating NMT1 and NMT2 has established that they have overlapping but distinct substrate specificities in vivo and in vitro [63], potentially translating into different roles in development and cellular processes such as apoptosis and proliferation. Young and co-workers investigated the functional redundancy of NMT isozymes in vivo by knocking out the NMT1 gene in mice via an insertional mutation process termed gene trapping [33]. They found that in normal mice NMT1 and NMT2 were expressed to a similar extent in a wide variety of tissues but that in embryos NMT1 was expressed more than NMT2.…”

“…NMT has been shown to be essential for the survival of S. cerevisiae [29], C. albicans [30], C. neoformans [31] and the bloodstream forms of the parasites L. major and T. brucei [32]. It is important in the development of Drosophila [27] and mice [33]. There is no precisely defined "myristoylation motif", although substrate specificity can be rationalised to a certain extent and the enzyme has an absolute requirement for a peptide or protein substrate with glycine at the N terminus.…”

Filamin B deficiency in mice results in skeletal malformations and impaired microvascular development

“…Frozen tissue pieces were sectioned and fixed by using 0.25% glutaraldehyde in PBS for 5-10 min. The procedure for X-Gal staining was similar to that described previously (22). Briefly, sections were incubated at 37°C in PBS supplemented with 1 mg/ml X-Gal (Sigma)/5 mM potassium ferricyanide/5 mM potassium ferrocyanide/2 mM MgCl 2 .…”

N-myristoyltransferase in the leukocytic development processes

“…Through the suppression of constitutive apoptosis, activated neutrophils live longer. NMT1 and NMT2 have reported roles in cell survival and embryogenesis (Wilcox et al 1987;Duronio et al 1989;Lodge et al 1994;Farazi et al 2001;Ntwasa et al 2001;Yang et al 2005). When probed for their roles in neutrophil longevity, the knockdown of either NMT1 or NMT2, both in the presence or absence of LPS, induces ~30% cell death compared with the control (Shrivastav et al 2010).…”

“…The NMT1 isoform is homologous to the NMT from lower eukaryotes and has been shown the rescue myistic acid auxotrophy in yeast (Duronio et al 1992). With respect to the functional importance of the NMT1 and NMT2 enzymes in vivo, it has been observed that during the embryonic development of mice, NMT2 is not able to rescue N-myristoylation of proteins for the proper development of the embryos in NMT1 −/− mice knockouts and the embryos die during early embryogenesis (Yang et al 2005). This clearly indicates the specific roles played by the individual NMTs and further suggests that the two isoforms cannot compensate for each other's specific functional roles.…”