Myristoylation (Molecular Biology)

N-Myristoylation is a cotranslational process in which eukaryotic or viral proteins become amide-linked to the 14-carbon saturated fatty acid, myristic acid (see Fig. 1 and Table 1). Myristoylation occurs on the a-amino group of the N-terminal glycine residue exposed after removal of the initiator methionine residue (see Translation). The reaction takes place in the cytosol, and is catalyzed by N-myristoyl transferase using myristoyl CoA as the myristic acid donor. Recent work suggests that N-myristoylation may also occur post-translationally but as yet there is only one instance of this type of reaction (1). Myristoylation provides some proteins with a relatively weak membrane anchor, and is important for correctly localizing them within the cell. Myristoylation also occurs on many proteins that do not appear to associate with membranes. The role of the myristoyl group in these proteins is less clear, but may involve stabilizing the protein structure, facilitating protein-protein interactions by binding to a site on another protein, and viral assembly.

Figure 1. Modification of an N-terminal glycine residue by a myristoyl group. The N-terminal glycine residue of the protein (outlined by the dotted line) is amide-linked via its exposed a-amino group to the 14-carbon saturated fatty acid, myristic acid.

Table 1. Examples of Myristoylated Proteins

In studies with the purified N-myristoyl transferase and synthetic peptide substrates, the only amino acid that is absolutely required for myristoylation is the N-terminal glycine (2, 3). This observation agrees well with the known distribution of myristoylation among naturally occurring proteins. On the other hand, the identity of residues at positions 2-8 can also have some influence on the efficiency of myristoylation. For example, substitution of residue Ser5 (in G-protein as subunits) with an Asp residue may be the reason that as subunits are not myristoylated, in contrast to most other a subunits.

A search of the yeast genome database suggests that about 1% of yeast ORFs will encode myristoylated proteins (4). Palmitoyl CoA is not a substrate for N-myristoyl transferase, which accounts for the relative lack of naturally occurring N-palmitoylated proteins. The transferase is less selective for shorter acyl chain lengths (eg, C^) or for degree of unsaturation (3). Detailed analysis of several myristoylated proteins reveals considerable acyl heterogeneity, and in some cases myristic acid is not even the most abundant molecular species.

That myristoylation occurs on proteins that do not bind to membranes suggests that the myristoyl group, unlike other lipid groups attached to proteins, may have functions other than membrane anchoring, but there is relatively little detailed information as to what these other functions might be. The amide linkage in N-myristoylated proteins is relatively resistant to hydrolysis, and is not degraded or turned over metabolically. Myristoylated and nonmyristoylated forms of the cyclic AMP-dependent protein kinase regulatory subunit have similar kinetic properties, and are equal in their ability to associate with the catalytic subunit. The myristate, however, does seem to increase thermal stability of the kinase, possibly by binding to an adjacent hydrophobic region of the protein (5). Myristoyl groups also seem to play a role in stabilizing interactions between protein molecules. This may be especially important for viral assembly, eg, the interaction between VP4 and VP3 on the inner surface of the polio virus capsid. The short length and lack of bulky side chains may make myristate particularly versatile and capable of binding to a membrane or to a protein site, a feature that is exploited for regulatory purposes as described below.

The myristoyl group has a relatively low affinity for membranes and by itself results only in weak and transient binding of proteins to membranes (6-8). It has been shown that membrane affinity of myristoylated proteins can be altered by three distinct mechanisms and may provide a way to regulate their distribution within the cell (for a general discussion of factors that affect membrane affinity of lipid-anchored proteins see Membrane Anchors):

1. Basic amino acid residues close to the N-terminus will bind to the negatively charged phospholipid bilayer and increase the membrane affinity, as proposed for the tyrosine kinase p60src and the MARCKS protein. The strength of these electrostatic interactions is reduced by phosphorylation of the MARCKS protein, causing it to redistribute within the cell (9).

2. Palmitoylation of a cysteine residue close to the amino terminus increases the affinity of myrisoylated proteins for intracellular membranes (10, 11). This occurs in some G protein a subunits (eg, ao) and Src family tyrosine kinases (eg, p59fn and p56lck). Palmitoylation of these proteins also depends on prior myristoylation, because the palmitoyltransferase activities are localized in membranes (see Palmitoylation).

3. The myristoyl group may also bind to a site on the protein itself, which prevents it from acting as a membrane anchor. The myristoyl group might be extruded by a conformational change that "closes" the binding site, causing the protein to bind to the membrane. This mechanism has been proposed for recoverin and hippocalcin (Ca -sensing proteins in the visual and nervous systems) and for ADP ribosylation factor, where the conformational changes occurs in response to Ca and GTP, respectively (12).