In vitro synthesis of pp60v-src: myristylation in a cell-free system.

Abstract

Covalent attachment of myristic acid to pp6o0-sc, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60 V-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp6o0-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.Covalent attachment of the 14-carbon fatty acid, myristic acid, has been shown to occur on a wide variety of viral and cellular proteins (11,18,22,23,35,41). One of the bestcharacterized myristylated proteins is pp6O-srC, the transforming protein of Rous sarcoma virus (RSV; 6, 14). Studies of numerous point and deletion mutants of pp6O-src which lack the myristate attachment site have established that myristylation is a necessary event for directing pp60v-sr to the plasma membrane. Mutant pp6O-src molecules which are not myristylated remain in the cytoplasm and do not transform cells (10, 15).The structural requirements for myristylation of a polypeptide chain have recently been elucidated. Myristate is attached through an amide linkage with a glycine residue at the amino terminus of the acceptor protein (7,24,34) by N-myristyl transferase (NMT). An amino-terminal glycine is essential but not sufficient for myristylation, and a serine or threonine residue is present at position 5 in nearly all myristylated proteins (39, 40). Additional sequence determinants are important, since deletions within the first 10 amino acids of the mature pp6v-vsrc polypeptide sequence abolish myristylation of the resultant protein (26,27). However, no clear consensus sequence seems to exist among all of the known myristylated proteins.Myristylation of both viral and cellular proteins has been shown to be an early event during protein biosynthesis. Treatment of cells with cycloheximide, an inhibitor of protein synthesis, results in immediate cessation of myristylation (5,18,20,22). Moreover, nascent polypeptide chains which are covalently associated with tRNA are already myristylated (44), implying that myristylation occurs cotranslationally. Exactly when t...

Molecular characterization of Calymmin, a novel notochord sheath‐associated extracellular matrix protein in the zebrafish embryo

“…The detection of a broader reactive band corresponding to Calymmin, in both in vitro transcription-translation and embryo protein extracts, may suggest the existence of posttranslational modifications of the peptide both in vivo and in cell-free systems. This would agree with the various N-and mucin type Oglycosylation conserved motifs, and the high number of potential N-myristoylation sites found in the amino acid sequence of Calymmin (e.g., Deichaite et al, 1988).…”

Amino Acid Residue Penultimate to the Amino-terminal Gly Residue Strongly Affects Two Cotranslational Protein Modifications, N-Myristoylation andN-Acetylation

“…Since both NMTs and methionine aminopeptidases function cotranslationally while the nascent polypeptide chain is still attached to the ribosomes (20,36,37), it might be possible to speculate that these two enzymes may share similar restrictions with respect to their substrate specificities.…”

“…Since the in vitro translation system using rabbit reticulocyte lysate contains the components involved in cotranslational protein N-myristoylation and N-acetylation (17,19,20), the use of this system to study cotranslational protein modification seems to be appropriate. In fact, we previously demonstrated that tumor necrosis factor (TNF), a nonmyristoylated model protein, could be efficiently myristoylated in the in vitro trans-lation system when an N-myristoylation motif of Rasheed leukemia virus-Gag protein or G i1 ␣ protein was linked to the mature domain of TNF (21,22).…”

Conserved Domains of the Nullo Protein Required for Cell-Surface Localization and Formation of Adherens Junctions

“…Because the mechanism for N-terminal myristoylation appears to be conserved in Drosophila (Deichaite et al, 1988;Ntwasa et al, 1997;Benting et al, 2000), it is likely that Nullo exists as a myristoylprotein in vivo.…”

“…First, the consensus target for NMT activity appears to be well conserved between Drosophila and other organisms. Both Drosophila embryonic extracts and Schnieder cells have been shown to have an NMT activity that modifies myristoyl proteins from other organisms in a specific manner, and the corresponding NMT gene has been shown to be expressed before cellularization (Deichaite et al, 1988;Ntwasa et al, 1997;Benting et al, 2000). Second, the glycine to alanine mutation which blocks myristoylation in vitro has a striking effect on the subcellular localization of Nullo in vivo, suggesting that the N-myristoylation site is important for the targeting of Nullo to the plasma membrane.…”